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Sample records for macromolecular crystallography mad

  1. The New Macromolecular Crystallography Stations At MAX-lab: The MAD Station

    SciTech Connect

    Ursby, Thomas; Svensson, Christer; Sommarin, Bengt; Mammen, Christian B.; Als-Nielsen, Jens; Cerenius, Yngve; Fodje, Michel N.; Logan, Derek T.; Thunnissen, Marjolein M. G. M.; Liljas, Anders; Larsen, Sine

    2004-05-12

    A new beamline, Cassiopeia, at MAX II is about to come into operation. It consists of an energy-tunable station and four side stations intended for macromolecular crystallography. The X-ray source is a 3.5 T superconducting multipole wiggler installed in the 1.5 GeV MAX II storage ring. The energy-tunable station use grazing incidence Rh-coated silicon mirrors and an internally water-cooled Si(111) double-crystal monochromator while the four side stations use bent diamond and germanium monochromators and multilayer mirrors. This paper concentrates on the optics design of the energy-tunable station and also briefly describes other beamline components.

  2. A decade of user operation on the macromolecular crystallography MAD beamline ID14-4 at the ESRF

    PubMed Central

    McCarthy, Andrew A.; Brockhauser, Sandor; Nurizzo, Didier; Theveneau, Pascal; Mairs, Trevor; Spruce, Darren; Guijarro, Matias; Lesourd, Marc; Ravelli, Raimond B. G.; McSweeney, Sean

    2009-01-01

    ID14-4 at the ESRF is the first tunable undulator-based macromolecular crystallography beamline that can celebrate a decade of user service. During this time ID14-4 has not only been instrumental in the determination of the structures of biologically important molecules but has also contributed significantly to the development of various instruments, novel data collection schemes and pioneering radiation damage studies on biological samples. Here, the evolution of ID14-4 over the last decade is presented, and some of the major improvements that were carried out in order to maintain its status as one of the most productive macromolecular crystallography beamlines are highlighted. The experimental hutch has been upgraded to accommodate a high-precision diffractometer, a sample changer and a large CCD detector. More recently, the optical hutch has been refurbished in order to improve the X-ray beam quality on ID14-4 and to incorporate the most modern and robust optical elements used at other ESRF beamlines. These new optical elements will be described and their effect on beam stability discussed. These studies may be useful in the design, construction and maintenance of future X-ray beamlines for macromolecular crystallography and indeed other applications, such as those planned for the ESRF upgrade. PMID:19844017

  3. Microgravity and Macromolecular Crystallography

    NASA Technical Reports Server (NTRS)

    Kundrot, Craig E.; Judge, Russell A.; Pusey, Marc L.; Snell, Edward H.; Rose, M. Franklin (Technical Monitor)

    2000-01-01

    Macromolecular crystal growth has been seen as an ideal experiment to make use of the reduced acceleration environment provided by an orbiting spacecraft. The experiments are small, simply operated and have a high potential scientific and economic impact. In this review we examine the theoretical reasons why microgravity should be a beneficial environment for crystal growth and survey the history of experiments on the Space Shuttle Orbiter, on unmanned spacecraft, and on the Mir space station. Finally we outline the direction for optimizing the future use of orbiting platforms.

  4. Automated data collection for macromolecular crystallography.

    PubMed

    Winter, Graeme; McAuley, Katherine E

    2011-09-01

    An overview, together with some practical advice, is presented of the current status of the automation of macromolecular crystallography (MX) data collection, with a focus on MX beamlines at Diamond Light Source, UK.

  5. In situ macromolecular crystallography using microbeams.

    PubMed

    Axford, Danny; Owen, Robin L; Aishima, Jun; Foadi, James; Morgan, Ann W; Robinson, James I; Nettleship, Joanne E; Owens, Raymond J; Moraes, Isabel; Fry, Elizabeth E; Grimes, Jonathan M; Harlos, Karl; Kotecha, Abhay; Ren, Jingshan; Sutton, Geoff; Walter, Thomas S; Stuart, David I; Evans, Gwyndaf

    2012-05-01

    Despite significant progress in high-throughput methods in macromolecular crystallography, the production of diffraction-quality crystals remains a major bottleneck. By recording diffraction in situ from crystals in their crystallization plates at room temperature, a number of problems associated with crystal handling and cryoprotection can be side-stepped. Using a dedicated goniometer installed on the microfocus macromolecular crystallography beamline I24 at Diamond Light Source, crystals have been studied in situ with an intense and flexible microfocus beam, allowing weakly diffracting samples to be assessed without a manual crystal-handling step but with good signal to noise, despite the background scatter from the plate. A number of case studies are reported: the structure solution of bovine enterovirus 2, crystallization screening of membrane proteins and complexes, and structure solution from crystallization hits produced via a high-throughput pipeline. These demonstrate the potential for in situ data collection and structure solution with microbeams.

  6. EIGER detector: application in macromolecular crystallography

    PubMed Central

    Casanas, Arnau; Warshamanage, Rangana; Finke, Aaron D.; Panepucci, Ezequiel; Olieric, Vincent; Nöll, Anne; Tampé, Robert; Brandstetter, Stefan; Förster, Andreas; Mueller, Marcus; Schulze-Briese, Clemens; Bunk, Oliver; Wang, Meitian

    2016-01-01

    The development of single-photon-counting detectors, such as the PILATUS, has been a major recent breakthrough in macromolecular crystallography, enabling noise-free detection and novel data-acquisition modes. The new EIGER detector features a pixel size of 75 × 75 µm, frame rates of up to 3000 Hz and a dead time as low as 3.8 µs. An EIGER 1M and EIGER 16M were tested on Swiss Light Source beamlines X10SA and X06SA for their application in macromolecular crystallography. The combination of fast frame rates and a very short dead time allows high-quality data acquisition in a shorter time. The ultrafine φ-slicing data-collection method is introduced and validated and its application in finding the optimal rotation angle, a suitable rotation speed and a sufficient X-ray dose are presented. An improvement of the data quality up to slicing at one tenth of the mosaicity has been observed, which is much finer than expected based on previous findings. The influence of key data-collection parameters on data quality is discussed. PMID:27599736

  7. Fifteen years of the Protein Crystallography Station: The coming of age of macromolecular neutron crystallography

    DOE PAGES

    Chen, Julian C.-H.; Unkefer, Clifford Jay

    2017-01-01

    The Protein Crystallography Station (PCS), located at the Los Alamos Neutron Scattering Center (LANSCE), was the first macromolecular crystallography beamline to be built at a spallation neutron source. Following testing and commissioning, the PCS user program was funded by the Biology and Environmental Research program of the Department of Energy Office of Science (DOE-OBER) for 13 years (2002–2014). The PCS remained the only dedicated macromolecular neutron crystallography station in North America until the construction and commissioning of the MaNDi and IMAGINE instruments at Oak Ridge National Laboratory, which started in 2012. The instrument produced a number of research and technicalmore » outcomes that have contributed to the field, clearly demonstrating the power of neutron crystallography in helping scientists to understand enzyme reaction mechanisms, hydrogen bonding and visualization of H-atom positions, which are critical to nearly all chemical reactions. During this period, neutron crystallography became a technique that increasingly gained traction, and became more integrated into macromolecular crystallography through software developments led by investigators at the PCS. As a result, this review highlights the contributions of the PCS to macromolecular neutron crystallography, and gives an overview of the history of neutron crystallography and the development of macromolecular neutron crystallography from the 1960s to the 1990s and onwards through the 2000s.« less

  8. Fifteen years of the Protein Crystallography Station: the coming of age of macromolecular neutron crystallography.

    PubMed

    Chen, Julian C-H; Unkefer, Clifford J

    2017-01-01

    The Protein Crystallography Station (PCS), located at the Los Alamos Neutron Scattering Center (LANSCE), was the first macromolecular crystallography beamline to be built at a spallation neutron source. Following testing and commissioning, the PCS user program was funded by the Biology and Environmental Research program of the Department of Energy Office of Science (DOE-OBER) for 13 years (2002-2014). The PCS remained the only dedicated macromolecular neutron crystallography station in North America until the construction and commissioning of the MaNDi and IMAGINE instruments at Oak Ridge National Laboratory, which started in 2012. The instrument produced a number of research and technical outcomes that have contributed to the field, clearly demonstrating the power of neutron crystallo-graphy in helping scientists to understand enzyme reaction mechanisms, hydrogen bonding and visualization of H-atom positions, which are critical to nearly all chemical reactions. During this period, neutron crystallography became a technique that increasingly gained traction, and became more integrated into macromolecular crystallography through software developments led by investigators at the PCS. This review highlights the contributions of the PCS to macromolecular neutron crystallography, and gives an overview of the history of neutron crystallography and the development of macromolecular neutron crystallography from the 1960s to the 1990s and onwards through the 2000s.

  9. Fifteen years of the Protein Crystallography Station: The coming of age of macromolecular neutron crystallography

    SciTech Connect

    Chen, Julian C.-H.; Unkefer, Clifford Jay

    2017-01-01

    The Protein Crystallography Station (PCS), located at the Los Alamos Neutron Scattering Center (LANSCE), was the first macromolecular crystallography beamline to be built at a spallation neutron source. Following testing and commissioning, the PCS user program was funded by the Biology and Environmental Research program of the Department of Energy Office of Science (DOE-OBER) for 13 years (2002–2014). The PCS remained the only dedicated macromolecular neutron crystallography station in North America until the construction and commissioning of the MaNDi and IMAGINE instruments at Oak Ridge National Laboratory, which started in 2012. The instrument produced a number of research and technical outcomes that have contributed to the field, clearly demonstrating the power of neutron crystallography in helping scientists to understand enzyme reaction mechanisms, hydrogen bonding and visualization of H-atom positions, which are critical to nearly all chemical reactions. During this period, neutron crystallography became a technique that increasingly gained traction, and became more integrated into macromolecular crystallography through software developments led by investigators at the PCS. As a result, this review highlights the contributions of the PCS to macromolecular neutron crystallography, and gives an overview of the history of neutron crystallography and the development of macromolecular neutron crystallography from the 1960s to the 1990s and onwards through the 2000s.

  10. Fifteen years of the Protein Crystallography Station: the coming of age of macromolecular neutron crystallography

    PubMed Central

    Chen, Julian C.-H.

    2017-01-01

    The Protein Crystallography Station (PCS), located at the Los Alamos Neutron Scattering Center (LANSCE), was the first macromolecular crystallography beamline to be built at a spallation neutron source. Following testing and commissioning, the PCS user program was funded by the Biology and Environmental Research program of the Department of Energy Office of Science (DOE-OBER) for 13 years (2002–2014). The PCS remained the only dedicated macromolecular neutron crystallography station in North America until the construction and commissioning of the MaNDi and IMAGINE instruments at Oak Ridge National Laboratory, which started in 2012. The instrument produced a number of research and technical outcomes that have contributed to the field, clearly demonstrating the power of neutron crystallo­graphy in helping scientists to understand enzyme reaction mechanisms, hydrogen bonding and visualization of H-atom positions, which are critical to nearly all chemical reactions. During this period, neutron crystallography became a technique that increasingly gained traction, and became more integrated into macromolecular crystallography through software developments led by investigators at the PCS. This review highlights the contributions of the PCS to macromolecular neutron crystallography, and gives an overview of the history of neutron crystallography and the development of macromolecular neutron crystallography from the 1960s to the 1990s and onwards through the 2000s. PMID:28250943

  11. Temperature-dependent macromolecular X-ray crystallography

    SciTech Connect

    Weik, Martin Colletier, Jacques-Philippe

    2010-04-01

    The dynamical behaviour of crystalline macromolecules and their surrounding solvent as a function of cryo-temperature is reviewed. X-ray crystallography provides structural details of biological macromolecules. Whereas routine data are collected close to 100 K in order to mitigate radiation damage, more exotic temperature-controlled experiments in a broader temperature range from 15 K to room temperature can provide both dynamical and structural insights. Here, the dynamical behaviour of crystalline macromolecules and their surrounding solvent as a function of cryo-temperature is reviewed. Experimental strategies of kinetic crystallography are discussed that have allowed the generation and trapping of macromolecular intermediate states by combining reaction initiation in the crystalline state with appropriate temperature profiles. A particular focus is on recruiting X-ray-induced changes for reaction initiation, thus unveiling useful aspects of radiation damage, which otherwise has to be minimized in macromolecular crystallography.

  12. Neutron Crystallography for Macromolecular Structure Analysis

    NASA Astrophysics Data System (ADS)

    Kuroki, Ryota

    Hydrogen atoms in proteins as well as protein-bound water molecules play a significant role in many chemical reaction processes in living systems, such as catalytic reaction and molecular recognition. Neutron crystallography is a powerful tool to identify locations of light atoms like hydrogen. In the field of neutron crystallography, the development of diffractometers and techniques for preparation and crystallization of target samples has been developed to complement the low flux of neutron sources. In Japan, single-crystal diffractometers named BIX-3 and BIX-4 have been developed, and contribute to the effective collection of neutron diffraction data. Recent developments on the complementary use of neutron and X-ray diffraction data have begun solving previously undetermined problems of protein function. Further efforts to acquire higher measurement performance are now in progress to increase the application of neutron crystallographic studies.

  13. Graphical tools for macromolecular crystallography in PHENIX

    PubMed Central

    Echols, Nathaniel; Grosse-Kunstleve, Ralf W.; Afonine, Pavel V.; Bunkóczi, Gábor; Chen, Vincent B.; Headd, Jeffrey J.; McCoy, Airlie J.; Moriarty, Nigel W.; Read, Randy J.; Richardson, David C.; Richardson, Jane S.; Terwilliger, Thomas C.; Adams, Paul D.

    2012-01-01

    A new Python-based graphical user interface for the PHENIX suite of crystallography software is described. This interface unifies the command-line programs and their graphical displays, simplifying the development of new interfaces and avoiding duplication of function. With careful design, graphical interfaces can be displayed automatically, instead of being manually constructed. The resulting package is easily maintained and extended as new programs are added or modified. PMID:22675231

  14. Upgrade of IMCA-CAT Bending Magnet Beamline 17-BM for Macromolecular Crystallography at the Advanced Photon Source

    SciTech Connect

    Koshelev, I.; Huang, R.; Graber, T.; Meron, M.; Muir, J.L.; Lavender, W.; Battaile, K.; Mulichak, A.M.; Keefe, L.J.

    2007-05-15

    Pharmaceutical research depends on macromolecular crystallography as a tool in drug design and development. To solve the de novo three-dimensional atomic structure of a protein, it is essential to know the phases of the X-rays scattered by a protein crystal. Experimental phases can be obtained from multiwavelength anomalous dispersion (MAD) experiments. Dedicated to macromolecular crystallography, the IMCA-CAT bending magnet beamline at sector 17 of the Advanced Photon Source (APS) was upgraded to provide the energy resolution required to successfully perform synchrotron radiation-based MAD phasing of protein crystal structures. A collimating mirror was inserted into the beam path upstream of a double-crystal monochromator, thus increasing the monochromatic beam throughput in a particular bandwidth without sacrificing the energy resolution of the system. The beam is focused horizontally by a sagittally bent crystal and vertically by a cylindrically bent mirror, delivering a beam at the sample of 130 {micro}m (vertically) x 250 {micro}m (horizontally) FWHM. As a result of the upgrade, the beamline now operates with an energy range of 7.5 x 17.5 keV, delivers 8 x 10{sup +11} photons/sec at 12.398 keV at the sample, and has an energy resolution of {delta}E/E = 1.45 x 10{sup -4} at 10 keV, which is suitable for MAD experiments.

  15. Upgrade of IMCA-CAT Bending Magnet Beamline 17-BM for Macromolecular Crystallography at the Advanced Photon Source

    SciTech Connect

    Koshelev, I.; Huang, R.; Muir, J. L.; Battaile, K.; Mulichak, A. M.; Keefe, L. J.; Graber, T.; Meron, M.; Lavender, W.

    2007-01-19

    Pharmaceutical research depends on macromolecular crystallography as a tool in drug design and development. To solve the de novo three-dimensional atomic structure of a protein, it is essential to know the phases of the X-rays scattered by a protein crystal. Experimental phases can be obtained from multiwavelength anomalous dispersion (MAD) experiments. Dedicated to macromolecular crystallography, the IMCA-CAT bending magnet beamline at sector 17 of the Advanced Photon Source (APS) was upgraded to provide the energy resolution required to successfully perform synchrotron radiation-based MAD phasing of protein crystal structures. A collimating mirror was inserted into the beam path upstream of a double-crystal monochromator, thus increasing the monochromatic beam throughput in a particular bandwidth without sacrificing the energy resolution of the system. The beam is focused horizontally by a sagittally bent crystal and vertically by a cylindrically bent mirror, delivering a beam at the sample of 130 {mu}m (vertically) x 250 {mu}m (horizontally) FWHM. As a result of the upgrade, the beamline now operates with an energy range of 7.5x17.5 keV, delivers 8 x 10+11 photons/sec at 12.398 keV at the sample, and has an energy resolution of {delta}E/E = 1.45 x 10-4 at 10 keV, which is suitable for MAD experiments.

  16. In-vacuum long-wavelength macromolecular crystallography.

    PubMed

    Wagner, Armin; Duman, Ramona; Henderson, Keith; Mykhaylyk, Vitaliy

    2016-03-01

    Structure solution based on the weak anomalous signal from native (protein and DNA) crystals is increasingly being attempted as part of synchrotron experiments. Maximizing the measurable anomalous signal by collecting diffraction data at longer wavelengths presents a series of technical challenges caused by the increased absorption of X-rays and larger diffraction angles. A new beamline at Diamond Light Source has been built specifically for collecting data at wavelengths beyond the capability of other synchrotron macromolecular crystallography beamlines. Here, the theoretical considerations in support of the long-wavelength beamline are outlined and the in-vacuum design of the endstation is discussed, as well as other hardware features aimed at enhancing the accuracy of the diffraction data. The first commissioning results, representing the first in-vacuum protein structure solution, demonstrate the promising potential of the beamline.

  17. Large-volume protein crystal growth for neutron macromolecular crystallography

    SciTech Connect

    Ng, Joseph D.; Baird, James K.; Coates, Leighton; Garcia-Ruiz, Juan M.; Hodge, Teresa A.; Huang, Sijay

    2015-03-30

    Neutron macromolecular crystallography (NMC) is the prevailing method for the accurate determination of the positions of H atoms in macromolecules. As neutron sources are becoming more available to general users, finding means to optimize the growth of protein crystals to sizes suitable for NMC is extremely important. Historically, much has been learned about growing crystals for X-ray diffraction. However, owing to new-generation synchrotron X-ray facilities and sensitive detectors, protein crystal sizes as small as in the nano-range have become adequate for structure determination, lessening the necessity to grow large crystals. Here, some of the approaches, techniques and considerations for the growth of crystals to significant dimensions that are now relevant to NMC are revisited. We report that these include experimental strategies utilizing solubility diagrams, ripening effects, classical crystallization techniques, microgravity and theoretical considerations.

  18. Large-volume protein crystal growth for neutron macromolecular crystallography

    DOE PAGES

    Ng, Joseph D.; Baird, James K.; Coates, Leighton; ...

    2015-03-30

    Neutron macromolecular crystallography (NMC) is the prevailing method for the accurate determination of the positions of H atoms in macromolecules. As neutron sources are becoming more available to general users, finding means to optimize the growth of protein crystals to sizes suitable for NMC is extremely important. Historically, much has been learned about growing crystals for X-ray diffraction. However, owing to new-generation synchrotron X-ray facilities and sensitive detectors, protein crystal sizes as small as in the nano-range have become adequate for structure determination, lessening the necessity to grow large crystals. Here, some of the approaches, techniques and considerations for themore » growth of crystals to significant dimensions that are now relevant to NMC are revisited. We report that these include experimental strategies utilizing solubility diagrams, ripening effects, classical crystallization techniques, microgravity and theoretical considerations.« less

  19. In-vacuum long-wavelength macromolecular crystallography

    PubMed Central

    Wagner, Armin; Duman, Ramona; Henderson, Keith; Mykhaylyk, Vitaliy

    2016-01-01

    Structure solution based on the weak anomalous signal from native (protein and DNA) crystals is increasingly being attempted as part of synchrotron experiments. Maximizing the measurable anomalous signal by collecting diffraction data at longer wavelengths presents a series of technical challenges caused by the increased absorption of X-rays and larger diffraction angles. A new beamline at Diamond Light Source has been built specifically for collecting data at wavelengths beyond the capability of other synchrotron macromolecular crystallography beamlines. Here, the theoretical considerations in support of the long-wavelength beamline are outlined and the in-vacuum design of the endstation is discussed, as well as other hardware features aimed at enhancing the accuracy of the diffraction data. The first commissioning results, representing the first in-vacuum protein structure solution, demonstrate the promising potential of the beamline. PMID:26960130

  20. Outrunning free radicals in room-temperature macromolecular crystallography

    PubMed Central

    Owen, Robin L.; Axford, Danny; Nettleship, Joanne E.; Owens, Raymond J.; Robinson, James I.; Morgan, Ann W.; Doré, Andrew S.; Lebon, Guillaume; Tate, Christopher G.; Fry, Elizabeth E.; Ren, Jingshan; Stuart, David I.; Evans, Gwyndaf

    2012-01-01

    A significant increase in the lifetime of room-temperature macromolecular crystals is reported through the use of a high-brilliance X-ray beam, reduced exposure times and a fast-readout detector. This is attributed to the ability to collect diffraction data before hydroxyl radicals can propagate through the crystal, fatally disrupting the lattice. Hydroxyl radicals are shown to be trapped in amorphous solutions at 100 K. The trend in crystal lifetime was observed in crystals of a soluble protein (immunoglobulin γ Fc receptor IIIa), a virus (bovine enterovirus serotype 2) and a membrane protein (human A2A adenosine G-protein coupled receptor). The observation of a similar effect in all three systems provides clear evidence for a common optimal strategy for room-temperature data collection and will inform the design of future synchrotron beamlines and detectors for macro­molecular crystallography. PMID:22751666

  1. Outrunning free radicals in room-temperature macromolecular crystallography

    SciTech Connect

    Owen, Robin L. Axford, Danny; Nettleship, Joanne E.; Owens, Raymond J.; Robinson, James I.; Morgan, Ann W.; Doré, Andrew S.; Lebon, Guillaume; Tate, Christopher G.; Fry, Elizabeth E.; Ren, Jingshan; Stuart, David I.; Evans, Gwyndaf

    2012-06-15

    A systematic increase in lifetime is observed in room-temperature protein and virus crystals through the use of reduced exposure times and a fast detector. A significant increase in the lifetime of room-temperature macromolecular crystals is reported through the use of a high-brilliance X-ray beam, reduced exposure times and a fast-readout detector. This is attributed to the ability to collect diffraction data before hydroxyl radicals can propagate through the crystal, fatally disrupting the lattice. Hydroxyl radicals are shown to be trapped in amorphous solutions at 100 K. The trend in crystal lifetime was observed in crystals of a soluble protein (immunoglobulin γ Fc receptor IIIa), a virus (bovine enterovirus serotype 2) and a membrane protein (human A{sub 2A} adenosine G-protein coupled receptor). The observation of a similar effect in all three systems provides clear evidence for a common optimal strategy for room-temperature data collection and will inform the design of future synchrotron beamlines and detectors for macromolecular crystallography.

  2. Synchrotron radiation macromolecular crystallography: science and spin-offs

    PubMed Central

    Helliwell, John R.; Mitchell, Edward P.

    2015-01-01

    A current overview of synchrotron radiation (SR) in macromolecular crystallography (MX) instrumentation, methods and applications is presented. Automation has been and remains a central development in the last decade, as have the rise of remote access and of industrial service provision. Results include a high number of Protein Data Bank depositions, with an increasing emphasis on the successful use of microcrystals. One future emphasis involves pushing the frontiers of using higher and lower photon energies. With the advent of X-ray free-electron lasers, closely linked to SR developments, the use of ever smaller samples such as nanocrystals, nanoclusters and single molecules is anticipated, as well as the opening up of femtosecond time-resolved diffraction structural studies. At SR sources, a very high-throughput assessment for the best crystal samples and the ability to tackle just a few micron and sub-micron crystals will become widespread. With higher speeds and larger detectors, diffraction data volumes are becoming long-term storage and archiving issues; the implications for today and the future are discussed. Together with the rise of the storage ring to its current pre-eminence in MX data provision, the growing tendency of central facility sites to offer other centralized facilities complementary to crystallography, such as cryo-electron microscopy and NMR, is a welcome development. PMID:25866664

  3. JBluIce-EPICS control system for macromolecular crystallography.

    PubMed

    Stepanov, Sergey; Makarov, Oleg; Hilgart, Mark; Pothineni, Sudhir Babu; Urakhchin, Alex; Devarapalli, Satish; Yoder, Derek; Becker, Michael; Ogata, Craig; Sanishvili, Ruslan; Venugopalan, Nagarajan; Smith, Janet L; Fischetti, Robert F

    2011-03-01

    The trio of macromolecular crystallography beamlines constructed by the General Medicine and Cancer Institutes Collaborative Access Team (GM/CA-CAT) in Sector 23 of the Advanced Photon Source (APS) have been in growing demand owing to their outstanding beam quality and capacity to measure data from crystals of only a few micrometres in size. To take full advantage of the state-of-the-art mechanical and optical design of these beamlines, a significant effort has been devoted to designing fast, convenient, intuitive and robust beamline controls that could easily accommodate new beamline developments. The GM/CA-CAT beamline controls are based on the power of EPICS for distributed hardware control, the rich Java graphical user interface of Eclipse RCP and the task-oriented philosophy as well as the look and feel of the successful SSRL BluIce graphical user interface for crystallography. These beamline controls feature a minimum number of software layers, the wide use of plug-ins that can be written in any language and unified motion controls that allow on-the-fly scanning and optimization of any beamline component. This paper describes the ways in which BluIce was combined with EPICS and converted into the Java-based JBluIce, discusses the solutions aimed at streamlining and speeding up operations and gives an overview of the tools that are provided by this new open-source control system for facilitating crystallographic experiments, especially in the field of microcrystallography.

  4. JBluIce-EPICS control system for macromolecular crystallography.

    SciTech Connect

    Stepanov, S.; Makarov, O.; Hilgart, M.; Pothineni, S.; Urakhchin, A.; Devarapalli, S.; Yoder, D.; Becker, M.; Ogata, C.; Sanishvili, R.; Nagarajan, V.; Smith, J. L.; Fischetti, R. F.

    2011-01-01

    The trio of macromolecular crystallography beamlines constructed by the General Medicine and Cancer Institutes Collaborative Access Team (GM/CA-CAT) in Sector 23 of the Advanced Photon Source (APS) have been in growing demand owing to their outstanding beam quality and capacity to measure data from crystals of only a few micrometres in size. To take full advantage of the state-of-the-art mechanical and optical design of these beamlines, a significant effort has been devoted to designing fast, convenient, intuitive and robust beamline controls that could easily accommodate new beamline developments. The GM/CA-CAT beamline controls are based on the power of EPICS for distributed hardware control, the rich Java graphical user interface of Eclipse RCP and the task-oriented philosophy as well as the look and feel of the successful SSRL BluIce graphical user interface for crystallography. These beamline controls feature a minimum number of software layers, the wide use of plug-ins that can be written in any language and unified motion controls that allow on-the-fly scanning and optimization of any beamline component. This paper describes the ways in which BluIce was combined with EPICS and converted into the Java-based JBluIce, discusses the solutions aimed at streamlining and speeding up operations and gives an overview of the tools that are provided by this new open-source control system for facilitating crystallographic experiments, especially in the field of microcrystallography.

  5. Microbeam MAD Beamline for Challenging Protein Crystallography in TPS

    NASA Astrophysics Data System (ADS)

    Liu, D. G.; Chao, C. H.; Chang, C. H.; Juang, J. M.; Liu, C. Y.; Chang, S. H.; Chang, C. F.; Chou, C. K.; Tseng, C. C.; Chiang, C. H.; Jean, Y. C.; Tang, M. T.; Chung, S. C.; Chang, S. L.

    2013-03-01

    The TPS-05A beamline is the first X-ray beamline at NSRRC built for micro protein crystallography experiment as well as one of the seven ID beamlines in phase I at the TPS synchrotron facility. A 2-meter in-vacuum undulator (IU22) serves as the photon source from which the harmonics #3 to #9 will provide brilliance of 1018-1020 photons s-1 mrad-2 mm-2 (0.1% bandwidth)-1 and photon flux of 1013-1014 photons s-1 (0.1% bandwidth)-1 in the required energy range of 5.7-20 keV (2.175-0.620 Å) to cover MAD phasing experiments at 1 Å and SAD phasing experiments at 2 Å. The beamline optics consists of a cryo-cooled double crystal monochromator (DCM) and a pair of focusing K-B mirrors. Requirements from the user group include a target focus size of 50 μm × 50 μm (H × V) at the sample position, photon flux greater than 2 × 1012 photons s-1 at Se K-edge (0.9795 Å), pinholes for adjusting the beam size down to 5 μm. Calculation of heat load for the first optical element, i.e. the first crystal of DCM, is included in this paper.

  6. Radiation damage to nucleoprotein complexes in macromolecular crystallography

    PubMed Central

    Bury, Charles; Garman, Elspeth F.; Ginn, Helen Mary; Ravelli, Raimond B. G.; Carmichael, Ian; Kneale, Geoff; McGeehan, John E.

    2015-01-01

    Significant progress has been made in macromolecular crystallography over recent years in both the understanding and mitigation of X-ray induced radiation damage when collecting diffraction data from crystalline proteins. In contrast, despite the large field that is productively engaged in the study of radiation chemistry of nucleic acids, particularly of DNA, there are currently very few X-ray crystallographic studies on radiation damage mechanisms in nucleic acids. Quantitative comparison of damage to protein and DNA crystals separately is challenging, but many of the issues are circumvented by studying pre-formed biological nucleoprotein complexes where direct comparison of each component can be made under the same controlled conditions. Here a model protein–DNA complex C.Esp1396I is employed to investigate specific damage mechanisms for protein and DNA in a biologically relevant complex over a large dose range (2.07–44.63 MGy). In order to allow a quantitative analysis of radiation damage sites from a complex series of macromolecular diffraction data, a computational method has been developed that is generally applicable to the field. Typical specific damage was observed for both the protein on particular amino acids and for the DNA on, for example, the cleavage of base-sugar N1—C and sugar-phosphate C—O bonds. Strikingly the DNA component was determined to be far more resistant to specific damage than the protein for the investigated dose range. At low doses the protein was observed to be susceptible to radiation damage while the DNA was far more resistant, damage only being observed at significantly higher doses. PMID:25723923

  7. Radiation damage to nucleoprotein complexes in macromolecular crystallography

    DOE PAGES

    Bury, Charles; Garman, Elspeth F.; Ginn, Helen Mary; ...

    2015-01-30

    Significant progress has been made in macromolecular crystallography over recent years in both the understanding and mitigation of X-ray induced radiation damage when collecting diffraction data from crystalline proteins. Despite the large field that is productively engaged in the study of radiation chemistry of nucleic acids, particularly of DNA, there are currently very few X-ray crystallographic studies on radiation damage mechanisms in nucleic acids. Quantitative comparison of damage to protein and DNA crystals separately is challenging, but many of the issues are circumvented by studying pre-formed biological nucleoprotein complexes where direct comparison of each component can be made under themore » same controlled conditions. A model protein–DNA complex C.Esp1396I is employed to investigate specific damage mechanisms for protein and DNA in a biologically relevant complex over a large dose range (2.07–44.63 MGy). In order to allow a quantitative analysis of radiation damage sites from a complex series of macromolecular diffraction data, a computational method has been developed that is generally applicable to the field. Typical specific damage was observed for both the protein on particular amino acids and for the DNA on, for example, the cleavage of base-sugar N1—C and sugar-phosphate C—O bonds. Strikingly the DNA component was determined to be far more resistant to specific damage than the protein for the investigated dose range. We observed the protein at low doses and found that they were susceptible to radiation damage while the DNA was far more resistant, damage only being observed at significantly higher doses.« less

  8. Radiation damage to nucleoprotein complexes in macromolecular crystallography

    SciTech Connect

    Bury, Charles; Garman, Elspeth F.; Ginn, Helen Mary; Ravelli, Raimond B. G.; Carmichael, Ian; Kneale, Geoff; McGeehan, John E.

    2015-01-30

    Significant progress has been made in macromolecular crystallography over recent years in both the understanding and mitigation of X-ray induced radiation damage when collecting diffraction data from crystalline proteins. Despite the large field that is productively engaged in the study of radiation chemistry of nucleic acids, particularly of DNA, there are currently very few X-ray crystallographic studies on radiation damage mechanisms in nucleic acids. Quantitative comparison of damage to protein and DNA crystals separately is challenging, but many of the issues are circumvented by studying pre-formed biological nucleoprotein complexes where direct comparison of each component can be made under the same controlled conditions. A model protein–DNA complex C.Esp1396I is employed to investigate specific damage mechanisms for protein and DNA in a biologically relevant complex over a large dose range (2.07–44.63 MGy). In order to allow a quantitative analysis of radiation damage sites from a complex series of macromolecular diffraction data, a computational method has been developed that is generally applicable to the field. Typical specific damage was observed for both the protein on particular amino acids and for the DNA on, for example, the cleavage of base-sugar N1—C and sugar-phosphate C—O bonds. Strikingly the DNA component was determined to be far more resistant to specific damage than the protein for the investigated dose range. We observed the protein at low doses and found that they were susceptible to radiation damage while the DNA was far more resistant, damage only being observed at significantly higher doses.

  9. JBluIce–EPICS control system for macromolecular crystallography

    PubMed Central

    Stepanov, Sergey; Makarov, Oleg; Hilgart, Mark; Pothineni, Sudhir Babu; Urakhchin, Alex; Devarapalli, Satish; Yoder, Derek; Becker, Michael; Ogata, Craig; Sanishvili, Ruslan; Venugopalan, Nagarajan; Smith, Janet L.; Fischetti, Robert F.

    2011-01-01

    The trio of macromolecular crystallography beamlines constructed by the General Medicine and Cancer Institutes Collaborative Access Team (GM/CA-CAT) in Sector 23 of the Advanced Photon Source (APS) have been in growing demand owing to their outstanding beam quality and capacity to measure data from crystals of only a few micrometres in size. To take full advantage of the state-of-the-art mechanical and optical design of these beamlines, a significant effort has been devoted to designing fast, convenient, intuitive and robust beamline controls that could easily accommodate new beamline developments. The GM/CA-CAT beamline controls are based on the power of EPICS for distributed hardware control, the rich Java graphical user interface of Eclipse RCP and the task-oriented philosophy as well as the look and feel of the successful SSRL BluIce graphical user interface for crystallography. These beamline controls feature a minimum number of software layers, the wide use of plug-ins that can be written in any language and unified motion controls that allow on-the-fly scanning and optimization of any beamline com­ponent. This paper describes the ways in which BluIce was combined with EPICS and converted into the Java-based JBluIce, discusses the solutions aimed at streamlining and speeding up operations and gives an overview of the tools that are provided by this new open-source control system for facilitating crystallo­graphic experiments, especially in the field of microcrystallo­graphy. PMID:21358048

  10. Macromolecular Crystallography and Structural Biology Databases at NIST

    PubMed Central

    Gilliland, Gary L.

    2001-01-01

    In the late 1970s, macromolecular crystallography at NIST began with collaboration between NIST and NIH to establish a single-crystal neutron diffractometer. This instrument was constructed and employed to solve a number of crystal structures: bovine ribonuclease A, bovine-ribonuclease-uridine vanadate complex, and porcine insulin. In the mid 1980s a Biomolecular Structure Group was created establishing NIST capabilities in biomolecular singe-crystal x-ray diffraction. The group worked on a variety of structural problems until joining the NIST/UMBI Center for Advanced Research in Biotechnology (CARB) in 1987. Crystallographic studies at CARB were then focused on protein engineering efforts that included among others chymosin, subtilisin BPN', interleukin 1β, and glutathione S-transferase. Recently, the structural biology efforts have centered on enzymes in the chorismate metabolic pathways involved in amino acid biosynthesis and in structural genomics that involves determining the structures of “hypothetical” proteins to aid in assigning function. In addition to crystallographic studies, structural biology database activities began with the formal establishment of the Biological Macro-molecule Crystallization Database in 1989. Later, in 1997, NIST in partnership with Rutgers and UCSD formed the Research Collaboratory for Structural Bioinformatics that successfully acquired the Protein Data Bank. The NIST efforts in these activities have focused on data uniformity, establishing and maintaining the physical archive, and working with the NMR community. PMID:27500071

  11. Macromolecular Crystallography and Structural Biology Databases at NIST.

    PubMed

    Gilliland, G L

    2001-01-01

    In the late 1970s, macromolecular crystallography at NIST began with collaboration between NIST and NIH to establish a single-crystal neutron diffractometer. This instrument was constructed and employed to solve a number of crystal structures: bovine ribonuclease A, bovine-ribonuclease-uridine vanadate complex, and porcine insulin. In the mid 1980s a Biomolecular Structure Group was created establishing NIST capabilities in biomolecular singe-crystal x-ray diffraction. The group worked on a variety of structural problems until joining the NIST/UMBI Center for Advanced Research in Biotechnology (CARB) in 1987. Crystallographic studies at CARB were then focused on protein engineering efforts that included among others chymosin, subtilisin BPN', interleukin 1β, and glutathione S-transferase. Recently, the structural biology efforts have centered on enzymes in the chorismate metabolic pathways involved in amino acid biosynthesis and in structural genomics that involves determining the structures of "hypothetical" proteins to aid in assigning function. In addition to crystallographic studies, structural biology database activities began with the formal establishment of the Biological Macro-molecule Crystallization Database in 1989. Later, in 1997, NIST in partnership with Rutgers and UCSD formed the Research Collaboratory for Structural Bioinformatics that successfully acquired the Protein Data Bank. The NIST efforts in these activities have focused on data uniformity, establishing and maintaining the physical archive, and working with the NMR community.

  12. New paradigm for macromolecular crystallography experiments at SSRL: automated crystal screening and remote data collection

    PubMed Central

    Soltis, S. Michael; Cohen, Aina E.; Deacon, Ashley; Eriksson, Thomas; González, Ana; McPhillips, Scott; Chui, Hsui; Dunten, Pete; Hollenbeck, Michael; Mathews, Irimpan; Miller, Mitch; Moorhead, Penjit; Phizackerley, R. Paul; Smith, Clyde; Song, Jinhu; van dem Bedem, Henry; Ellis, Paul; Kuhn, Peter; McPhillips, Timothy; Sauter, Nicholas; Sharp, Kenneth; Tsyba, Irina; Wolf, Guenter

    2008-01-01

    Complete automation of the macromolecular crystallography experiment has been achieved at SSRL through the combination of robust mechanized experimental hardware and a flexible control system with an intuitive user interface. These highly reliable systems have enabled crystallography experiments to be carried out from the researchers’ home institutions and other remote locations while retaining complete control over even the most challenging systems. A breakthrough component of the system, the Stanford Auto-Mounter (SAM), has enabled the efficient mounting of cryocooled samples without human intervention. Taking advantage of this automation, researchers have successfully screened more than 200 000 samples to select the crystals with the best diffraction quality for data collection as well as to determine optimal crystallization and cryocooling conditions. These systems, which have been deployed on all SSRL macromolecular crystallography beamlines and several beamlines worldwide, are used by more than 80 research groups in remote locations, establishing a new paradigm for macromolecular crystallo­graphy experimentation. PMID:19018097

  13. Impact of synchrotron radiation on macromolecular crystallography: a personal view

    PubMed Central

    Dauter, Zbigniew; Jaskolski, Mariusz; Wlodawer, Alexander

    2010-01-01

    The introduction of synchrotron radiation sources almost four decades ago has led to a revolutionary change in the way that diffraction data from macromolecular crystals are being collected. Here a brief history of the development of methodologies that took advantage of the availability of synchrotron sources are presented, and some personal experiences with the utilization of synchrotrons in the early days are recalled. PMID:20567074

  14. The R-factor gap in macromolecular crystallography: an untapped potential for insights on accurate structures

    PubMed Central

    Holton, James M; Classen, Scott; Frankel, Kenneth A; Tainer, John A

    2014-01-01

    In macromolecular crystallography, the agreement between observed and predicted structure factors (Rcryst and Rfree) is seldom better than 20%. This is much larger than the estimate of experimental error (Rmerge). The difference between Rcryst and Rmerge is the R-factor gap. There is no such gap in small-molecule crystallography, for which calculated structure factors are generally considered more accurate than the experimental measurements. Perhaps the true noise level of macromolecular data is higher than expected? Or is the gap caused by inaccurate phases that trap refined models in local minima? By generating simulated diffraction patterns using the program MLFSOM, and including every conceivable source of experimental error, we show that neither is the case. Processing our simulated data yielded values that were indistinguishable from those of real data for all crystallographic statistics except the final Rcryst and Rfree. These values decreased to 3.8% and 5.5% for simulated data, suggesting that the reason for high R-factors in macromolecular crystallography is neither experimental error nor phase bias, but rather an underlying inadequacy in the models used to explain our observations. The present inability to accurately represent the entire macromolecule with both its flexibility and its protein-solvent interface may be improved by synergies between small-angle X-ray scattering, computational chemistry and crystallography. The exciting implication of our finding is that macromolecular data contain substantial hidden and untapped potential to resolve ambiguities in the true nature of the nanoscale, a task that the second century of crystallography promises to fulfill. Database Coordinates and structure factors for the real data have been submitted to the Protein Data Bank under accession 4tws. PMID:25040949

  15. Cryosalts: suppression of ice formation in macromolecular crystallography.

    PubMed

    Rubinson, K A; Ladner, J E; Tordova, M; Gilliland, G L

    2000-08-01

    Quality data collection for macromolecular cryocrystallography requires suppressing the formation of crystalline or microcrystalline ice that may result from flash-freezing crystals. Described here is the use of lithium formate, lithium chloride and other highly soluble salts for forming ice-ring-free aqueous glasses upon cooling from ambient temperature to 100 K. These cryosalts are a new class of cryoprotectants that are shown to be effective with a variety of commonly used crystallization solutions and with proteins crystallized under different conditions. The influence of cryosalts on crystal mosaicity and diffraction resolution is comparable with or superior to traditional organic cryoprotectants.

  16. Remote Access to the PXRR Macromolecular Crystallography Facilities at the NSLS

    SciTech Connect

    Soares, A.S.; Schneider, D. K.; Skinner, J. M.; Cowan, M.; Buono, R.; Robinson, H. H.; Heroux, A.; Carlucci-Dayton, M.; Saxena, A.; Sweet, R. M.

    2008-09-01

    The most recent surge of innovations that have simplified and streamlined the process of determining macromolecular structures by crystallography owes much to the efforts of the structural genomics community. However, this was only the last step in a long evolution that saw the metamorphosis of crystallography from an heroic effort that involved years of dedication and skill into a straightforward measurement that is occasionally almost trivial. Many of the steps in this remarkable odyssey involved reducing the physical labor that is demanded of experimenters in the field. Other steps reduced the technical expertise required for conducting those experiments.

  17. Remote Access to the PXRR Macromolecular Crystallography Facilities at the NSLS

    SciTech Connect

    A Soares; D Schneider; J Skinner; M Cowan; R Buono; H Robinson; A Heroux; M Carlucci-Dayton; A Saxena; R Sweet

    2011-12-31

    The most recent surge of innovations that have simplified and streamlined the process of determining macromolecular structures by crystallography owes much to the efforts of the structural genomics community. However, this was only the last step in a long evolution that saw the metamorphosis of crystallography from an heroic effort that involved years of dedication and skill into a straightforward measurement that is occasionally almost trivial. Many of the steps in this remarkable odyssey involved reducing the physical labor that is demanded of experimenters in the field. Other steps reduced the technical expertise required for conducting those experiments.

  18. A history of experimental phasing in macromolecular crystallography

    PubMed Central

    Isaacs, Neil

    2016-01-01

    It was just over a century ago that W. L. Bragg published a paper describing the first crystal structures to be determined using X-ray diffraction data. These structures were obtained from considerations of X-ray diffraction (Bragg equation), crystallography (crystal lattices and symmetry) and the scattering power of different atoms. Although W. H. Bragg proposed soon afterwards, in 1915, that the periodic electron density in crystals could be analysed using Fourier transforms, it took some decades before experimental phasing methods were developed. Many scientists contributed to this development and this paper presents the author’s own perspective on this history. There will be other perspectives, so what follows is a history, rather than the history, of experimental phasing. PMID:26960116

  19. A history of experimental phasing in macromolecular crystallography.

    PubMed

    Isaacs, Neil

    2016-03-01

    It was just over a century ago that W. L. Bragg published a paper describing the first crystal structures to be determined using X-ray diffraction data. These structures were obtained from considerations of X-ray diffraction (Bragg equation), crystallography (crystal lattices and symmetry) and the scattering power of different atoms. Although W. H. Bragg proposed soon afterwards, in 1915, that the periodic electron density in crystals could be analysed using Fourier transforms, it took some decades before experimental phasing methods were developed. Many scientists contributed to this development and this paper presents the author's own perspective on this history. There will be other perspectives, so what follows is a history, rather than the history, of experimental phasing.

  20. Protein crystallography for aspiring crystallographers or how to avoid pitfalls and traps in macromolecular structure determination

    PubMed Central

    Wlodawer, Alexander; Minor, Wladek; Dauter, Zbigniew; Jaskolski, Mariusz

    2014-01-01

    The number of macromolecular structures deposited in the Protein Data Bank now approaches 100 000, with the vast majority of them determined by crystallographic methods. Thousands of papers describing such structures have been published in the scientific literature, and 20 Nobel Prizes in chemistry or medicine have been awarded for discoveries based on macromolecular crystallography. New hardware and software tools have made crystallography appear to be an almost routine (but still far from being analytical) technique and many structures are now being determined by scientists with very limited experience in the practical aspects of the field. However, this apparent ease is sometimes illusory and proper procedures need to be followed to maintain high standards of structure quality. In addition, many noncrystallographers may have problems with the critical evaluation and interpretation of structural results published in the scientific literature. The present review provides an outline of the technical aspects of crystallography for less experienced practitioners, as well as information that might be useful for users of macromolecular structures, aiming to show them how to interpret (but not overinterpret) the information present in the coordinate files and in their description. A discussion of the extent of information that can be gleaned from the atomic coordinates of structures solved at different resolution is provided, as well as problems and pitfalls encountered in structure determination and interpretation. PMID:24034303

  1. Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7.

    PubMed

    Langer, Gerrit; Cohen, Serge X; Lamzin, Victor S; Perrakis, Anastassis

    2008-01-01

    ARP/wARP is a software suite to build macromolecular models in X-ray crystallography electron density maps. Structural genomics initiatives and the study of complex macromolecular assemblies and membrane proteins all rely on advanced methods for 3D structure determination. ARP/wARP meets these needs by providing the tools to obtain a macromolecular model automatically, with a reproducible computational procedure. ARP/wARP 7.0 tackles several tasks: iterative protein model building including a high-level decision-making control module; fast construction of the secondary structure of a protein; building flexible loops in alternate conformations; fully automated placement of ligands, including a choice of the best-fitting ligand from a 'cocktail'; and finding ordered water molecules. All protocols are easy to handle by a nonexpert user through a graphical user interface or a command line. The time required is typically a few minutes although iterative model building may take a few hours.

  2. The collection of MicroED data for macromolecular crystallography.

    PubMed

    Shi, Dan; Nannenga, Brent L; de la Cruz, M Jason; Liu, Jinyang; Sawtelle, Steven; Calero, Guillermo; Reyes, Francis E; Hattne, Johan; Gonen, Tamir

    2016-05-01

    The formation of large, well-ordered crystals for crystallographic experiments remains a crucial bottleneck to the structural understanding of many important biological systems. To help alleviate this problem in crystallography, we have developed the MicroED method for the collection of electron diffraction data from 3D microcrystals and nanocrystals of radiation-sensitive biological material. In this approach, liquid solutions containing protein microcrystals are deposited on carbon-coated electron microscopy grids and are vitrified by plunging them into liquid ethane. MicroED data are collected for each selected crystal using cryo-electron microscopy, in which the crystal is diffracted using very few electrons as the stage is continuously rotated. This protocol gives advice on how to identify microcrystals by light microscopy or by negative-stain electron microscopy in samples obtained from standard protein crystallization experiments. The protocol also includes information about custom-designed equipment for controlling crystal rotation and software for recording experimental parameters in diffraction image metadata. Identifying microcrystals, preparing samples and setting up the microscope for diffraction data collection take approximately half an hour for each step. Screening microcrystals for quality diffraction takes roughly an hour, and the collection of a single data set is ∼10 min in duration. Complete data sets and resulting high-resolution structures can be obtained from a single crystal or by merging data from multiple crystals.

  3. The collection of MicroED data for macromolecular crystallography

    PubMed Central

    Shi, Dan; Nannenga, Brent L; de la Cruz, M Jason; Liu, Jinyang; Sawtelle, Steven; Calero, Guillermo; Reyes, Francis E; Hattne, Johan; Gonen, Tamir

    2017-01-01

    The formation of large, well-ordered crystals for crystallographic experiments remains a crucial bottleneck to the structural understanding of many important biological systems. To help alleviate this problem in crystallography, we have developed the MicroED method for the collection of electron diffraction data from 3D microcrystals and nanocrystals of radiation-sensitive biological material. In this approach, liquid solutions containing protein microcrystals are deposited on carbon-coated electron microscopy grids and are vitrified by plunging them into liquid ethane. MicroED data are collected for each selected crystal using cryo-electron microscopy, in which the crystal is diffracted using very few electrons as the stage is continuously rotated. This protocol gives advice on how to identify microcrystals by light microscopy or by negative-stain electron microscopy in samples obtained from standard protein crystallization experiments. The protocol also includes information about custom-designed equipment for controlling crystal rotation and software for recording experimental parameters in diffraction image metadata. Identifying microcrystals, preparing samples and setting up the microscope for diffraction data collection take approximately half an hour for each step. Screening microcrystals for quality diffraction takes roughly an hour, and the collection of a single data set is ~10 min in duration. Complete data sets and resulting high-resolution structures can be obtained from a single crystal or by merging data from multiple crystals. PMID:27077331

  4. The use of a mini-κ goniometer head in macromolecular crystallography diffraction experiments

    SciTech Connect

    Brockhauser, Sandor; Ravelli, Raimond B. G.; McCarthy, Andrew A.

    2013-07-01

    Hardware and software solutions for MX data-collection strategies using the EMBL/ESRF miniaturized multi-axis goniometer head are presented. Most macromolecular crystallography (MX) diffraction experiments at synchrotrons use a single-axis goniometer. This markedly contrasts with small-molecule crystallography, in which the majority of the diffraction data are collected using multi-axis goniometers. A novel miniaturized κ-goniometer head, the MK3, has been developed to allow macromolecular crystals to be aligned. It is available on the majority of the structural biology beamlines at the ESRF, as well as elsewhere. In addition, the Strategy for the Alignment of Crystals (STAC) software package has been developed to facilitate the use of the MK3 and other similar devices. Use of the MK3 and STAC is streamlined by their incorporation into online analysis tools such as EDNA. The current use of STAC and MK3 on the MX beamlines at the ESRF is discussed. It is shown that the alignment of macromolecular crystals can result in improved diffraction data quality compared with data obtained from randomly aligned crystals.

  5. The Stanford Automated Mounter: Pushing the limits of sample exchange at the SSRL macromolecular crystallography beamlines

    SciTech Connect

    Russi, Silvia; Song, Jinhu; McPhillips, Scott E.; Cohen, Aina E.

    2016-02-24

    The Stanford Automated Mounter System, a system for mounting and dismounting cryo-cooled crystals, has been upgraded to increase the throughput of samples on the macromolecular crystallography beamlines at the Stanford Synchrotron Radiation Lightsource. This upgrade speeds up robot maneuvers, reduces the heating/drying cycles, pre-fetches samples and adds an air-knife to remove frost from the gripper arms. As a result, sample pin exchange during automated crystal quality screening now takes about 25 s, five times faster than before this upgrade.

  6. The Stanford Automated Mounter: Pushing the limits of sample exchange at the SSRL macromolecular crystallography beamlines

    DOE PAGES

    Russi, Silvia; Song, Jinhu; McPhillips, Scott E.; ...

    2016-02-24

    The Stanford Automated Mounter System, a system for mounting and dismounting cryo-cooled crystals, has been upgraded to increase the throughput of samples on the macromolecular crystallography beamlines at the Stanford Synchrotron Radiation Lightsource. This upgrade speeds up robot maneuvers, reduces the heating/drying cycles, pre-fetches samples and adds an air-knife to remove frost from the gripper arms. As a result, sample pin exchange during automated crystal quality screening now takes about 25 s, five times faster than before this upgrade.

  7. The Stanford Automated Mounter: pushing the limits of sample exchange at the SSRL macromolecular crystallography beamlines

    PubMed Central

    Russi, Silvia; Song, Jinhu; McPhillips, Scott E.; Cohen, Aina E.

    2016-01-01

    The Stanford Automated Mounter System, a system for mounting and dismounting cryo-cooled crystals, has been upgraded to increase the throughput of samples on the macromolecular crystallography beamlines at the Stanford Synchrotron Radiation Lightsource. This upgrade speeds up robot maneuvers, reduces the heating/drying cycles, pre-fetches samples and adds an air-knife to remove frost from the gripper arms. Sample pin exchange during automated crystal quality screening now takes about 25 s, five times faster than before this upgrade. PMID:27047309

  8. Radiation damage within nucleoprotein complexes studied by macromolecular X-ray crystallography

    NASA Astrophysics Data System (ADS)

    Bury, Charles S.; Carmichael, Ian; McGeehan, John E.; Garman, Elspeth F.

    2016-11-01

    In X-ray crystallography, for the determination of the 3-D structure of macromolecules, radiation damage is still an inherent problem at modern third generation synchrotron sources, even when utilising cryo-crystallographic techniques (sample held at 100 K). At doses of just several MGy, at which a typical diffraction dataset is collected, site-specific radiation-induced chemical changes are known to manifest within protein crystals, and a wide body of literature is now devoted to understanding the mechanisms behind such damage. Far less is known regarding radiation-induced damage to crystalline nucleic acids and the wider class of nucleoprotein complexes during macromolecular X-ray crystallography (MX) data collection. As the MX structural biology community now strives to solve structures for increasingly larger and complex macromolecular assemblies, it essential to understand how such structures are affected by the X-ray radiation used to solve them. The purpose of this review is to summarise advances in the field of specific damage to nucleoprotein complexes and to present case studies of MX damage investigations on both protein-DNA (C.Esp1396I) and protein-RNA (TRAP) complexes. To motivate further investigations into MX damage mechanisms within nucleoprotein complexes, current and emerging protocols for investigating specific damage within Fobs(n)-Fobs(1) electron density difference maps are discussed.

  9. Recent Major Improvements to the ALS Sector 5 MacromolecularCrystallography Beamlines

    SciTech Connect

    Morton, Simon A.; Glossinger, James; Smith-Baumann, Alexis; McKean, John P.; Trame, Christine; Dickert, Jeff; Rozales, Anthony; Dauz,Azer; Taylor, John; Zwart, Petrus; Duarte, Robert; Padmore, Howard; McDermott, Gerry; Adams, Paul

    2007-07-01

    Although the Advanced Light Source (ALS) was initially conceived primarily as a low energy (1.9GeV) 3rd generation source of VUV and soft x-ray radiation it was realized very early in the development of the facility that a multipole wiggler source coupled with high quality, (brightness preserving), optics would result in a beamline whose performance across the optimal energy range (5-15keV) for macromolecular crystallography (MX) would be comparable to, or even exceed, that of many existing crystallography beamlines at higher energy facilities. Hence, starting in 1996, a suite of three beamlines, branching off a single wiggler source, was constructed, which together formed the ALS Macromolecular Crystallography Facility. From the outset this facility was designed to cater equally to the needs of both academic and industrial users with a heavy emphasis placed on the development and introduction of high throughput crystallographic tools, techniques, and facilities--such as large area CCD detectors, robotic sample handling and automounting facilities, a service crystallography program, and a tightly integrated, centralized, and highly automated beamline control environment for users. This facility was immediately successful, with the primary Multiwavelength Anomalous Diffraction beamline (5.0.2) in particular rapidly becoming one of the foremost crystallographic facilities in the US--responsible for structures such as the 70S ribosome. This success in-turn triggered enormous growth of the ALS macromolecular crystallography community and spurred the development of five additional ALS MX beamlines all utilizing the newly developed superconducting bending magnets ('superbends') as sources. However in the years since the original Sector 5.0 beamlines were built the performance demands of macromolecular crystallography users have become ever more exacting; with growing emphasis placed on studying larger complexes, more difficult structures, weakly diffracting or smaller

  10. A technique for determining the deuterium/hydrogen contrast map in neutron macromolecular crystallography.

    PubMed

    Chatake, Toshiyuki; Fujiwara, Satoru

    2016-01-01

    A difference in the neutron scattering length between hydrogen and deuterium leads to a high density contrast in neutron Fourier maps. In this study, a technique for determining the deuterium/hydrogen (D/H) contrast map in neutron macromolecular crystallography is developed and evaluated using ribonuclease A. The contrast map between the D2O-solvent and H2O-solvent crystals is calculated in real space, rather than in reciprocal space as performed in previous neutron D/H contrast crystallography. The present technique can thus utilize all of the amplitudes of the neutron structure factors for both D2O-solvent and H2O-solvent crystals. The neutron D/H contrast maps clearly demonstrate the powerful detectability of H/D exchange in proteins. In fact, alternative protonation states and alternative conformations of hydroxyl groups are observed at medium resolution (1.8 Å). Moreover, water molecules can be categorized into three types according to their tendency towards rotational disorder. These results directly indicate improvement in the neutron crystal structure analysis. This technique is suitable for incorporation into the standard structure-determination process used in neutron protein crystallography; consequently, more precise and efficient determination of the D-atom positions is possible using a combination of this D/H contrast technique and standard neutron structure-determination protocols.

  11. AutoDrug: fully automated macromolecular crystallography workflows for fragment-based drug discovery

    SciTech Connect

    Tsai, Yingssu; McPhillips, Scott E.; González, Ana; McPhillips, Timothy M.; Zinn, Daniel; Cohen, Aina E.; Feese, Michael D.; Bushnell, David; Tiefenbrunn, Theresa; Stout, C. David; Ludaescher, Bertram; Hedman, Britt; Hodgson, Keith O.; Soltis, S. Michael

    2013-05-01

    New software has been developed for automating the experimental and data-processing stages of fragment-based drug discovery at a macromolecular crystallography beamline. A new workflow-automation framework orchestrates beamline-control and data-analysis software while organizing results from multiple samples. AutoDrug is software based upon the scientific workflow paradigm that integrates the Stanford Synchrotron Radiation Lightsource macromolecular crystallography beamlines and third-party processing software to automate the crystallography steps of the fragment-based drug-discovery process. AutoDrug screens a cassette of fragment-soaked crystals, selects crystals for data collection based on screening results and user-specified criteria and determines optimal data-collection strategies. It then collects and processes diffraction data, performs molecular replacement using provided models and detects electron density that is likely to arise from bound fragments. All processes are fully automated, i.e. are performed without user interaction or supervision. Samples can be screened in groups corresponding to particular proteins, crystal forms and/or soaking conditions. A single AutoDrug run is only limited by the capacity of the sample-storage dewar at the beamline: currently 288 samples. AutoDrug was developed in conjunction with RestFlow, a new scientific workflow-automation framework. RestFlow simplifies the design of AutoDrug by managing the flow of data and the organization of results and by orchestrating the execution of computational pipeline steps. It also simplifies the execution and interaction of third-party programs and the beamline-control system. Modeling AutoDrug as a scientific workflow enables multiple variants that meet the requirements of different user groups to be developed and supported. A workflow tailored to mimic the crystallography stages comprising the drug-discovery pipeline of CoCrystal Discovery Inc. has been deployed and successfully

  12. The kinetic dose limit in room-temperature time-resolved macromolecular crystallography

    PubMed Central

    Schmidt, M.; Šrajer, V.; Purwar, N.; Tripathi, S.

    2012-01-01

    Protein X-ray structures are determined with ionizing radiation that damages the protein at high X-ray doses. As a result, diffraction patterns deteriorate with the increased absorbed dose. Several strategies such as sample freezing or scavenging of X-ray-generated free radicals are currently employed to minimize this damage. However, little is known about how the absorbed X-ray dose affects time-resolved Laue data collected at physiological temperatures where the protein is fully functional in the crystal, and how the kinetic analysis of such data depends on the absorbed dose. Here, direct evidence for the impact of radiation damage on the function of a protein is presented using time-resolved macromolecular crystallography. The effect of radiation damage on the kinetic analysis of time-resolved X-ray data is also explored. PMID:22338689

  13. The macromolecular crystallography beamline I911-3 at the MAX IV laboratory

    PubMed Central

    Ursby, Thomas; Unge, Johan; Appio, Roberto; Logan, Derek T.; Fredslund, Folmer; Svensson, Christer; Larsson, Krister; Labrador, Ana; Thunnissen, Marjolein M. G. M.

    2013-01-01

    The macromolecular crystallography beamline I911-3, part of the Cassiopeia/I911 suite of beamlines, is based on a superconducting wiggler at the MAX II ring of the MAX IV Laboratory in Lund, Sweden. The beamline is energy-tunable within a range between 6 and 18 keV. I911-3 opened for users in 2005. In 2010–2011 the experimental station was completely rebuilt and refurbished such that it has become a state-of-the-art experimental station with better possibilities for rapid throughput, crystal screening and work with smaller samples. This paper describes the complete I911-3 beamline and how it is embedded in the Cassiopeia suite of beamlines. PMID:23765310

  14. Clustering procedures for the optimal selection of data sets from multiple crystals in macromolecular crystallography.

    PubMed

    Foadi, James; Aller, Pierre; Alguel, Yilmaz; Cameron, Alex; Axford, Danny; Owen, Robin L; Armour, Wes; Waterman, David G; Iwata, So; Evans, Gwyndaf

    2013-08-01

    The availability of intense microbeam macromolecular crystallography beamlines at third-generation synchrotron sources has enabled data collection and structure solution from microcrystals of <10 µm in size. The increased likelihood of severe radiation damage where microcrystals or particularly sensitive crystals are used forces crystallographers to acquire large numbers of data sets from many crystals of the same protein structure. The associated analysis and merging of multi-crystal data is currently a manual and time-consuming step. Here, a computer program, BLEND, that has been written to assist with and automate many of the steps in this process is described. It is demonstrated how BLEND has successfully been used in the solution of a novel membrane protein.

  15. D3, the new diffractometer for the macromolecular crystallography beamlines of the Swiss Light Source

    PubMed Central

    Fuchs, Martin R.; Pradervand, Claude; Thominet, Vincent; Schneider, Roman; Panepucci, Ezequiel; Grunder, Marcel; Gabadinho, Jose; Dworkowski, Florian S. N.; Tomizaki, Takashi; Schneider, Jörg; Mayer, Aline; Curtin, Adrian; Olieric, Vincent; Frommherz, Uli; Kotrle, Goran; Welte, Jörg; Wang, Xinyu; Maag, Stephan; Schulze-Briese, Clemens; Wang, Meitian

    2014-01-01

    A new diffractometer for microcrystallography has been developed for the three macromolecular crystallography beamlines of the Swiss Light Source. Building upon and critically extending previous developments realised for the high-resolution endstations of the two undulator beamlines X06SA and X10SA, as well as the super-bend dipole beamline X06DA, the new diffractometer was designed to the following core design goals. (i) Redesign of the goniometer to a sub-micrometer peak-to-peak cylinder of confusion for the horizontal single axis. Crystal sizes down to at least 5 µm and advanced sample-rastering and scanning modes are supported. In addition, it can accommodate the new multi-axis goniometer PRIGo (Parallel Robotics Inspired Goniometer). (ii) A rapid-change beam-shaping element system with aperture sizes down to a minimum of 10 µm for microcrystallography measurements. (iii) Integration of the on-axis microspectrophotometer MS3 for microscopic sample imaging with 1 µm image resolution. Its multi-mode optical spectroscopy module is always online and supports in situ UV/Vis absorption, fluorescence and Raman spectroscopy. (iv) High stability of the sample environment by a mineral cast support construction and by close containment of the cryo-stream. Further features are the support for in situ crystallization plate screening and a minimal achievable detector distance of 120 mm for the Pilatus 6M, 2M and the macromolecular crystallography group’s planned future area detector Eiger 16M. PMID:24562555

  16. Optimized beamline design for macromolecular crystallography at the Cornell High Energy Synchrotron Source (CHESS) (abstract)

    NASA Astrophysics Data System (ADS)

    Schildkamp, Wilfried; Bilderback, Donald; Moffat, Keith

    1989-07-01

    The A1 station on the CHESS wiggler beamline has been the workhorse for most macromolecular crystallographic experiments. This station is equipped with a fixed energy focusing germanium (111) monochromator and a focusing total reflection mirror. Our macromolecular crystallographers made full use of the high flux of more than 1012 photons/s/mm2 and the stable beam conditions, both in position and energy resolution. As a result, the A1 station was heavily oversubscribed. CHESS is presently expanding its capabilities and a new diffraction station for macromolecular crystallography is under construction. This beamline will be powered by a 24-pole hybrid permanent magnet wiggler with a critical energy of 25 keV. A focusing monochromator, which handles a specific heat load of 10 W/mm2, will have a range of tunability which covers all relevant absorption edges from 7 to 15 keV using a Ge(111) crystal. The energy resolution and the focusing properties remain constant within a factor of 2 over the entire tunability range. We expect a brilliance of about 1013 photons/s/mm2/mrad2/0.1% bandpass. The diffraction station will be equipped with an oscillation camera which can be used with x-ray film of 5×5 or 8×10 in. size or alternatively with Kodak storage phosphors. A wide variety of clamp-on accessories, like crystal coolers, fast shutters, helium pathways, polarimeter, etc. are available. The station will contain a beampipe system, which can also be used for small angle scattering experiments with sample-to-detector distances of up to 3000 mm. The entire diffraction station, its control area, a biological preparation area, and a darkroom are to be embedded in a biological safety containment of the level BL3. This will allow diffraction studies of virulent strains of viruses and other biohazards, which could not previously be studied at synchrotron radiation sources before without causing major disruption to the normal laboratory procedure.

  17. Room-temperature macromolecular crystallography using a micro-patterned silicon chip with minimal background scattering

    PubMed Central

    Roedig, Philip; Duman, Ramona; Sanchez-Weatherby, Juan; Vartiainen, Ismo; Burkhardt, Anja; Warmer, Martin; David, Christian; Wagner, Armin; Meents, Alke

    2016-01-01

    Recent success at X-ray free-electron lasers has led to serial crystallography experiments staging a comeback at synchrotron sources as well. With crystal lifetimes typically in the millisecond range and the latest-generation detector technologies with high framing rates up to 1 kHz, fast sample exchange has become the bottleneck for such experiments. A micro-patterned chip has been developed from single-crystalline silicon, which acts as a sample holder for up to several thousand microcrystals at a very low background level. The crystals can be easily loaded onto the chip and excess mother liquor can be efficiently removed. Dehydration of the crystals is prevented by keeping them in a stream of humidified air during data collection. Further sealing of the sample holder, for example with Kapton, is not required. Room-temperature data collection from insulin crystals loaded onto the chip proves the applicability of the chip for macromolecular crystallography. Subsequent structure refinements reveal no radiation-damage-induced structural changes for insulin crystals up to a dose of 565.6 kGy, even though the total diffraction power of the crystals has on average decreased to 19.1% of its initial value for the same dose. A decay of the diffracting power by half is observed for a dose of D 1/2 = 147.5 ± 19.1 kGy, which is about 1/300 of the dose before crystals show a similar decay at cryogenic temperatures. PMID:27275143

  18. AutoDrug: fully automated macromolecular crystallography workflows for fragment-based drug discovery

    PubMed Central

    Tsai, Yingssu; McPhillips, Scott E.; González, Ana; McPhillips, Timothy M.; Zinn, Daniel; Cohen, Aina E.; Feese, Michael D.; Bushnell, David; Tiefenbrunn, Theresa; Stout, C. David; Ludaescher, Bertram; Hedman, Britt; Hodgson, Keith O.; Soltis, S. Michael

    2013-01-01

    AutoDrug is software based upon the scientific workflow paradigm that integrates the Stanford Synchrotron Radiation Lightsource macromolecular crystallography beamlines and third-party processing software to automate the crystallo­graphy steps of the fragment-based drug-discovery process. AutoDrug screens a cassette of fragment-soaked crystals, selects crystals for data collection based on screening results and user-specified criteria and determines optimal data-collection strategies. It then collects and processes diffraction data, performs molecular replacement using provided models and detects electron density that is likely to arise from bound fragments. All processes are fully automated, i.e. are performed without user interaction or supervision. Samples can be screened in groups corresponding to particular proteins, crystal forms and/or soaking conditions. A single AutoDrug run is only limited by the capacity of the sample-storage dewar at the beamline: currently 288 samples. AutoDrug was developed in conjunction with RestFlow, a new scientific workflow-automation framework. RestFlow simplifies the design of AutoDrug by managing the flow of data and the organization of results and by orchestrating the execution of computational pipeline steps. It also simplifies the execution and interaction of third-party programs and the beamline-control system. Modeling AutoDrug as a scientific workflow enables multiple variants that meet the requirements of different user groups to be developed and supported. A workflow tailored to mimic the crystallography stages comprising the drug-discovery pipeline of CoCrystal Discovery Inc. has been deployed and successfully demonstrated. This workflow was run once on the same 96 samples that the group had examined manually and the workflow cycled successfully through all of the samples, collected data from the same samples that were selected manually and located the same peaks of unmodeled density in the resulting difference

  19. Development of an online UV-visible microspectrophotometer for a macromolecular crystallography beamline.

    PubMed

    Shimizu, Nobutaka; Shimizu, Tetsuya; Baba, Seiki; Hasegawa, Kazuya; Yamamoto, Masaki; Kumasaka, Takashi

    2013-11-01

    Measurement of the UV-visible absorption spectrum is a convenient technique for detecting chemical changes of proteins, and it is therefore useful to combine spectroscopy and diffraction studies. An online microspectrophotometer for the UV-visible region was developed and installed on the macromolecular crystallography beamline, BL38B1, at SPring-8. This spectrophotometer is equipped with a difference dispersive double monochromator, a mercury-xenon lamp as the light source, and a photomultiplier as the detector. The optical path is mostly constructed using mirrors, in order to obtain high brightness in the UV region, and the confocal optics are assembled using a cross-slit diaphragm like an iris to eliminate stray light. This system can measure optical densities up to a maximum of 4.0. To study the effect of radiation damage, preliminary measurements of glucose isomerase and thaumatin crystals were conducted in the UV region. Spectral changes dependent on X-ray dose were observed at around 280 nm, suggesting that structural changes involving Trp or Tyr residues occurred in the protein crystal. In the case of the thaumatin crystal, a broad peak around 400 nm was also generated after X-ray irradiation, suggesting the cleavage of a disulfide bond. Dose-dependent spectral changes were also observed in cryo-solutions alone, and these changes differed with the composition of the cryo-solution. These responses in the UV region are informative regarding the state of the sample; consequently, this device might be useful for X-ray crystallography.

  20. 08B1-1: an automated beamline for macromolecular crystallography experiments at the Canadian Light Source.

    PubMed

    Fodje, Michel; Grochulski, Pawel; Janzen, Kathryn; Labiuk, Shaunivan; Gorin, James; Berg, Russ

    2014-05-01

    Beamline 08B1-1 is a recently commissioned bending-magnet beamline at the Canadian Light Source. The beamline is designed for automation and remote access. Together with the undulator-based beamline 08ID-1, they constitute the Canadian Macromolecular Crystallography Facility. This paper describes the design, specifications, hardware and software of beamline 08B1-1. A few scientific results using data obtained at the beamline will be highlighted.

  1. The status of the macromolecular crystallography beamlines at the European Synchrotron Radiation Facility

    NASA Astrophysics Data System (ADS)

    Mueller-Dieckmann, Christoph; Bowler, Matthew W.; Carpentier, Philippe; Flot, David; McCarthy, Andrew A.; Nanao, Max H.; Nurizzo, Didier; Pernot, Petra; Popov, Alexander; Round, Adam; Royant, Antoine; de Sanctis, Daniele; von Stetten, David; Leonard, Gordon A.

    2015-04-01

    The European Synchrotron Radiation Facility (ESRF) is the oldest and most powerful 3rd generation synchrotron in Europe, providing X-rays to more than 40 experimental stations welcoming several thousand researchers per year. A major success story has been the ESRF's facilities for macromolecular crystallography (MX). These are grouped around 3 straight sections: On ID23 canted undulators accommodate ID23-1, a mini-focus tuneable energy end station and ID23-2, the world's first micro-focus beamline dedicated to MX; ID29 houses a single, mini-focus, tuneable energy end station; ID30 will provide three end stations for MX due in operation from mid-2014 to early 2015. Here, one branch of a canted X-ray source feeds two fixed-energy end stations (MASSIF-1, MASSIF-3). The second feeds ID30B, a variable focus, tuneable energy beamline. MASSIF-1 is optimised for automatic high-throughput experiments requiring a relatively large beam size at the sample position, MASSIF-3 is a high-intensity, micro-focus facility designed to complement ID23-2. All end stations are highly automated, equipped with sample mounting robots and large area, fast-readout photon-counting detectors. Experiment control and tracking is achieved via a combination of the MXCuBE2 graphical user interface and the ISPyB database, the former allowing user-friendly control of all beamline components, the latter providing data tracking before, after and during experiments.

  2. Diffraction cartography: applying microbeams to macromolecular crystallography sample evaluation and data collection.

    PubMed

    Bowler, Matthew W; Guijarro, Matias; Petitdemange, Sebastien; Baker, Isabel; Svensson, Olof; Burghammer, Manfred; Mueller-Dieckmann, Christoph; Gordon, Elspeth J; Flot, David; McSweeney, Sean M; Leonard, Gordon A

    2010-08-01

    Crystals of biological macromolecules often exhibit considerable inter-crystal and intra-crystal variation in diffraction quality. This requires the evaluation of many samples prior to data collection, a practice that is already widespread in macromolecular crystallography. As structural biologists move towards tackling ever more ambitious projects, new automated methods of sample evaluation will become crucial to the success of many projects, as will the availability of synchrotron-based facilities optimized for high-throughput evaluation of the diffraction characteristics of samples. Here, two examples of the types of advanced sample evaluation that will be required are presented: searching within a sample-containing loop for microcrystals using an X-ray beam of 5 microm diameter and selecting the most ordered regions of relatively large crystals using X-ray beams of 5-50 microm in diameter. A graphical user interface developed to assist with these screening methods is also presented. For the case in which the diffraction quality of a relatively large crystal is probed using a microbeam, the usefulness and implications of mapping diffraction-quality heterogeneity (diffraction cartography) are discussed. The implementation of these techniques in the context of planned upgrades to the ESRF's structural biology beamlines is also presented.

  3. RoboDiff: combining a sample changer and goniometer for highly automated macromolecular crystallography experiments

    PubMed Central

    Nurizzo, Didier; Bowler, Matthew W.; Caserotto, Hugo; Dobias, Fabien; Giraud, Thierry; Surr, John; Guichard, Nicolas; Papp, Gergely; Guijarro, Matias; Mueller-Dieckmann, Christoph; Flot, David; McSweeney, Sean; Cipriani, Florent; Theveneau, Pascal; Leonard, Gordon A.

    2016-01-01

    Automation of the mounting of cryocooled samples is now a feature of the majority of beamlines dedicated to macromolecular crystallography (MX). Robotic sample changers have been developed over many years, with the latest designs increasing capacity, reliability and speed. Here, the development of a new sample changer deployed at the ESRF beamline MASSIF-1 (ID30A-1), based on an industrial six-axis robot, is described. The device, named RoboDiff, includes a high-capacity dewar, acts as both a sample changer and a high-accuracy goniometer, and has been designed for completely unattended sample mounting and diffraction data collection. This aim has been achieved using a high level of diagnostics at all steps of the process from mounting and characterization to data collection. The RoboDiff has been in service on the fully automated endstation MASSIF-1 at the ESRF since September 2014 and, at the time of writing, has processed more than 20 000 samples completely automatically. PMID:27487827

  4. The use of workflows in the design and implementation of complex experiments in macromolecular crystallography

    PubMed Central

    Brockhauser, Sandor; Svensson, Olof; Bowler, Matthew W.; Nanao, Max; Gordon, Elspeth; Leal, Ricardo M. F.; Popov, Alexander; Gerring, Matthew; McCarthy, Andrew A.; Gotz, Andy

    2012-01-01

    The automation of beam delivery, sample handling and data analysis, together with increasing photon flux, diminishing focal spot size and the appearance of fast-readout detectors on synchrotron beamlines, have changed the way that many macromolecular crystallography experiments are planned and executed. Screening for the best diffracting crystal, or even the best diffracting part of a selected crystal, has been enabled by the development of microfocus beams, precise goniometers and fast-readout detectors that all require rapid feedback from the initial processing of images in order to be effective. All of these advances require the coupling of data feedback to the experimental control system and depend on immediate online data-analysis results during the experiment. To facilitate this, a Data Analysis WorkBench (DAWB) for the flexible creation of complex automated protocols has been developed. Here, example workflows designed and implemented using DAWB are presented for enhanced multi-step crystal characterizations, experiments involving crystal re­orientation with kappa goniometers, crystal-burning experiments for empirically determining the radiation sensitivity of a crystal system and the application of mesh scans to find the best location of a crystal to obtain the highest diffraction quality. Beamline users interact with the prepared workflows through a specific brick within the beamline-control GUI MXCuBE. PMID:22868763

  5. Facilities for macromolecular crystallography at the Helmholtz-Zentrum Berlin.

    PubMed

    Mueller, Uwe; Darowski, Nora; Fuchs, Martin R; Förster, Ronald; Hellmig, Michael; Paithankar, Karthik S; Pühringer, Sandra; Steffien, Michael; Zocher, Georg; Weiss, Manfred S

    2012-05-01

    Three macromolecular crystallography (MX) beamlines at the Helmholtz-Zentrum Berlin (HZB) are available for the regional, national and international structural biology user community. The state-of-the-art synchrotron beamlines for MX BL14.1, BL14.2 and BL14.3 are located within the low-β section of the BESSY II electron storage ring. All beamlines are fed from a superconducting 7 T wavelength-shifter insertion device. BL14.1 and BL14.2 are energy tunable in the range 5-16 keV, while BL14.3 is a fixed-energy side station operated at 13.8 keV. All three beamlines are equipped with CCD detectors. BL14.1 and BL14.2 are in regular user operation providing about 200 beam days per year and about 600 user shifts to approximately 50 research groups across Europe. BL14.3 has initially been used as a test facility and was brought into regular user mode operation during the year 2010. BL14.1 has recently been upgraded with a microdiffractometer including a mini-κ goniometer and an automated sample changer. Additional user facilities include office space adjacent to the beamlines, a sample preparation laboratory, a biology laboratory (safety level 1) and high-end computing resources. In this article the instrumentation of the beamlines is described, and a summary of the experimental possibilities of the beamlines and the provided ancillary equipment for the user community is given.

  6. The use of workflows in the design and implementation of complex experiments in macromolecular crystallography

    SciTech Connect

    Brockhauser, Sandor; Svensson, Olof; Bowler, Matthew W.; Nanao, Max; Gordon, Elspeth; Leal, Ricardo M. F.; Popov, Alexander; Gerring, Matthew; McCarthy, Andrew A.; Gotz, Andy

    2012-08-01

    A powerful and easy-to-use workflow environment has been developed at the ESRF for combining experiment control with online data analysis on synchrotron beamlines. This tool provides the possibility of automating complex experiments without the need for expertise in instrumentation control and programming, but rather by accessing defined beamline services. The automation of beam delivery, sample handling and data analysis, together with increasing photon flux, diminishing focal spot size and the appearance of fast-readout detectors on synchrotron beamlines, have changed the way that many macromolecular crystallography experiments are planned and executed. Screening for the best diffracting crystal, or even the best diffracting part of a selected crystal, has been enabled by the development of microfocus beams, precise goniometers and fast-readout detectors that all require rapid feedback from the initial processing of images in order to be effective. All of these advances require the coupling of data feedback to the experimental control system and depend on immediate online data-analysis results during the experiment. To facilitate this, a Data Analysis WorkBench (DAWB) for the flexible creation of complex automated protocols has been developed. Here, example workflows designed and implemented using DAWB are presented for enhanced multi-step crystal characterizations, experiments involving crystal reorientation with kappa goniometers, crystal-burning experiments for empirically determining the radiation sensitivity of a crystal system and the application of mesh scans to find the best location of a crystal to obtain the highest diffraction quality. Beamline users interact with the prepared workflows through a specific brick within the beamline-control GUI MXCuBE.

  7. Facilities for macromolecular crystallography at the Helmholtz-Zentrum Berlin

    PubMed Central

    Mueller, Uwe; Darowski, Nora; Fuchs, Martin R.; Förster, Ronald; Hellmig, Michael; Paithankar, Karthik S.; Pühringer, Sandra; Steffien, Michael; Zocher, Georg; Weiss, Manfred S.

    2012-01-01

    Three macromolecular crystallography (MX) beamlines at the Helmholtz-Zentrum Berlin (HZB) are available for the regional, national and international structural biology user community. The state-of-the-art synchrotron beamlines for MX BL14.1, BL14.2 and BL14.3 are located within the low-β section of the BESSY II electron storage ring. All beamlines are fed from a superconducting 7 T wavelength-shifter insertion device. BL14.1 and BL14.2 are energy tunable in the range 5–16 keV, while BL14.3 is a fixed-energy side station operated at 13.8 keV. All three beamlines are equipped with CCD detectors. BL14.1 and BL14.2 are in regular user operation providing about 200 beam days per year and about 600 user shifts to approximately 50 research groups across Europe. BL14.3 has initially been used as a test facility and was brought into regular user mode operation during the year 2010. BL14.1 has recently been upgraded with a microdiffractometer including a mini-κ goniometer and an automated sample changer. Additional user facilities include office space adjacent to the beamlines, a sample preparation laboratory, a biology laboratory (safety level 1) and high-end computing resources. In this article the instrumentation of the beamlines is described, and a summary of the experimental possibilities of the beamlines and the provided ancillary equipment for the user community is given. PMID:22514183

  8. AR-NE3A, a New Macromolecular Crystallography Beamline for Pharmaceutical Applications at the Photon Factory

    SciTech Connect

    Yamada, Yusuke; Hiraki, Masahiko; Sasajima, Kumiko; Matsugaki, Naohiro; Igarashi, Noriyuki; Kikuchi, Takashi; Mori, Takeharu; Toyoshima, Akio; Kishimoto, Shunji; Wakatsuki, Soichi; Amano, Yasushi; Warizaya, Masaichi; Sakashita, Hitoshi

    2010-06-23

    Recent advances in high-throughput techniques for macromolecular crystallography have highlighted the importance of structure-based drug design (SBDD), and the demand for synchrotron use by pharmaceutical researchers has increased. Thus, in collaboration with Astellas Pharma Inc., we have constructed a new high-throughput macromolecular crystallography beamline, AR-NE3A, which is dedicated to SBDD. At AR-NE3A, a photon flux up to three times higher than those at existing high-throughput beams at the Photon Factory, AR-NW12A and BL-5A, can be realized at the same sample positions. Installed in the experimental hutch are a high-precision diffractometer, fast-readout, high-gain CCD detector, and sample exchange robot capable of handling more than two hundred cryo-cooled samples stored in a Dewar. To facilitate high-throughput data collection required for pharmaceutical research, fully automated data collection and processing systems have been developed. Thus, sample exchange, centering, data collection, and data processing are automatically carried out based on the user's pre-defined schedule. Although Astellas Pharma Inc. has a priority access to AR-NE3A, the remaining beam time is allocated to general academic and other industrial users.

  9. AR-NE3A, a New Macromolecular Crystallography Beamline for Pharmaceutical Applications at the Photon Factory

    NASA Astrophysics Data System (ADS)

    Yamada, Yusuke; Hiraki, Masahiko; Sasajima, Kumiko; Matsugaki, Naohiro; Igarashi, Noriyuki; Amano, Yasushi; Warizaya, Masaichi; Sakashita, Hitoshi; Kikuchi, Takashi; Mori, Takeharu; Toyoshima, Akio; Kishimoto, Shunji; Wakatsuki, Soichi

    2010-06-01

    Recent advances in high-throughput techniques for macromolecular crystallography have highlighted the importance of structure-based drug design (SBDD), and the demand for synchrotron use by pharmaceutical researchers has increased. Thus, in collaboration with Astellas Pharma Inc., we have constructed a new high-throughput macromolecular crystallography beamline, AR-NE3A, which is dedicated to SBDD. At AR-NE3A, a photon flux up to three times higher than those at existing high-throughput beams at the Photon Factory, AR-NW12A and BL-5A, can be realized at the same sample positions. Installed in the experimental hutch are a high-precision diffractometer, fast-readout, high-gain CCD detector, and sample exchange robot capable of handling more than two hundred cryo-cooled samples stored in a Dewar. To facilitate high-throughput data collection required for pharmaceutical research, fully automated data collection and processing systems have been developed. Thus, sample exchange, centering, data collection, and data processing are automatically carried out based on the user's pre-defined schedule. Although Astellas Pharma Inc. has a priority access to AR-NE3A, the remaining beam time is allocated to general academic and other industrial users.

  10. In crystallo optical spectroscopy (icOS) as a complementary tool on the macromolecular crystallography beamlines of the ESRF

    SciTech Connect

    Stetten, David von; Giraud, Thierry; Carpentier, Philippe; Sever, Franc; Terrien, Maxime; Dobias, Fabien; Juers, Douglas H.; Flot, David; Mueller-Dieckmann, Christoph; Leonard, Gordon A.; Sanctis, Daniele de; Royant, Antoine

    2015-01-01

    The current version of the Cryobench in crystallo optical spectroscopy facility of the ESRF is presented. The diverse experiments that can be performed at the Cryobench are also reviewed. The analysis of structural data obtained by X-ray crystallography benefits from information obtained from complementary techniques, especially as applied to the crystals themselves. As a consequence, optical spectroscopies in structural biology have become instrumental in assessing the relevance and context of many crystallographic results. Since the year 2000, it has been possible to record such data adjacent to, or directly on, the Structural Biology Group beamlines of the ESRF. A core laboratory featuring various spectrometers, named the Cryobench, is now in its third version and houses portable devices that can be directly mounted on beamlines. This paper reports the current status of the Cryobench, which is now located on the MAD beamline ID29 and is thus called the ID29S-Cryobench (where S stands for ‘spectroscopy’). It also reviews the diverse experiments that can be performed at the Cryobench, highlighting the various scientific questions that can be addressed.

  11. Precise Manipulation and Patterning of Protein Crystals for Macromolecular Crystallography Using Surface Acoustic Waves.

    PubMed

    Guo, Feng; Zhou, Weijie; Li, Peng; Mao, Zhangming; Yennawar, Neela H; French, Jarrod B; Huang, Tony Jun

    2015-06-01

    Advances in modern X-ray sources and detector technology have made it possible for crystallographers to collect usable data on crystals of only a few micrometers or less in size. Despite these developments, sample handling techniques have significantly lagged behind and often prevent the full realization of current beamline capabilities. In order to address this shortcoming, a surface acoustic wave-based method for manipulating and patterning crystals is developed. This method, which does not damage the fragile protein crystals, can precisely manipulate and pattern micrometer and submicrometer-sized crystals for data collection and screening. The technique is robust, inexpensive, and easy to implement. This method not only promises to significantly increase efficiency and throughput of both conventional and serial crystallography experiments, but will also make it possible to collect data on samples that were previously intractable.

  12. Precise Manipulation and Patterning of Protein Crystals for Macromolecular Crystallography using Surface Acoustic Waves

    PubMed Central

    Guo, Feng; Zhou, Weijie; Li, Peng; Mao, Zhangming; Yennawar, Neela; French, Jarrod B.; Jun Huang, Tony

    2015-01-01

    Advances in modern X-ray sources and detector technology have made it possible for crystallographers to collect usable data on crystals of only a few micrometers or less in size. Despite these developments, sample handling techniques have significantly lagged behind and often prevent the full realization of current beamline capabilities. In order to address this shortcoming we have developed a surface acoustic wave-based method for manipulating and patterning crystals. This method, which does not damage the fragile protein crystals, can precisely manipulate and pattern micrometer and sub-micrometer sized crystals for data collection and screening. The technique is robust, inexpensive, and easy to implement. This method not only promises to significantly increase efficiency and throughput of both conventional and serial crystallography experiments, but also will make it possible to collect data on samples that were previously intractable. PMID:25641793

  13. MolProbity: all-atom structure validation for macromolecular crystallography.

    PubMed

    Chen, Vincent B; Arendall, W Bryan; Headd, Jeffrey J; Keedy, Daniel A; Immormino, Robert M; Kapral, Gary J; Murray, Laura W; Richardson, Jane S; Richardson, David C

    2010-01-01

    MolProbity is a structure-validation web service that provides broad-spectrum solidly based evaluation of model quality at both the global and local levels for both proteins and nucleic acids. It relies heavily on the power and sensitivity provided by optimized hydrogen placement and all-atom contact analysis, complemented by updated versions of covalent-geometry and torsion-angle criteria. Some of the local corrections can be performed automatically in MolProbity and all of the diagnostics are presented in chart and graphical forms that help guide manual rebuilding. X-ray crystallography provides a wealth of biologically important molecular data in the form of atomic three-dimensional structures of proteins, nucleic acids and increasingly large complexes in multiple forms and states. Advances in automation, in everything from crystallization to data collection to phasing to model building to refinement, have made solving a structure using crystallography easier than ever. However, despite these improvements, local errors that can affect biological interpretation are widespread at low resolution and even high-resolution structures nearly all contain at least a few local errors such as Ramachandran outliers, flipped branched protein side chains and incorrect sugar puckers. It is critical both for the crystallographer and for the end user that there are easy and reliable methods to diagnose and correct these sorts of errors in structures. MolProbity is the authors' contribution to helping solve this problem and this article reviews its general capabilities, reports on recent enhancements and usage, and presents evidence that the resulting improvements are now beneficially affecting the global database.

  14. JBluIce-EPICS: a fast and flexible open-source beamline control system for macromolecular crystallography

    NASA Astrophysics Data System (ADS)

    Stepanov, S.; Hilgart, M.; Makarov, O.; Pothineni, S. B.; Yoder, D.; Ogata, C.; Sanishvili, R.; Venugopalan, N.; Becker, M.; Clift, M.; Smith, J. L.; Fischetti, R. F.

    2013-03-01

    This paper overviews recent advances in the JBluIce-EPICS open-source control system designed at the macromolecular crystallography beamlines of the National Institute of General Medical Sciences and National Cancer Institute at the Advanced Photon Source (GM/CA@APS). We discuss some technical highlights of this system distinguishing it from the competition, such as reduction of software layers to only two, possibility to operate JBluIce in parallel with other beamline controls, plugin-enabled architecture where the plugins can be written in any programming language, and utilization of the whole power of the Java integrated development environment in the Graphical User Interface. Then, we demonstrate how these highlights help to make JBluIce fast, easily adaptable to new beamline developments, and intuitive for users. In particular, we discuss several recent additions to the system including a bridge between crystal rastering and data collection, automatic detection of raster polygons from optical crystal centering, background data processing, and a pathway to a fully automated pipeline from crystal screening to solving crystal structure.

  15. One-Micron Beams for Macromolecular Crystallography at GM/CA-CAT

    SciTech Connect

    Yoder, D. W.; Sanishvili, R.; Xu, S.; Makarov, O.; Benn, R.; Corcoran, S.; Fischetti, R. F.; Vogt, S.

    2010-06-23

    GM/CA-CAT has developed a 1-{mu}m beam for challenging micro-diffraction experiments with macromolecular crystals (e.g. small crystals) and for radiation damage studies. Reflective (Kirkpatrick-Baez mirrors) and diffractive (Fresnel zone plates) optics have been used to focus the beam. Both cases are constrained by the need to maintain a small beam convergence. Using two different zone plates, 1.0x1.0 and 0.8x0.9 {mu}m{sup 2} (VxH,FWHM) beams were created at 15.2 keV and 18.5 keV, respectively. Additionally, by introducing a vertical focusing mirror upstream of the zone plate, a line focus at 15.2 keV was created (28x1.4 {mu}m{sup 2} VxH,FWHM) with the line oriented perpendicular to the X-ray polarization and the crystal rotation axis. Crystal-mounting stages with nanometer resolution have been assembled to profile these beams and to perform diffraction experiments.

  16. Web-Ice: Integrated Data Collection and Analysis for Macromolecular Crystallography

    SciTech Connect

    Gonzalez, Ana; Gonzalez, Ana; Moorhead, Penjit; McPhillips, Scott E.; Song, Jinhu; Sharp, Ken; Taylor, John R.; Adams, Paul D.; Sauter, Nicholas K.; Soltis, S. Michael

    2007-10-10

    New software tools are introduced to facilitate diffraction experiments involving largenumbers of crystals. While existing programs have long provided a framework for lattice indexing, Bragg spot integration, and symmetry determination, these initial data processing steps often require significant manual effort. This limits the timely availability of data analysis needed for high-throughput procedures, including the selection of the best crystals from a large sample pool, and the calculation of optimal data collection parameters to assure complete spot coverage with minimal radiation damage. To make these protocols more efficient, we developed a network of software applications and application servers, collectively known as Web-Ice. When the package is installed at a crystallography beamline, a programming interface allows the beamline control software (e.g., Blu-Ice / DCSS) to trigger data analysis automatically. Results are organized based on a list of samples that the user provides, and are examined within a Web page, accessible both locally at the beamline or remotely. Optional programming interfaces permit the user tocontrol data acquisition through the Web browser. The system as a whole is implemented to support multiple users and multiple processors, and can be expanded to provide additional scientific functionality. Web-Ice has a distributed architecture consisting of several stand-alone software components working together via a well defined interface. Other synchrotrons or institutions may integrate selected components or the whole of Web-Ice with their own data acquisition software. Updated information about current developments may be obtained at http://smb.slac.stanford.edu/research/developments/webice.

  17. Challenges of sulfur SAD phasing as a routine method in macromolecular crystallography

    SciTech Connect

    Doutch, James; Hough, Michael A.; Hasnain, S. Samar; Strange, Richard W.

    2011-11-29

    The sulfur SAD phasing method allows the determination of protein structuresde novowithout reference to derivatives such as Se-methionine. The feasibility for routine automated sulfur SAD phasing using a number of current protein crystallography beamlines at several synchrotrons was examined using crystals of trimericAchromobacter cycloclastesnitrite reductase (AcNiR), which contains a near average proportion of sulfur-containing residues and two Cu atoms per subunit. Experiments using X-ray wavelengths in the range 1.9–2.4 Å show that we are not yet at the level where sulfur SAD is routinely successful forautomatedstructure solution and model building using existing beamlines and current software tools. On the other hand, experiments using the shortest X-ray wavelengths available on existing beamlines could be routinely exploited to solve and produce unbiased structural models using the similarly weak anomalous scattering signals from the intrinsic metal atoms in proteins. The comparison of long-wavelength phasing (the Bijvoet ratio for nine S atoms and two Cu atoms is ~1.25% at ~2 Å) and copper phasing (the Bijvoet ratio for two Cu atoms is 0.81% at ~0.75Å) forAcNiR suggests that lower data multiplicity than is currently required for success should in general be possible for sulfur phasing if appropriate improvements to beamlines and data collection strategies can be implemented.

  18. Developments in optics and performance at BL13-XALOC, the macromolecular crystallography beamline at the Alba Synchrotron

    PubMed Central

    Juanhuix, Jordi; Gil-Ortiz, Fernando; Cuní, Guifré; Colldelram, Carles; Nicolás, Josep; Lidón, Julio; Boter, Eva; Ruget, Claude; Ferrer, Salvador; Benach, Jordi

    2014-01-01

    BL13-XALOC is currently the only macromolecular crystallography beamline at the 3 GeV ALBA synchrotron near Barcelona, Spain. The optics design is based on an in-vacuum undulator, a Si(111) channel-cut crystal monochromator and a pair of KB mirrors. It allows three main operation modes: a focused configuration, where both mirrors can focus the beam at the sample position to 52 µm × 5.5 µm FWHM (H × V); a defocused configuration that can match the size of the beam to the dimensions of the crystals or to focus the beam at the detector; and an unfocused configuration, where one or both mirrors are removed from the photon beam path. To achieve a uniform defocused beam, the slope errors of the mirrors were reduced down to 55 nrad RMS by employing a novel method that has been developed at the ALBA high-accuracy metrology laboratory. Thorough commissioning with X-ray beam and user operation has demonstrated an excellent energy and spatial stability of the beamline. The end-station includes a high-accuracy single-axis diffractometer, a removable mini-kappa stage, an automated sample-mounting robot and a photon-counting detector that allows shutterless operation. The positioning tables of the diffractometer and the detector are based on a novel and highly stable design. This equipment, together with the operation flexibility of the beamline, allows a large variety of types of crystals to be tackled, from medium-sized crystals with large unit-cell parameters to microcrystals. Several examples of data collections measured during beamline commissioning are described. The beamline started user operation on 18 July 2012. PMID:24971961

  19. Harvesting and cryo-cooling crystals of membrane proteins grown in lipidic mesophases for structure determination by macromolecular crystallography.

    PubMed

    Li, Dianfan; Boland, Coilín; Aragao, David; Walsh, Kilian; Caffrey, Martin

    2012-09-02

    An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases(1-5), has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field(6-21) (www.mpdb.tcd.ie). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting(22,23). Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)(24,25) are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection. The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies(4,26). The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been

  20. A new on-axis micro-spectrophotometer for combining Raman, fluorescence and UV/Vis absorption spectroscopy with macromolecular crystallography at the Swiss Light Source.

    PubMed

    Pompidor, Guillaume; Dworkowski, Florian S N; Thominet, Vincent; Schulze-Briese, Clemens; Fuchs, Martin R

    2013-09-01

    The combination of X-ray diffraction experiments with optical methods such as Raman, UV/Vis absorption and fluorescence spectroscopy greatly enhances and complements the specificity of the obtained information. The upgraded version of the in situ on-axis micro-spectrophotometer, MS2, at the macromolecular crystallography beamline X10SA of the Swiss Light Source is presented. The instrument newly supports Raman and resonance Raman spectroscopy, in addition to the previously available UV/Vis absorption and fluorescence modes. With the recent upgrades of the spectral bandwidth, instrument stability, detection efficiency and control software, the application range of the instrument and its ease of operation were greatly improved. Its on-axis geometry with collinear X-ray and optical axes to ensure optimal control of the overlap of sample volumes probed by each technique is still unique amongst comparable facilities worldwide and the instrument has now been in general user operation for over two years.

  1. A new on-axis micro-spectrophotometer for combining Raman, fluorescence and UV/Vis absorption spectroscopy with macromolecular crystallography at the Swiss Light Source

    PubMed Central

    Pompidor, Guillaume; Dworkowski, Florian S. N.; Thominet, Vincent; Schulze-Briese, Clemens; Fuchs, Martin R.

    2013-01-01

    The combination of X-ray diffraction experiments with optical methods such as Raman, UV/Vis absorption and fluorescence spectroscopy greatly enhances and complements the specificity of the obtained information. The upgraded version of the in situ on-axis micro-spectrophotometer, MS2, at the macromolecular crystallography beamline X10SA of the Swiss Light Source is presented. The instrument newly supports Raman and resonance Raman spectroscopy, in addition to the previously available UV/Vis absorption and fluorescence modes. With the recent upgrades of the spectral bandwidth, instrument stability, detection efficiency and control software, the application range of the instrument and its ease of operation were greatly improved. Its on-axis geometry with collinear X-ray and optical axes to ensure optimal control of the overlap of sample volumes probed by each technique is still unique amongst comparable facilities worldwide and the instrument has now been in general user operation for over two years. PMID:23955041

  2. A new on-axis multimode spectrometer for the macromolecular crystallography beamlines of the Swiss Light Source

    PubMed Central

    Owen, Robin L.; Pearson, Arwen R.; Meents, Alke; Boehler, Pirmin; Thominet, Vincent; Schulze-Briese, Clemens

    2009-01-01

    X-ray crystallography at third-generation synchrotron sources permits tremendous insight into the three-dimensional structure of macromolecules. Additional information is, however, often required to aid the transition from structure to function. In situ spectroscopic methods such as UV–Vis absorption and (resonance) Raman can provide this, and can also provide a means of detecting X-ray-induced changes. Here, preliminary results are introduced from an on-axis UV–Vis absorption and Raman multimode spectrometer currently being integrated into the beamline environment at X10SA of the Swiss Light Source. The continuing development of the spectrometer is also outlined. PMID:19240329

  3. A micro-patterned silicon chip as sample holder for macromolecular crystallography experiments with minimal background scattering

    PubMed Central

    Roedig, P.; Vartiainen, I.; Duman, R.; Panneerselvam, S.; Stübe, N.; Lorbeer, O.; Warmer, M.; Sutton, G.; Stuart, D. I.; Weckert, E.; David, C.; Wagner, A.; Meents, A.

    2015-01-01

    At low emittance synchrotron sources it has become possible to perform structure determinations from the measurement of multiple microcrystals which were previously considered too small for diffraction experiments. Conventional mounting techniques do not fulfill the requirements of these new experiments. They significantly contribute to background scattering and it is difficult to locate the crystals, making them incompatible with automated serial crystallography. We have developed a micro-fabricated sample holder from single crystalline silicon with micropores, which carries up to thousands of crystals and significantly reduces the background scattering level. For loading, the suspended microcrystals are pipetted onto the chip and excess mother liquor is subsequently soaked off through the micropores. Crystals larger than the pore size are retained and arrange themselves according to the micropore pattern. Using our chip we were able to collect 1.5 Å high resolution diffraction data from protein microcrystals with sizes of 4 micrometers and smaller. PMID:26022615

  4. Implementation and performance of SIBYLS: a dual endstation small-angle X-ray scattering and macromolecular crystallography beamline at the Advanced Light Source

    PubMed Central

    Classen, Scott; Hura, Greg L.; Holton, James M.; Rambo, Robert P.; Rodic, Ivan; McGuire, Patrick J.; Dyer, Kevin; Hammel, Michal; Meigs, George; Frankel, Kenneth A.; Tainer, John A.

    2013-01-01

    The SIBYLS beamline (12.3.1) of the Advanced Light Source at Lawrence Berkeley National Laboratory, supported by the US Department of Energy and the National Institutes of Health, is optimized for both small-angle X-ray scattering (SAXS) and macromolecular crystallography (MX), making it unique among the world’s mostly SAXS or MX dedicated beamlines. Since SIBYLS was commissioned, assessments of the limitations and advantages of a combined SAXS and MX beamline have suggested new strategies for integration and optimal data collection methods and have led to additional hardware and software enhancements. Features described include a dual mode monochromator [containing both Si(111) crystals and Mo/B4C multilayer elements], rapid beamline optics conversion between SAXS and MX modes, active beam stabilization, sample-loading robotics, and mail-in and remote data collection. These features allow users to gain valuable insights from both dynamic solution scattering and high-resolution atomic diffraction experiments performed at a single synchrotron beamline. Key practical issues considered for data collection and analysis include radiation damage, structural ensembles, alternative conformers and flexibility. SIBYLS develops and applies efficient combined MX and SAXS methods that deliver high-impact results by providing robust cost-effective routes to connect structures to biology and by performing experiments that aid beamline designs for next generation light sources. PMID:23396808

  5. P13, the EMBL macromolecular crystallography beamline at the low-emittance PETRA III ring for high- and low-energy phasing with variable beam focusing

    PubMed Central

    Cianci, Michele; Bourenkov, Gleb; Pompidor, Guillaume; Karpics, Ivars; Kallio, Johanna; Bento, Isabel; Roessle, Manfred; Cipriani, Florent; Fiedler, Stefan; Schneider, Thomas R.

    2017-01-01

    The macromolecular crystallography P13 beamline is part of the European Molecular Biology Laboratory Integrated Facility for Structural Biology at PETRA III (DESY, Hamburg, Germany) and has been in user operation since mid-2013. P13 is tunable across the energy range from 4 to 17.5 keV to support crystallographic data acquisition exploiting a wide range of elemental absorption edges for experimental phase determination. An adaptive Kirk­patrick–Baez focusing system provides an X-ray beam with a high photon flux and tunable focus size to adapt to diverse experimental situations. Data collections at energies as low as 4 keV (λ = 3.1 Å) are possible due to a beamline design minimizing background and maximizing photon flux particularly at low energy (up to 1011 photons s−1 at 4 keV), a custom calibration of the PILATUS 6M-F detector for use at low energies, and the availability of a helium path. At high energies, the high photon flux (5.4 × 1011 photons s−1 at 17.5 keV) combined with a large area detector mounted on a 2θ arm allows data collection to sub-atomic resolution (0.55 Å). A peak flux of about 8.0 × 1012 photons s−1 is reached at 11 keV. Automated sample mounting is available by means of the robotic sample changer ‘MARVIN’ with a dewar capacity of 160 samples. In close proximity to the beamline, laboratories have been set up for sample preparation and characterization; a laboratory specifically equipped for on-site heavy atom derivatization with a library of more than 150 compounds is available to beamline users. PMID:28009574

  6. Using macromolecular-crystallography beamline and microfluidic platform for small-angle diffraction studies of lipidic matrices for membrane-protein crystallization

    NASA Astrophysics Data System (ADS)

    Kondrashkina, E.; Khvostichenko, D. S.; Perry, S. L.; Von Osinski, J.; Kenis, P. J. A.; Brister, K.

    2013-03-01

    Macromolecular-crystallography (MX) beamlines routinely provide a possibility to change X-ray beam energy, focus the beam to a size of tens of microns, align a sample on a microdiffractometer using on-axis video microscope, and collect data with an area-detector positioned in three dimensions. These capabilities allow for running complementary measurements of small-angle X-ray scattering and diffraction (SAXS) at the same beamline with such additions to the standard MX setup as a vacuum path between the sample and the detector, a modified beam stop, and a custom sample cell. On the 21-ID-D MX beamline at the Advanced Photon Source we attach a vacuum flight tube to the area detector support and use the support motion for aligning a beam stop built into the rear end of the flight tube. At 8 KeV energy and 1 m sample-to-detector distance we can achieve a small-angle resolution of 0.01A-1 in the reciprocal space. Measuring SAXS with this setup, we have studied phase diagrams of lipidic mesophases used as matrices for membrane-protein crystallization. The outcome of crystallization trials is significantly affected by the structure of the lipidic mesophases, which is determined by the composition of the crystallization mixture. We use a microfluidic chip for the mesophase formulation and in situ SAXS data collection. Using the MX beamline and the microfluidic platform we have demonstrated the viability of the high-throughput SAXS studies facilitating screening of lipidic matrices for membrane-protein crystallization.

  7. Conceptual design of novel IP-conveyor-belt Weissenberg-mode data-collection system with multi-readers for macromolecular crystallography. A comparison between Galaxy and Super Galaxy.

    PubMed

    Sakabe, N; Sakabe, K; Sasaki, K

    2004-01-01

    Galaxy is a Weissenberg-type high-speed high-resolution and highly accurate fully automatic data-collection system using two cylindrical IP-cassettes each with a radius of 400 mm and a width of 450 mm. It was originally developed for static three-dimensional analysis using X-ray diffraction and was installed on bending-magnet beamline BL6C at the Photon Factory. It was found, however, that Galaxy was also very useful for time-resolved protein crystallography on a time scale of minutes. This has prompted us to design a new IP-conveyor-belt Weissenberg-mode data-collection system called Super Galaxy for time-resolved crystallography with improved time and crystallographic resolution over that achievable with Galaxy. Super Galaxy was designed with a half-cylinder-shaped cassette with a radius of 420 mm and a width of 690 mm. Using 1.0 A incident X-rays, these dimensions correspond to a maximum resolutions of 0.71 A in the vertical direction and 1.58 A in the horizontal. Upper and lower screens can be used to set the frame size of the recorded image. This function is useful not only to reduce the frame exchange time but also to save disk space on the data server. The use of an IP-conveyor-belt and many IP-readers make Super Galaxy well suited for time-resolved, monochromatic X-ray crystallography at a very intense third-generation SR beamline. Here, Galaxy and a conceptual design for Super Galaxy are described, and their suitability for use as data-collection systems for macromolecular time-resolved monochromatic X-ray crystallography are compared.

  8. Personal remarks on the future of protein crystallography and structural biology.

    PubMed

    Jaskolski, Mariusz

    2010-01-01

    Protein crystallography, the main experimental method of structural biology, has undergone in the recent past three revolutionary changes leading to its unexpected renaissance. They were connected with (i) the introduction of synchrotron radiation sources for X-ray diffraction experiments, (ii) implementation of Se-Met multiwavelength anomalous diffraction (MAD) for phasing, and (iii) initiation of structural genomics (SG) programs. It can be foreseen that in the next 10-15 years protein crystallography will continue to be in this revolutionary phase. We can expect not only an avalanche of protein crystal structures from SG centers, but also attacking of more demanding projects, such as the structure of membrane proteins and of very large macromolecular complexes. On the technological front, the introduction of X-ray radiation from free-electron lasers will revolutionize the experimental possibilities, making feasible even the imaging of single molecules and of intact biological cells.

  9. Automated macromolecular crystallization screening

    DOEpatents

    Segelke, Brent W.; Rupp, Bernhard; Krupka, Heike I.

    2005-03-01

    An automated macromolecular crystallization screening system wherein a multiplicity of reagent mixes are produced. A multiplicity of analysis plates is produced utilizing the reagent mixes combined with a sample. The analysis plates are incubated to promote growth of crystals. Images of the crystals are made. The images are analyzed with regard to suitability of the crystals for analysis by x-ray crystallography. A design of reagent mixes is produced based upon the expected suitability of the crystals for analysis by x-ray crystallography. A second multiplicity of mixes of the reagent components is produced utilizing the design and a second multiplicity of reagent mixes is used for a second round of automated macromolecular crystallization screening. In one embodiment the multiplicity of reagent mixes are produced by a random selection of reagent components.

  10. Comparing Chemistry to Outcome: The Development of a Chemical Distance Metric, Coupled with Clustering and Hierarchal Visualization Applied to Macromolecular Crystallography

    PubMed Central

    Bruno, Andrew E.; Ruby, Amanda M.; Luft, Joseph R.; Grant, Thomas D.; Seetharaman, Jayaraman; Montelione, Gaetano T.; Hunt, John F.; Snell, Edward H.

    2014-01-01

    Many bioscience fields employ high-throughput methods to screen multiple biochemical conditions. The analysis of these becomes tedious without a degree of automation. Crystallization, a rate limiting step in biological X-ray crystallography, is one of these fields. Screening of multiple potential crystallization conditions (cocktails) is the most effective method of probing a proteins phase diagram and guiding crystallization but the interpretation of results can be time-consuming. To aid this empirical approach a cocktail distance coefficient was developed to quantitatively compare macromolecule crystallization conditions and outcome. These coefficients were evaluated against an existing similarity metric developed for crystallization, the C6 metric, using both virtual crystallization screens and by comparison of two related 1,536-cocktail high-throughput crystallization screens. Hierarchical clustering was employed to visualize one of these screens and the crystallization results from an exopolyphosphatase-related protein from Bacteroides fragilis, (BfR192) overlaid on this clustering. This demonstrated a strong correlation between certain chemically related clusters and crystal lead conditions. While this analysis was not used to guide the initial crystallization optimization, it led to the re-evaluation of unexplained peaks in the electron density map of the protein and to the insertion and correct placement of sodium, potassium and phosphate atoms in the structure. With these in place, the resulting structure of the putative active site demonstrated features consistent with active sites of other phosphatases which are involved in binding the phosphoryl moieties of nucleotide triphosphates. The new distance coefficient, CDcoeff, appears to be robust in this application, and coupled with hierarchical clustering and the overlay of crystallization outcome, reveals information of biological relevance. While tested with a single example the potential applications

  11. The use of trimethylamine N-oxide as a primary precipitating agent and related methylamine osmolytes as cryoprotective agents for macromolecular crystallography

    SciTech Connect

    Marshall, Haley; Venkat, Murugappan; Hti Lar Seng, Nang San; Cahn, Jackson; Juers, Douglas H.

    2012-01-01

    The stabilizing osmolyte trimethylamine N-oxide (TMAO) is shown to be an efficient primary precipitant for protein crystal growth. In addition to TMAO, two other methylamine osmolytes, sarcosine and betaine, are shown to be effective cryoprotective agents for protein crystal cooling. Both crystallization and cryoprotection are often bottlenecks for high-resolution X-ray structure determination of macromolecules. Methylamine osmolytes are known stabilizers of protein structure. One such osmolyte, trimethylamine N-oxide (TMAO), has seen occasional use as an additive to improve macromolecular crystal quality and has recently been shown to be an effective cryoprotective agent for low-temperature data collection. Here, TMAO and the related osmolytes sarcosine and betaine are investigated as primary precipitating agents for protein crystal growth. Crystallization experiments were undertaken with 14 proteins. Using TMAO, seven proteins crystallized in a total of 13 crystal forms, including a new tetragonal crystal form of trypsin. The crystals diffracted well, and eight of the 13 crystal forms could be effectively cryocooled as grown with TMAO as an in situ cryoprotective agent. Sarcosine and betaine produced crystals of four and two of the 14 proteins, respectively. In addition to TMAO, sarcosine and betaine were effective post-crystallization cryoprotective agents for two different crystal forms of thermolysin. Precipitation reactions of TMAO with several transition-metal ions (Fe{sup 3+}, Co{sup 2+}, Cu{sup 2+} and Zn{sup 2+}) did not occur with sarcosine or betaine and were inhibited for TMAO at lower pH. Structures of proteins from TMAO-grown crystals and from crystals soaked in TMAO, sarcosine or betaine were determined, showing osmolyte binding in five of the 12 crystals tested. When an osmolyte was shown to bind, it did so near the protein surface, interacting with water molecules, side chains and backbone atoms, often at crystal contacts.

  12. Macromolecular Crystallization in Microgravity

    NASA Technical Reports Server (NTRS)

    Snell, Edward H.; Helliwell, John R.

    2004-01-01

    The key concepts that attracted crystal growers, macromolecular or solid state, to microgravity research is that density difference fluid flows and sedimentation of the growing crystals are greatly reduced. Thus, defects and flaws in the crystals can be reduced, even eliminated, and crystal volume can be increased. Macromolecular crystallography differs from the field of crystalline semiconductors. For the latter, crystals are harnessed for their electrical behaviors. A crystal of a biological macromolecule is used instead for diffraction experiments (X-ray or neutron) to determine the three-dimensional structure of the macromolecule. The better the internal order of the crystal of a biological macromolecule then the more molecular structure detail that can be extracted. This structural information that enables an understanding of how the molecule functions. This knowledge is changing the biological and chemical sciences with major potential in understanding disease pathologies. Macromolecular structural crystallography in general is a remarkable field where physics, biology, chemistry, and mathematics meet to enable insight to the basic fundamentals of life. In this review, we examine the use of microgravity as an environment to grow macromolecular crystals. We describe the crystallization procedures used on the ground, how the resulting crystals are studied and the knowledge obtained from those crystals. We address the features desired in an ordered crystal and the techniques used to evaluate those features in detail. We then introduce the microgravity environment, the techniques to access that environment, and the theory and evidence behind the use of microgravity for crystallization experiments. We describe how ground-based laboratory techniques have been adapted to microgravity flights and look at some of the methods used to analyze the resulting data. Several case studies illustrate the physical crystal quality improvements and the macromolecular structural

  13. Micro-crystallography comes of age

    PubMed Central

    Smith, Janet L.; Fischetti, Robert F.; Yamamoto, Masaki

    2012-01-01

    The latest revolution in macromolecular crystallography was incited by the development of dedicated, user friendly, micro-crystallography beamlines. Brilliant X-ray beams of diameter 20 microns or less, now available at most synchrotron sources, enable structure determination from samples that previously were inaccessible. Relative to traditional crystallography, crystals with one or more small dimensions have diffraction patterns with vastly improved signal-to-noise when recorded with an appropriately matched beam size. Structures can be solved from isolated, well diffracting regions within inhomogeneous samples. This review summarizes the technological requirements and approaches to producing micro-beams and how they continue to change the practice of crystallography. PMID:23021872

  14. Five-dimensional crystallography

    PubMed Central

    Schmidt, Marius; Graber, Tim; Henning, Robert; Srajer, Vukica

    2010-01-01

    A method for determining a comprehensive chemical kinetic mechanism in macromolecular reactions is presented. The method is based on five-dimensional crystallography, where, in addition to space and time, temperature is also taken into consideration and an analysis based on singular value decomposition is applied. First results of such a time-resolved crystallographic study are presented. Temperature-dependent time-resolved X-ray diffraction measurements were conducted on the newly upgraded BioCARS 14-ID-B beamline at the Advanced Photon Source and aimed at elucidating a comprehensive kinetic mechanism of the photoactive yellow protein photocycle. Extensive time series of crystallographic data were collected at two temperatures, 293 K and 303 K. Relaxation times of the reaction extracted from these time series exhibit measurable differences for the two temperatures, hence demonstrating that five-dimensional crystallography is feasible. PMID:20164643

  15. Neutron proton crystallography station (PCS)

    SciTech Connect

    Fisher, Zoe; Kovalevsky, Andrey; Johnson, Hannah; Mustyakimov, Marat

    2009-01-01

    The PCS (Protein Crystallography Station) at Los Alamos Neutron Science Center (LANSCE) is a unique facility in the USA that is designed and optimized for detecting and collecting neutron diffraction data from macromolecular crystals. PCS utilizes the 20 Hz spallation neutron source at LANSCE to enable time-of-flight measurements using 0.6-7.0 {angstrom} neutrons. This increases the neutron flux on the sample by using a wavelength range that is optimal for studying macromolecular crystal structures. The diagram below show a schematic of PCS and photos of the detector and instrument cave.

  16. What's Mad Cow Disease?

    MedlinePlus

    ... What Happens in the Operating Room? What's Mad Cow Disease? KidsHealth > For Kids > What's Mad Cow Disease? A A A You might have heard news reports about mad cow disease and wondered: What the heck is that? ...

  17. MACROMOLECULAR THERAPEUTICS

    PubMed Central

    Yang, Jiyuan; Kopeček, Jindřich

    2014-01-01

    This review covers water-soluble polymer-drug conjugates and macromolecules that possess biological activity without attached low molecular weight drugs. The main design principles of traditional and backbone degradable polymer-drug conjugates as well as the development of a new paradigm in nanomedicines – (low molecular weight) drug-free macromolecular therapeutics are discussed. To address the biological features of cancer, macromolecular therapeutics directed to stem/progenitor cells and the tumor microenvironment are deliberated. Finally, the future perspectives of the field are briefly debated. PMID:24747162

  18. Macromolecular therapeutics.

    PubMed

    Yang, Jiyuan; Kopeček, Jindřich

    2014-09-28

    This review covers water-soluble polymer-drug conjugates and macromolecules that possess biological activity without attached low molecular weight drugs. The main design principles of traditional and backbone degradable polymer-drug conjugates as well as the development of a new paradigm in nanomedicines - (low molecular weight) drug-free macromolecular therapeutics are discussed. To address the biological features of cancer, macromolecular therapeutics directed to stem/progenitor cells and the tumor microenvironment are deliberated. Finally, the future perspectives of the field are briefly debated.

  19. Facilities for small-molecule crystallography at synchrotron sources.

    PubMed

    Barnett, Sarah A; Nowell, Harriott; Warren, Mark R; Wilcox, Andrian; Allan, David R

    2016-01-01

    Although macromolecular crystallography is a widely supported technique at synchrotron radiation facilities throughout the world, there are, in comparison, only very few beamlines dedicated to small-molecule crystallography. This limited provision is despite the increasing demand for beamtime from the chemical crystallography community and the ever greater overlap between systems that can be classed as either small macromolecules or large small molecules. In this article, a very brief overview of beamlines that support small-molecule single-crystal diffraction techniques will be given along with a more detailed description of beamline I19, a dedicated facility for small-molecule crystallography at Diamond Light Source.

  20. Macromolecular recognition.

    PubMed

    Deremble, Cyril; Lavery, Richard

    2005-04-01

    Computational methods are being developed both to detect the binding surfaces of individual macromolecules and to predict the structure of binary macromolecular complexes. Speeding up and refining this process has required work on search algorithms, molecular representations and interaction potentials. Although backbone flexibility and solvent effects continue to pose problems, encouraging results have been obtained for both protein-protein and protein-DNA complexes.

  1. [Madness in Foucault: art and madness, madness and unreason].

    PubMed

    Providello, Guilherme Gonzaga Duarte; Yasui, Silvio

    2013-10-01

    After presenting the ideas on madness and its interface with art as expressed in the writings of Michel Foucault, Peter Pál Pelbart, and Gilles Deleuze, the article explores how these authors question the relationship between art and madness. It begins with the notion that madness does not tell the truth about art, and vice versa, but that there are links between both that must be delved into if we are to engage in deeper reflection on the topic. The text problematizes the statement that madness is the absence of an oeuvre and examines how this impacts the possibility of achieving an artistic oeuvre. It further problematizes the idea of madness as excluded language, that is, the idea that madness implies not only the exclusion of the body but also the disqualification of discourse.

  2. What's Mad Cow Disease?

    MedlinePlus

    ... Quizzes Kids' Dictionary of Medical Words En Español What Other Kids Are Reading Video: Am I Normal? ( ... Emergency Room? What Happens in the Operating Room? What's Mad Cow Disease? KidsHealth > For Kids > What's Mad ...

  3. Warm dense crystallography

    NASA Astrophysics Data System (ADS)

    Valenza, Ryan A.; Seidler, Gerald T.

    2016-03-01

    The intense femtosecond-scale pulses from x-ray free electron lasers (XFELs) are able to create and interrogate interesting states of matter characterized by long-lived nonequilibrium semicore or core electron occupancies or by the heating of dense phases via the relaxation cascade initiated by the photoelectric effect. We address here the latter case of "warm dense matter" (WDM) and investigate the observable consequences of x-ray heating of the electronic degrees of freedom in crystalline systems. We report temperature-dependent density functional theory calculations for the x-ray diffraction from crystalline LiF, graphite, diamond, and Be. We find testable, strong signatures of condensed-phase effects that emphasize the importance of wide-angle scattering to study nonequilibrium states. These results also suggest that the reorganization of the valence electron density at eV-scale temperatures presents a confounding factor to achieving atomic resolution in macromolecular serial femtosecond crystallography (SFX) studies at XFELs, as performed under the "diffract before destroy" paradigm.

  4. Data Mining of Macromolecular Structures.

    PubMed

    van Beusekom, Bart; Perrakis, Anastassis; Joosten, Robbie P

    2016-01-01

    The use of macromolecular structures is widespread for a variety of applications, from teaching protein structure principles all the way to ligand optimization in drug development. Applying data mining techniques on these experimentally determined structures requires a highly uniform, standardized structural data source. The Protein Data Bank (PDB) has evolved over the years toward becoming the standard resource for macromolecular structures. However, the process selecting the data most suitable for specific applications is still very much based on personal preferences and understanding of the experimental techniques used to obtain these models. In this chapter, we will first explain the challenges with data standardization, annotation, and uniformity in the PDB entries determined by X-ray crystallography. We then discuss the specific effect that crystallographic data quality and model optimization methods have on structural models and how validation tools can be used to make informed choices. We also discuss specific advantages of using the PDB_REDO databank as a resource for structural data. Finally, we will provide guidelines on how to select the most suitable protein structure models for detailed analysis and how to select a set of structure models suitable for data mining.

  5. Fragment-Based Screening by Protein Crystallography: Successes and Pitfalls

    PubMed Central

    Chilingaryan, Zorik; Yin, Zhou; Oakley, Aaron J.

    2012-01-01

    Fragment-based drug discovery (FBDD) concerns the screening of low-molecular weight compounds against macromolecular targets of clinical relevance. These compounds act as starting points for the development of drugs. FBDD has evolved and grown in popularity over the past 15 years. In this paper, the rationale and technology behind the use of X-ray crystallography in fragment based screening (FBS) will be described, including fragment library design and use of synchrotron radiation and robotics for high-throughput X-ray data collection. Some recent uses of crystallography in FBS will be described in detail, including interrogation of the drug targets β-secretase, phenylethanolamine N-methyltransferase, phosphodiesterase 4A and Hsp90. These examples provide illustrations of projects where crystallography is straightforward or difficult, and where other screening methods can help overcome the limitations of crystallography necessitated by diffraction quality. PMID:23202926

  6. International summer school on macromolecular crystallographic computing. Final report

    SciTech Connect

    1998-08-01

    The School was the seventh in a series of International Union of Crystallography (IUCr) Crystallographic Symposia. The format of the School was formal lectures in the morning, tutorials in the afternoon, and software demonstrations and more lectures in the evening. The full program which left both the organizers and attendees exhausted, reflects the current state of excitement in the field of macromolecular structure determination using the technique of X-ray crystallography. The new and improved technologies and techniques described in these Proceedings are contributing to that growth and at the same time, as pointed out in the paper given by Sussman, creating challenges for the Protein Data Bank (PDB). As the School progressed, the authors were struck by the similarities to events which took place in small molecule crystallography beginning some 20 to 25 years ago. Growth then was fueled by the advent of new algorithms, affordable computer hardware, and good software. So it is today for macromolecular crystallography, but with the added bonus of the Internet which is changing how scientist conduct their research. Flack presented this view as part of his on-going contribution to how crystallographers use the Internet. After presentations discussing structures en masse they returned to the more traditional mode of presentation which parallels the determination of a single macromolecular structure: data collection -- phasing -- model building and visualization -- refinement.

  7. The future of crystallography in drug discovery

    PubMed Central

    Zheng, Heping; Hou, Jing; Zimmerman, Matthew D; Wlodawer, Alexander; Minor, Wladek

    2014-01-01

    Introduction X-ray crystallography plays an important role in structure-based drug design (SBDD), and accurate analysis of crystal structures of target macromolecules and macromolecule–ligand complexes is critical at all stages. However, whereas there has been significant progress in improving methods of structural biology, particularly in X-ray crystallography, corresponding progress in the development of computational methods (such as in silico high-throughput screening) is still on the horizon. Crystal structures can be overinterpreted and thus bias hypotheses and follow-up experiments. As in any experimental science, the models of macromolecular structures derived from X-ray diffraction data have their limitations, which need to be critically evaluated and well understood for structure-based drug discovery. Areas covered This review describes how the validity, accuracy and precision of a protein or nucleic acid structure determined by X-ray crystallography can be evaluated from three different perspectives: i) the nature of the diffraction experiment; ii) the interpretation of an electron density map; and iii) the interpretation of the structural model in terms of function and mechanism. The strategies to optimally exploit a macromolecular structure are also discussed in the context of ‘Big Data’ analysis, biochemical experimental design and structure-based drug discovery. Expert opinion Although X-ray crystallography is one of the most detailed ‘microscopes’ available today for examining macromolecular structures, the authors would like to re-emphasize that such structures are only simplified models of the target macromolecules. The authors also wish to reinforce the idea that a structure should not be thought of as a set of precise coordinates but rather as a framework for generating hypotheses to be explored. Numerous biochemical and biophysical experiments, including new diffraction experiments, can and should be performed to verify or falsify

  8. Mads.jl

    SciTech Connect

    Vesselinov, Velimir; O'Malley, Daniel; Lin, Youzuo; Vesselinova, Neda

    2016-07-01

    Mads.jl (Model analysis and decision support in Julia) is a code that streamlines the process of using data and models for analysis and decision support. It is based on another open-source code developed at LANL and written in C/C++ (MADS; http://mads.lanl.gov; LA-CC-11- 035). Mads.jl can work with external models of arbitrary complexity as well as built-in models of flow and transport in porous media. It enables a number of data- and model-based analyses including model calibration, sensitivity analysis, uncertainty quantification, and decision analysis. The code also can use a series of alternative adaptive computational techniques for Bayesian sampling, Monte Carlo, and Bayesian Information-Gap Decision Theory. The code is implemented in the Julia programming language, and has high-performance (parallel) and memory management capabilities. The code uses a series of third party modules developed by others. The code development will also include contributions to the existing third party modules written in Julia; this contributions will be important for the efficient implementation of the algorithm used by Mads.jl. The code also uses a series of LANL developed modules that are developed by Dan O'Malley; these modules will be also a part of the Mads.jl release. Mads.jl will be released under GPL V3 license. The code will be distributed as a Git repo at gitlab.com and github.com. Mads.jl manual and documentation will be posted at madsjulia.lanl.gov.

  9. MADS Users' Guide

    NASA Technical Reports Server (NTRS)

    Moerder, Daniel D.

    2014-01-01

    MADS (Minimization Assistant for Dynamical Systems) is a trajectory optimization code in which a user-specified performance measure is directly minimized, subject to constraints placed on a low-order discretization of user-supplied plant ordinary differential equations. This document describes the mathematical formulation of the set of trajectory optimization problems for which MADS is suitable, and describes the user interface. Usage examples are provided.

  10. MAD phasing with krypton.

    PubMed

    Cohen, A; Ellis, P; Kresge, N; Soltis, S M

    2001-02-01

    Experiments demonstrating the feasibility of Kr-edge MAD on frozen crystals as a routine method for structure determination are reported. Approximately 50% of protein crystals can be successfully derivatized by pressurization with the noble gases xenon or krypton. While Xe has produced many useful derivatives for MIR phasing over the last several years, the Xe edges (K edge = 34.6 keV, L(I) = 5.5 keV) are not easily accessible for MAD studies. As the Kr K edge (14.3 keV) is accessible on most MAD beamlines, Kr derivatization provides the additional opportunity to conduct a MAD experiment and obtain phases using only a single crystal. This paper describes the phasing of two proteins using Kr MAD: the 17 kDa Fe protein myoglobin (Mb) from sperm whale (Physeter catodon) and an 18 kDa protein (SP18) from green abalone (Haliotis fulgens). Three-wavelength data were collected at SSRL beamline 9-2 from crystals of Mb and SP18 incubated in 2.76 MPa of Kr gas for 2 min, depressurized and then flash-frozen in a stream of nitrogen gas at 100 K. MAD phases were calculated using the program SHARP and the resulting density improved with wARP. The final maps for both Mb and SP18 were of excellent quality.

  11. Operation of the Australian Store.Synchrotron for macromolecular crystallography

    SciTech Connect

    Meyer, Grischa R.; Aragão, David; Mudie, Nathan J.; Caradoc-Davies, Tom T.; McGowan, Sheena; Bertling, Philip J.; Groenewegen, David; Quenette, Stevan M.; Bond, Charles S.; Buckle, Ashley M.; Androulakis, Steve

    2014-10-01

    The Store.Synchrotron service, a fully functional, cloud computing-based solution to raw X-ray data archiving and dissemination at the Australian Synchrotron, is described. The Store.Synchrotron service, a fully functional, cloud computing-based solution to raw X-ray data archiving and dissemination at the Australian Synchrotron, is described. The service automatically receives and archives raw diffraction data, related metadata and preliminary results of automated data-processing workflows. Data are able to be shared with collaborators and opened to the public. In the nine months since its deployment in August 2013, the service has handled over 22.4 TB of raw data (∼1.7 million diffraction images). Several real examples from the Australian crystallographic community are described that illustrate the advantages of the approach, which include real-time online data access and fully redundant, secure storage. Discoveries in biological sciences increasingly require multidisciplinary approaches. With this in mind, Store.Synchrotron has been developed as a component within a greater service that can combine data from other instruments at the Australian Synchrotron, as well as instruments at the Australian neutron source ANSTO. It is therefore envisaged that this will serve as a model implementation of raw data archiving and dissemination within the structural biology research community.

  12. Operation of the Australian Store.Synchrotron for macromolecular crystallography

    PubMed Central

    Meyer, Grischa R.; Aragão, David; Mudie, Nathan J.; Caradoc-Davies, Tom T.; McGowan, Sheena; Bertling, Philip J.; Groenewegen, David; Quenette, Stevan M.; Bond, Charles S.; Buckle, Ashley M.; Androulakis, Steve

    2014-01-01

    The Store.Synchrotron service, a fully functional, cloud computing-based solution to raw X-ray data archiving and dissemination at the Australian Synchrotron, is described. The service automatically receives and archives raw diffraction data, related metadata and preliminary results of automated data-processing workflows. Data are able to be shared with collaborators and opened to the public. In the nine months since its deployment in August 2013, the service has handled over 22.4 TB of raw data (∼1.7 million diffraction images). Several real examples from the Australian crystallographic community are described that illustrate the advantages of the approach, which include real-time online data access and fully redundant, secure storage. Discoveries in biological sciences increasingly require multidisciplinary approaches. With this in mind, Store.Synchrotron has been developed as a component within a greater service that can combine data from other instruments at the Australian Synchrotron, as well as instruments at the Australian neutron source ANSTO. It is therefore envisaged that this will serve as a model implementation of raw data archiving and dissemination within the structural biology research community. PMID:25286837

  13. Operation of the Australian Store.Synchrotron for macromolecular crystallography.

    PubMed

    Meyer, Grischa R; Aragão, David; Mudie, Nathan J; Caradoc-Davies, Tom T; McGowan, Sheena; Bertling, Philip J; Groenewegen, David; Quenette, Stevan M; Bond, Charles S; Buckle, Ashley M; Androulakis, Steve

    2014-10-01

    The Store.Synchrotron service, a fully functional, cloud computing-based solution to raw X-ray data archiving and dissemination at the Australian Synchrotron, is described. The service automatically receives and archives raw diffraction data, related metadata and preliminary results of automated data-processing workflows. Data are able to be shared with collaborators and opened to the public. In the nine months since its deployment in August 2013, the service has handled over 22.4 TB of raw data (∼1.7 million diffraction images). Several real examples from the Australian crystallographic community are described that illustrate the advantages of the approach, which include real-time online data access and fully redundant, secure storage. Discoveries in biological sciences increasingly require multidisciplinary approaches. With this in mind, Store.Synchrotron has been developed as a component within a greater service that can combine data from other instruments at the Australian Synchrotron, as well as instruments at the Australian neutron source ANSTO. It is therefore envisaged that this will serve as a model implementation of raw data archiving and dissemination within the structural biology research community.

  14. Center for Macromolecular Crystallography, University of Alabama in Birmingham

    NASA Technical Reports Server (NTRS)

    Navia, Manuel A.

    1991-01-01

    Porcine pancreatic elastase (PPE) crystals grown under microgravity conditions on mission STS-26 of the Space Shuttle Discovery were shown to diffract to considerably higher resolution than the best PPE crystals grown by us on the ground. We have now independently refined both the microgravity and ground-based data. Preliminary results of these refinements are summarized. These results show nearly a doubling of experimental diffraction data for this structure, exceeding 1.3 A resolution. Improved phase information derived from the refined structure of PPE based on this microgravity data has allowed us to interpret previously-uninterpretable electron density obtained from ground-based crystals of a complex of PPE with a chemically-reactive inhibitor. Intermediate stages in the enzyme-inhibitor reaction mechanism in the crystal can now be directly observed. Further refinement of PPE structures is in progress.

  15. Method for removing atomic-model bias in macromolecular crystallography

    DOEpatents

    Terwilliger, Thomas C.

    2006-08-01

    Structure factor bias in an electron density map for an unknown crystallographic structure is minimized by using information in a first electron density map to elicit expected structure factor information. Observed structure factor amplitudes are combined with a starting set of crystallographic phases to form a first set of structure factors. A first electron density map is then derived and features of the first electron density map are identified to obtain expected distributions of electron density. Crystallographic phase probability distributions are established for possible crystallographic phases of reflection k, and the process is repeated as k is indexed through all of the plurality of reflections. An updated electron density map is derived from the crystallographic phase probability distributions for each one of the reflections. The entire process is then iterated to obtain a final set of crystallographic phases with minimum bias from known electron density maps.

  16. Macromolecular Crystallography conventional and high-throughput methods

    SciTech Connect

    Wasserman, Stephen R.; Smith, David W.; D'Amico, Kevin L.; Koss, John W.; Morisco, Laura L.; Burley, Stephen K.

    2007-09-27

    High-throughput data collection requires the seamless interoperation of various hardware components. User-supplied descriptions of protein crystals must also be directly linked with the diffraction data. Such linkages can be achieved efficiently with computer databases. A database that tracks production of the protein samples, crystallization, and diffraction from the resultant crystals serves as the glue that holds the entire gene-to-structure process together. This chapter begins by discussing data collection processes and hardware. It then illustrates how a well-constructed database ensures information flow through the steps of data acquisition. Such a database allows synchrotron beamline measurements to be directly and efficiently integrated into the process of protein crystallographic structure determination.

  17. Fast native-SAD phasing for routine macromolecular structure determination.

    PubMed

    Weinert, Tobias; Olieric, Vincent; Waltersperger, Sandro; Panepucci, Ezequiel; Chen, Lirong; Zhang, Hua; Zhou, Dayong; Rose, John; Ebihara, Akio; Kuramitsu, Seiki; Li, Dianfan; Howe, Nicole; Schnapp, Gisela; Pautsch, Alexander; Bargsten, Katja; Prota, Andrea E; Surana, Parag; Kottur, Jithesh; Nair, Deepak T; Basilico, Federica; Cecatiello, Valentina; Pasqualato, Sebastiano; Boland, Andreas; Weichenrieder, Oliver; Wang, Bi-Cheng; Steinmetz, Michel O; Caffrey, Martin; Wang, Meitian

    2015-02-01

    We describe a data collection method that uses a single crystal to solve X-ray structures by native SAD (single-wavelength anomalous diffraction). We solved the structures of 11 real-life examples, including a human membrane protein, a protein-DNA complex and a 266-kDa multiprotein-ligand complex, using this method. The data collection strategy is suitable for routine structure determination and can be implemented at most macromolecular crystallography synchrotron beamlines.

  18. Molecular modeling and expression analysis of a MADS-box cDNA from mango (Mangifera indica L.).

    PubMed

    Pacheco-Sánchez, Magda A; Contreras-Vergara, Carmen A; Hernandez-Navarro, Eduardo; Yepiz-Plascencia, Gloria; Martínez-Téllez, Miguel A; Casas-Flores, Sergio; Arvizu-Flores, Aldo A; Islas-Osuna, Maria A

    2014-08-01

    MADS-box genes are a large family of transcription factors initially discovered for their role during development of flowers and fruits. The MADS-box transcription factors from animals have been studied by X-ray protein crystallography but those from plants remain to be studied. In this work, a MADS-box cDNA from mango encoding a protein of 254 residues was obtained and compared. Based on phylogenetic analysis, it is proposed that the MADS-box transcription factor expressed in mango fruit (MiMADS1) belongs to the SEP clade of MADS-box proteins. MiMADS1 mRNA steady-state levels did not changed during mango fruit development and were up-regulated, when mango fruits reached physiological maturity as assessed by qRT-PCR. Thus, MiMADS1 could have a role during development and ripening of this fruit. The theoretical structural model of MiMADS1 showed the DNA-binding domain folding bound to a double-stranded DNA. Therefore, MiMADS1 is an interesting model for understanding DNA-binding for transcriptional regulation.

  19. A public database of macromolecular diffraction experiments.

    PubMed

    Grabowski, Marek; Langner, Karol M; Cymborowski, Marcin; Porebski, Przemyslaw J; Sroka, Piotr; Zheng, Heping; Cooper, David R; Zimmerman, Matthew D; Elsliger, Marc André; Burley, Stephen K; Minor, Wladek

    2016-11-01

    The low reproducibility of published experimental results in many scientific disciplines has recently garnered negative attention in scientific journals and the general media. Public transparency, including the availability of `raw' experimental data, will help to address growing concerns regarding scientific integrity. Macromolecular X-ray crystallography has led the way in requiring the public dissemination of atomic coordinates and a wealth of experimental data, making the field one of the most reproducible in the biological sciences. However, there remains no mandate for public disclosure of the original diffraction data. The Integrated Resource for Reproducibility in Macromolecular Crystallography (IRRMC) has been developed to archive raw data from diffraction experiments and, equally importantly, to provide related metadata. Currently, the database of our resource contains data from 2920 macromolecular diffraction experiments (5767 data sets), accounting for around 3% of all depositions in the Protein Data Bank (PDB), with their corresponding partially curated metadata. IRRMC utilizes distributed storage implemented using a federated architecture of many independent storage servers, which provides both scalability and sustainability. The resource, which is accessible via the web portal at http://www.proteindiffraction.org, can be searched using various criteria. All data are available for unrestricted access and download. The resource serves as a proof of concept and demonstrates the feasibility of archiving raw diffraction data and associated metadata from X-ray crystallographic studies of biological macromolecules. The goal is to expand this resource and include data sets that failed to yield X-ray structures in order to facilitate collaborative efforts that will improve protein structure-determination methods and to ensure the availability of `orphan' data left behind for various reasons by individual investigators and/or extinct structural genomics

  20. Status of the crystallography beamlines at PETRA III

    NASA Astrophysics Data System (ADS)

    Burkhardt, Anja; Pakendorf, Tim; Reime, Bernd; Meyer, Jan; Fischer, Pontus; Stübe, Nicolas; Panneerselvam, Saravanan; Lorbeer, Olga; Stachnik, Karolina; Warmer, Martin; Rödig, Philip; Göries, Dennis; Meents, Alke

    2016-03-01

    Since 2013, three beamlines for macromolecular crystallography are available to users at the third-generation synchrotron PETRA III in Hamburg: P11, P13 and P14, the latter two operated by EMBL. Beamline P11 is operated by DESY and is equipped with a Pilatus 6M detector. Together with the photon flux of 2× 10^{13} ph/s provided by the very brilliant X-ray source of PETRA III, a full data set can be typically collected in less than 2min. P11 provides state-of-the-art microfocusing capabilities with beam sizes down to 1× 1 μ m2, which makes the beamline ideally suited for investigation of microcrystals and serial crystallography experiments. An automatic sample changer allows fast sample exchange in less than 20s, which enables high-throughput crystallography and fast crystal screening. For sample preparation, an S2 biosafety laboratory is available in close proximity to the beamline.

  1. Sub-atomic resolution X-ray crystallography and neutron crystallography: promise, challenges and potential.

    PubMed

    Blakeley, Matthew P; Hasnain, Samar S; Antonyuk, Svetlana V

    2015-07-01

    The International Year of Crystallography saw the number of macromolecular structures deposited in the Protein Data Bank cross the 100000 mark, with more than 90000 of these provided by X-ray crystallography. The number of X-ray structures determined to sub-atomic resolution (i.e. ≤1 Å) has passed 600 and this is likely to continue to grow rapidly with diffraction-limited synchrotron radiation sources such as MAX-IV (Sweden) and Sirius (Brazil) under construction. A dozen X-ray structures have been deposited to ultra-high resolution (i.e. ≤0.7 Å), for which precise electron density can be exploited to obtain charge density and provide information on the bonding character of catalytic or electron transfer sites. Although the development of neutron macromolecular crystallography over the years has been far less pronounced, and its application much less widespread, the availability of new and improved instrumentation, combined with dedicated deuteration facilities, are beginning to transform the field. Of the 83 macromolecular structures deposited with neutron diffraction data, more than half (49/83, 59%) were released since 2010. Sub-mm(3) crystals are now regularly being used for data collection, structures have been determined to atomic resolution for a few small proteins, and much larger unit-cell systems (cell edges >100 Å) are being successfully studied. While some details relating to H-atom positions are tractable with X-ray crystallography at sub-atomic resolution, the mobility of certain H atoms precludes them from being located. In addition, highly polarized H atoms and protons (H(+)) remain invisible with X-rays. Moreover, the majority of X-ray structures are determined from cryo-cooled crystals at 100 K, and, although radiation damage can be strongly controlled, especially since the advent of shutterless fast detectors, and by using limited doses and crystal translation at micro-focus beams, radiation damage can still take place. Neutron

  2. Sub-atomic resolution X-ray crystallography and neutron crystallography: promise, challenges and potential

    PubMed Central

    Blakeley, Matthew P.; Hasnain, Samar S.; Antonyuk, Svetlana V.

    2015-01-01

    The International Year of Crystallography saw the number of macromolecular structures deposited in the Protein Data Bank cross the 100000 mark, with more than 90000 of these provided by X-ray crystallography. The number of X-ray structures determined to sub-atomic resolution (i.e. ≤1 Å) has passed 600 and this is likely to continue to grow rapidly with diffraction-limited synchrotron radiation sources such as MAX-IV (Sweden) and Sirius (Brazil) under construction. A dozen X-ray structures have been deposited to ultra-high resolution (i.e. ≤0.7 Å), for which precise electron density can be exploited to obtain charge density and provide information on the bonding character of catalytic or electron transfer sites. Although the development of neutron macromolecular crystallography over the years has been far less pronounced, and its application much less widespread, the availability of new and improved instrumentation, combined with dedicated deuteration facilities, are beginning to transform the field. Of the 83 macromolecular structures deposited with neutron diffraction data, more than half (49/83, 59%) were released since 2010. Sub-mm3 crystals are now regularly being used for data collection, structures have been determined to atomic resolution for a few small proteins, and much larger unit-cell systems (cell edges >100 Å) are being successfully studied. While some details relating to H-atom positions are tractable with X-ray crystallography at sub-atomic resolution, the mobility of certain H atoms precludes them from being located. In addition, highly polarized H atoms and protons (H+) remain invisible with X-rays. Moreover, the majority of X-ray structures are determined from cryo-cooled crystals at 100 K, and, although radiation damage can be strongly controlled, especially since the advent of shutterless fast detectors, and by using limited doses and crystal translation at micro-focus beams, radiation damage can still take place. Neutron

  3. Energy optimization of a regular macromolecular crystallography beamline for ultra-high-resolution crystallography

    SciTech Connect

    Rosenbaum, Gerd; Ginell, Stephan L.; Chen, Julian C.-H.

    2015-01-01

    In this study, a practical method for operating existing undulator synchrotron beamlines at photon energies considerably higher than their standard operating range is described and applied at beamline 19-ID of the Structural Biology Center at the Advanced Photon Source enabling operation at 30 keV. Adjustments to the undulator spectrum were critical to enhance the 30 keV flux while reducing the lower- and higher-energy harmonic contamination. A Pd-coated mirror and Al attenuators acted as effective low- and high-bandpass filters. The resulting flux at 30 keV, although significantly lower than with X-ray optics designed and optimized for this energy, allowed for accurate data collection on crystals of the small protein crambin to 0.38 Å resolution.

  4. Energy optimization of a regular macromolecular crystallography beamline for ultra-high-resolution crystallography

    DOE PAGES

    Rosenbaum, Gerd; Ginell, Stephan L.; Chen, Julian C.-H.

    2015-01-01

    In this study, a practical method for operating existing undulator synchrotron beamlines at photon energies considerably higher than their standard operating range is described and applied at beamline 19-ID of the Structural Biology Center at the Advanced Photon Source enabling operation at 30 keV. Adjustments to the undulator spectrum were critical to enhance the 30 keV flux while reducing the lower- and higher-energy harmonic contamination. A Pd-coated mirror and Al attenuators acted as effective low- and high-bandpass filters. The resulting flux at 30 keV, although significantly lower than with X-ray optics designed and optimized for this energy, allowed for accuratemore » data collection on crystals of the small protein crambin to 0.38 Å resolution.« less

  5. MAD - Monitoring ALICE Dataflow

    NASA Astrophysics Data System (ADS)

    Chibante Barroso, V.; Costa, F.; Grigoras, C.; Wegrzynek, A.

    2015-12-01

    ALICE (A Large Ion Collider Experiment) is the heavy-ion detector designed to study the physics of strongly interacting matter and the quark-gluon plasma at the CERN Large Hadron Collider (LHC). Following a successful Run 1, which ended in February 2013, the ALICE data acquisition (DAQ) entered a consolidation phase to prepare for Run 2 which will start in the beginning of 2015. A new software tool has been developed by the data acquisition project to improve the monitoring of the experiment's dataflow, from the data readout in the DAQ farm up to its shipment to CERN's main computer centre. This software, called ALICE MAD (Monitoring ALICE Dataflow), uses the MonALISA framework as core module to gather, process, aggregate and distribute monitoring values from the different processes running in the distributed DAQ farm. Data are not only pulled from the data sources to MAD but can also be pushed by dedicated data collectors or the data source processes. A large set of monitored metrics (from the backpressure status on the readout links to event counters in each of the DAQ nodes and aggregated data rates for the whole data acquisition) is needed to provide a comprehensive view of the DAQ status. MAD also injects alarms in the Orthos alarm system whenever abnormal conditions are detected. The MAD web-based GUI uses WebSockets to provide dynamic and on-time status displays for the ALICE shift crew. Designed as a widget-based system, MAD supports an easy integration of new visualization blocks and also customization of the information displayed to the shift crew based on the ALICE activities.

  6. Protein Crystallography from the Perspective of Technology Developments

    PubMed Central

    Su, Xiao-Dong; Zhang, Heng; Terwilliger, Thomas C.; Liljas, Anders; Xiao, Junyu; Dong, Yuhui

    2015-01-01

    Early on, crystallography was a domain of mineralogy and mathematics and dealt mostly with symmetry properties and imaginary crystal lattices. This changed when Wilhelm Conrad Röntgen discovered X-rays in 1895, and in 1912 Max von Laue and his associates discovered X-ray irradiated salt crystals would produce diffraction patterns that could reveal the internal atomic periodicity of the crystals. In the same year the father-and-son team, Henry and Lawrence Bragg successfully solved the first crystal structure of sodium chloride and the era of modern crystallography began. Protein crystallography (PX) started some 20 years later with the pioneering work of British crystallographers. In the past 50-60 years, the achievements of modern crystallography and particularly those in protein crystallography have been due to breakthroughs in theoretical and technical advancements such as phasing and direct methods; to more powerful X-ray sources such as synchrotron radiation (SR); to more sensitive and efficient X-ray detectors; to ever faster computers and to improvements in software. The exponential development of protein crystallography has been accelerated by the invention and applications of recombinant DNA technology that can yield nearly any protein of interest in large amounts and with relative ease. Novel methods, informatics platforms, and technologies for automation and high-throughput have allowed the development of large-scale, high efficiency macromolecular crystallography efforts in the field of structural genomics (SG). Very recently, the X-ray free-electron laser (XFEL) sources and its applications in protein crystallography have shown great potential for revolutionizing the whole field again in the near future. PMID:25983389

  7. Macromolecular diffractive imaging using imperfect crystals

    PubMed Central

    Ayyer, Kartik; Yefanov, Oleksandr; Oberthür, Dominik; Roy-Chowdhury, Shatabdi; Galli, Lorenzo; Mariani, Valerio; Basu, Shibom; Coe, Jesse; Conrad, Chelsie E.; Fromme, Raimund; Schaffer, Alexander; Dörner, Katerina; James, Daniel; Kupitz, Christopher; Metz, Markus; Nelson, Garrett; Lourdu Xavier, Paulraj; Beyerlein, Kenneth R.; Schmidt, Marius; Sarrou, Iosifina; Spence, John C. H.; Weierstall, Uwe; White, Thomas A.; Yang, Jay-How; Zhao, Yun; Liang, Mengning; Aquila, Andrew; Hunter, Mark S.; Robinson, Joseph S.; Koglin, Jason E.; Boutet, Sébastien; Fromme, Petra; Barty, Anton; Chapman, Henry N.

    2016-01-01

    The three-dimensional structures of macromolecules and their complexes are predominantly elucidated by X-ray protein crystallography. A major limitation is access to high-quality crystals, to ensure X-ray diffraction extends to sufficiently large scattering angles and hence yields sufficiently high-resolution information that the crystal structure can be solved. The observation that crystals with shrunken unit-cell volumes and tighter macromolecular packing often produce higher-resolution Bragg peaks1,2 hints that crystallographic resolution for some macromolecules may be limited not by their heterogeneity but rather by a deviation of strict positional ordering of the crystalline lattice. Such displacements of molecules from the ideal lattice give rise to a continuous diffraction pattern, equal to the incoherent sum of diffraction from rigid single molecular complexes aligned along several discrete crystallographic orientations and hence with an increased information content3. Although such continuous diffraction patterns have long been observed—and are of interest as a source of information about the dynamics of proteins4 —they have not been used for structure determination. Here we show for crystals of the integral membrane protein complex photosystem II that lattice disorder increases the information content and the resolution of the diffraction pattern well beyond the 4.5 Å limit of measurable Bragg peaks, which allows us to directly phase5 the pattern. With the molecular envelope conventionally determined at 4.5 Å as a constraint, we then obtain a static image of the photosystem II dimer at 3.5 Å resolution. This result shows that continuous diffraction can be used to overcome long-supposed resolution limits of macromolecular crystallography, with a method that puts great value in commonly encountered imperfect crystals and opens up the possibility for model-free phasing6,7. PMID:26863980

  8. Macromolecular diffractive imaging using imperfect crystals

    NASA Astrophysics Data System (ADS)

    Ayyer, Kartik; Yefanov, Oleksandr M.; Oberthür, Dominik; Roy-Chowdhury, Shatabdi; Galli, Lorenzo; Mariani, Valerio; Basu, Shibom; Coe, Jesse; Conrad, Chelsie E.; Fromme, Raimund; Schaffer, Alexander; Dörner, Katerina; James, Daniel; Kupitz, Christopher; Metz, Markus; Nelson, Garrett; Xavier, Paulraj Lourdu; Beyerlein, Kenneth R.; Schmidt, Marius; Sarrou, Iosifina; Spence, John C. H.; Weierstall, Uwe; White, Thomas A.; Yang, Jay-How; Zhao, Yun; Liang, Mengning; Aquila, Andrew; Hunter, Mark S.; Robinson, Joseph S.; Koglin, Jason E.; Boutet, Sébastien; Fromme, Petra; Barty, Anton; Chapman, Henry N.

    2016-02-01

    The three-dimensional structures of macromolecules and their complexes are mainly elucidated by X-ray protein crystallography. A major limitation of this method is access to high-quality crystals, which is necessary to ensure X-ray diffraction extends to sufficiently large scattering angles and hence yields information of sufficiently high resolution with which to solve the crystal structure. The observation that crystals with reduced unit-cell volumes and tighter macromolecular packing often produce higher-resolution Bragg peaks suggests that crystallographic resolution for some macromolecules may be limited not by their heterogeneity, but by a deviation of strict positional ordering of the crystalline lattice. Such displacements of molecules from the ideal lattice give rise to a continuous diffraction pattern that is equal to the incoherent sum of diffraction from rigid individual molecular complexes aligned along several discrete crystallographic orientations and that, consequently, contains more information than Bragg peaks alone. Although such continuous diffraction patterns have long been observed—and are of interest as a source of information about the dynamics of proteins—they have not been used for structure determination. Here we show for crystals of the integral membrane protein complex photosystem II that lattice disorder increases the information content and the resolution of the diffraction pattern well beyond the 4.5-ångström limit of measurable Bragg peaks, which allows us to phase the pattern directly. Using the molecular envelope conventionally determined at 4.5 ångströms as a constraint, we obtain a static image of the photosystem II dimer at a resolution of 3.5 ångströms. This result shows that continuous diffraction can be used to overcome what have long been supposed to be the resolution limits of macromolecular crystallography, using a method that exploits commonly encountered imperfect crystals and enables model-free phasing.

  9. Crystallography: Sources of inspiration

    NASA Astrophysics Data System (ADS)

    McSweeney, Sean; Fromme, Petra

    2014-01-01

    Synchrotrons have long been the preferred X-ray sources for crystallography, but competition has arrived with the advent of X-ray free-electron lasers. A synchrotron expert and an advocate of free-electron lasers discuss the prospects of the respective source types for applications in structural biology.

  10. CLIMS: crystallography laboratory information management system.

    PubMed

    Fulton, Kate F; Ervine, Shaun; Faux, Noel; Forster, Richard; Jodun, Rachel A; Ly, Wayson; Robilliard, Lee; Sonsini, Jai; Whelan, Dan; Whisstock, James C; Buckle, Ashley M

    2004-09-01

    Macromolecular crystallography requires simple yet effective means of organizing and managing the large amounts of data generated by crystallization experiments. There are several freely available web-based Laboratory Information Management Systems (LIMS) that assist in these tasks. These, however, rely on the limited user interfaces allowed in HTML-based web pages. To address this limitation, a new LIMS for protein crystallization, which features a novel rich graphical user interface (GUI) to a relational database, has been developed. This application, which is called CLIMS (Crystallography LIMS), assists in all aspects of protein-crystallization projects: protein expression, handling, crystallization optimization, visualization of results and preliminary diffraction data. Extensive use of templates, particularly for commercial screens and common optimization grid screens, exploits the redundancy in experimental setups. The crystallization tray is the central focus of the graphical interface, thus facilitating rapid visualization and annotation of results. CLIMS was developed specifically to cater for the needs of individual laboratories requiring an intuitive and robust system for managing crystallization experiments and is freely available.

  11. SPINE-compatible ‘carboloops’: a new microshaped vitreous carbon sample mount for X-ray and neutron crystallography

    PubMed Central

    Romoli, Filippo; Mossou, Estelle; Cuypers, Maxime; van der Linden, Peter; Carpentier, Philippe; Mason, Sax A.; Forsyth, V. Trevor; McSweeney, Sean

    2014-01-01

    A novel vitreous carbon mount for macromolecular crystallography, suitable for neutron and X-ray crystallographic studies, has been developed. The technology described here is compatible both with X-ray and neutron cryo-crystallography. The mounts have low density and low background scattering for both neutrons and X-rays. They are prepared by laser cutting, allowing high standards of production quality, the ability to custom-design the mount to specific crystal sizes and large-scale production. PMID:24817737

  12. [Travelers, mad, wandering].

    PubMed

    Vaschetto, Emilio

    2014-01-01

    This article explores the notion of "wandering" through the use of some phenomena enrolled at the dawn of modernity such as the Rousseau dromomanie's philosopher and writer, the origin of the first mad traveler (Albert Dadas), epidemics of mad travelers Europe and romantic tourism (with renewed acquires significance in the "beat generation" of the twentieth century). These historical facts are "mounting" as play contemporary manifestations such as loss, disorientation, to lose one's way, and wandering without reducing them only to clinical psychosis. Readings of classic psychiatrists such as Régis, Foville, Sérieux and Capgras, Tissié, go hand in hand with the current readings of the philosopher Ian Hacking and critics of pop culture as S. Reynolds and D. Diederichsen, illustrating how the travel's phenomenon can make different subjective configurations depending on historical times. In conclusion it is noted that not only psychosis exposes the wandering soul of suffering but there are also subject positions (as will be exemplified in a clinical case) and go no further nesting wandering into human existence.

  13. Blueprinting macromolecular electronics.

    PubMed

    Palma, Carlos-Andres; Samorì, Paolo

    2011-06-01

    Recently, by mastering either top-down or bottom-up approaches, tailor-made macromolecular nano-objects with semiconducting properties have been fabricated. These engineered nanostructures for organic electronics are based on conjugated systems predominantly made up of sp²-hybridized carbon, such as graphene nanoribbons. Here, we describe developments in a selection of these nanofabrication techniques, which include graphene carving, stimulus-induced synthesis of conjugated polymers and surface-assisted synthesis. We also assess their potential to reproduce chemically and spatially precise molecular arrangements, that is, molecular blueprints. In a broad context, the engineering of a molecular blueprint represents the fabrication of an integrated all-organic macromolecular electronic circuit. In this Perspective, we suggest chemical routes, as well as convergent on-surface synthesis and microfabrication approaches, for the ultimate goal of bringing the field closer to technology.

  14. Practical macromolecular cryocrystallography

    SciTech Connect

    Pflugrath, J. W.

    2015-05-27

    Current methods, reagents and experimental hardware for successfully and reproducibly flash-cooling macromolecular crystals to cryogenic temperatures for X-ray diffraction data collection are reviewed. Cryocrystallography is an indispensable technique that is routinely used for single-crystal X-ray diffraction data collection at temperatures near 100 K, where radiation damage is mitigated. Modern procedures and tools to cryoprotect and rapidly cool macromolecular crystals with a significant solvent fraction to below the glass-transition phase of water are reviewed. Reagents and methods to help prevent the stresses that damage crystals when flash-cooling are described. A method of using isopentane to assess whether cryogenic temperatures have been preserved when dismounting screened crystals is also presented.

  15. Seeing the chemistry in biology with neutron crystallography

    PubMed Central

    Langan, Paul; Chen, Julian C.-H.

    2017-01-01

    New developments in macromolecular neutron crystallography have led to an increasing number of structures published over the last decade. Hydrogen atoms, normally invisible in most X-ray crystal structures, become visible in neutron structures. Using X-rays allows one to see structure, while neutrons allow one to reveal the chemistry inherent in these macromolecular structures. A number of surprising and sometimes controversial results have emerged from recent neutron structures; because it is difficult to see or predict hydrogen atoms in X-ray structures, when they are seen by neutrons they can be in unexpected locations with important chemical and biological consequences. Here we describe examples of chemistry seen with neutrons for the first time in biological macromolecules over the past few years. PMID:23852376

  16. Seeing the chemistry in biology with neutron crystallography.

    PubMed

    Langan, Paul; Chen, Julian C-H

    2013-09-07

    New developments in macromolecular neutron crystallography have led to an increasing number of structures published over the last decade. Hydrogen atoms, normally invisible in most X-ray crystal structures, become visible with neutrons. Using X-rays allows one to see structure, while neutrons allow one to reveal the chemistry inherent in these macromolecular structures. A number of surprising and sometimes controversial results have emerged; because it is difficult to see or predict hydrogen atoms in X-ray structures, when they are seen by neutrons they can be in unexpected locations with important chemical and biological consequences. Here we describe examples of chemistry seen with neutrons for the first time in biological macromolecules over the past few years.

  17. Screening Madness in American Culture.

    PubMed

    Rohr, Susanne

    2015-09-01

    This two-step argument first establishes that the majority of recent American films dealing with mental illness draw on a traditional iconography of madness as it has been established over the centuries in Western culture. In this vocabulary of images, the mad are typically seen as wise fools, as dangerous villains or as gifted geniuses. The author then argues that some of these new films add a fourth category in which the mad are defined as normal and the person with autism as the embodiment of this normalcy. A close examination of the films then suggests that high functioning autism has become the embodiment of America's current cultural condition.

  18. Practical macromolecular cryocrystallography

    PubMed Central

    Pflugrath, J. W.

    2015-01-01

    Cryocrystallography is an indispensable technique that is routinely used for single-crystal X-ray diffraction data collection at temperatures near 100 K, where radiation damage is mitigated. Modern procedures and tools to cryoprotect and rapidly cool macromolecular crystals with a significant solvent fraction to below the glass-transition phase of water are reviewed. Reagents and methods to help prevent the stresses that damage crystals when flash-cooling are described. A method of using isopentane to assess whether cryogenic temperatures have been preserved when dismounting screened crystals is also presented. PMID:26057787

  19. Practical macromolecular cryocrystallography.

    PubMed

    Pflugrath, J W

    2015-06-01

    Cryocrystallography is an indispensable technique that is routinely used for single-crystal X-ray diffraction data collection at temperatures near 100 K, where radiation damage is mitigated. Modern procedures and tools to cryoprotect and rapidly cool macromolecular crystals with a significant solvent fraction to below the glass-transition phase of water are reviewed. Reagents and methods to help prevent the stresses that damage crystals when flash-cooling are described. A method of using isopentane to assess whether cryogenic temperatures have been preserved when dismounting screened crystals is also presented.

  20. Annealing macromolecular crystals.

    PubMed

    Hanson, B Leif; Bunick, Gerard J

    2007-01-01

    The process of crystal annealing has been used to improve the quality of diffraction from crystals that would otherwise be discarded for displaying unsatisfactory diffraction after flash cooling. Although techniques and protocols vary, macromolecular crystals are annealed by warming the flash-cooled crystal, then flash cooling it again. To apply macromolecular crystal annealing, a flash-cooled crystal displaying unacceptably high mosaicity or diffraction from ice is removed from the goniometer and immediately placed in cryoprotectant buffer. The crystal is incubated in the buffer at either room temperature or the temperature at which the crystal was grown. After about 3 min, the crystal is remounted in the loop and flash cooled. In situ annealing techniques, where the cold stream is diverted and the crystal allowed to warm on the loop prior to flash cooling, are variations of annealing that appears to work best when large solvent channels are not present in the crystal lattice or the solvent content of the crystal is relatively low.

  1. Spindle assembly checkpoint robustness requires Tpr-mediated regulation of Mad1/Mad2 proteostasis

    PubMed Central

    Schweizer, Nina; Ferrás, Cristina; Kern, David M.; Logarinho, Elsa; Cheeseman, Iain M.

    2013-01-01

    Tpr is a conserved nuclear pore complex (NPC) protein implicated in the spindle assembly checkpoint (SAC) by an unknown mechanism. Here, we show that Tpr is required for normal SAC response by stabilizing Mad1 and Mad2 before mitosis. Tpr coimmunoprecipitated with Mad1 and Mad2 (hereafter designated as Tpr/Mad1/Mad2 or TM2 complex) during interphase and mitosis, and is required for Mad1–c-Mad2 recruitment to NPCs. Interestingly, Tpr was normally undetectable at kinetochores and dispensable for Mad1, but not for Mad2, kinetochore localization, which suggests that SAC robustness depends on Mad2 levels at kinetochores. Protein half-life measurements demonstrate that Tpr stabilizes Mad1 and Mad2, ensuring normal Mad1–c-Mad2 production in an mRNA- and kinetochore-independent manner. Overexpression of GFP-Mad2 restored normal SAC response and Mad2 kinetochore levels in Tpr-depleted cells. Mechanistically, we provide evidence that Tpr might spatially regulate SAC proteostasis through the SUMO-isopeptidases SENP1 and SENP2 at NPCs. Thus, Tpr is a kinetochore-independent, rate-limiting factor required to mount and sustain a robust SAC response. PMID:24344181

  2. X-ray crystallography facility for the international space station

    NASA Astrophysics Data System (ADS)

    McDdonald, William T.; Lewis, Johanna L.; Smith, Craig D.; Delucas, Lawrence J.

    1997-01-01

    Directed by NASA's Office of Space Access and Technology (OSAT), the University of Alabama at Birmingham (UAB) Center for Macromolecular Crystallography (CMC) recently completed a Design Feasibility Study for the X-ray Crystallography Facility (XCF) for the International Space Station (ISS). The XCF is a facility for growing macromolecular protein crystals; harvesting, selecting, and mounting sample crystals, and snap-freezing the samples, if necessary; performing x-ray diffraction; and downlinking the diffraction data to the ground. Knowledge of the structure of protein molecules is essential for the development of pharmaceuticals by structure-based drug design techniques. Currently, x-ray diffraction of high quality protein crystals is the only method of determining the structure of these macromolecules. High quality protein crystals have been grown in microgravity onboard the Space Shuttle Orbiter for more than 10 years, but these crystals always have been returned to Earth for x-ray diffraction. The XCF will allow crystal growth, harvesting, mounting, and x-ray diffraction onboard the ISS, maximizing diffraction data quality and timeliness. This paper presents the XCF design concept, describing key feasibility issues for the ISS application and advanced technologies and operational features which resolve those issues. The conclusion is that the XCF design is feasible and can be operational onboard the ISS by early in 2002.

  3. MADS about MOSS

    PubMed Central

    Singer, SD

    2009-01-01

    Classic MIKC-type MADS-box genes (MIKCc) play diverse and crucial roles in angiosperm development, the most studied and best understood of which is the specification of floral organ identities. To shed light on how the flower evolved, phylogenetic and functional analyses of genes involved in its ontogeny, such as the MIKCc genes, must be undertaken in as broad a selection as possible of plants with disparate ancestries. Since little is known about the functions of these genes in non-seed plants, we investigated the developmental roles of a subset of the MIKCc genes present in the moss, Physcomitrella patens, which is positioned informatively near the base of the land plant evolutionary tree. We observed that transgenic lines possessing an antisense copy of a MIKCc gene characteristically displayed knocked-down expression of the corresponding native MIKCc gene as well as multiple diverse phenotypic alterations to the haploid gametophytic and diploid sporophytic generations of the life cycle.1 In this addendum, we re-examine our findings in the light of recent pertinent literature and provide additional data concerning the effects of simultaneously knocking out multiple MIKCc genes in this moss. PMID:19649183

  4. Macromolecular crystal growing system

    NASA Technical Reports Server (NTRS)

    Snyder, Robert S. (Inventor); Herren, Blair J. (Inventor); Carter, Daniel C. (Inventor); Yost, Vaughn H. (Inventor); Bugg, Charles E. (Inventor); Delucas, Lawrence J. (Inventor); Suddath, Fred L. (Inventor)

    1991-01-01

    A macromolecular crystal growing system especially designed for growing crystals in the low gravity of space as well as the gravity of earth includes at least one tray assembly, a carrier assembly which receives the tray, and a refrigeration-incubation module in which the carrier assembly is received. The tray assembly includes a plurality of sealed chambers with a plastic syringe and a plug means for the double tip of the syringe provided therein. Ganging mechanisms operate the syringes and plugs simultaneously in a precise and smooth operation. Preferably, the tray assemblies are mounted on ball bearing slides for smooth operation in inserting and removing the tray assemblies into the carrier assembly. The plugging mechanism also includes a loading control mechanism. A mechanism for leaving a syringe unplugged is also provided.

  5. Relationship with BSE (Mad Cow Disease)

    MedlinePlus

    ... Related Links Prion Diseases Creutzfeldt-Jakob Disease (CJD) Bovine Spongiform Encephalopathy (BSE), or Mad Cow Disease Chronic Wasting Disease ( ... Related Links Prion Diseases Creutzfeldt-Jakob Disease (CJD) Bovine Spongiform Encephalopathy (BSE), or Mad Cow Disease Chronic Wasting Disease ( ...

  6. Accurate macromolecular structures using minimal measurements from X-ray free-electron lasers.

    PubMed

    Hattne, Johan; Echols, Nathaniel; Tran, Rosalie; Kern, Jan; Gildea, Richard J; Brewster, Aaron S; Alonso-Mori, Roberto; Glöckner, Carina; Hellmich, Julia; Laksmono, Hartawan; Sierra, Raymond G; Lassalle-Kaiser, Benedikt; Lampe, Alyssa; Han, Guangye; Gul, Sheraz; DiFiore, Dörte; Milathianaki, Despina; Fry, Alan R; Miahnahri, Alan; White, William E; Schafer, Donald W; Seibert, M Marvin; Koglin, Jason E; Sokaras, Dimosthenis; Weng, Tsu-Chien; Sellberg, Jonas; Latimer, Matthew J; Glatzel, Pieter; Zwart, Petrus H; Grosse-Kunstleve, Ralf W; Bogan, Michael J; Messerschmidt, Marc; Williams, Garth J; Boutet, Sébastien; Messinger, Johannes; Zouni, Athina; Yano, Junko; Bergmann, Uwe; Yachandra, Vittal K; Adams, Paul D; Sauter, Nicholas K

    2014-05-01

    X-ray free-electron laser (XFEL) sources enable the use of crystallography to solve three-dimensional macromolecular structures under native conditions and without radiation damage. Results to date, however, have been limited by the challenge of deriving accurate Bragg intensities from a heterogeneous population of microcrystals, while at the same time modeling the X-ray spectrum and detector geometry. Here we present a computational approach designed to extract meaningful high-resolution signals from fewer diffraction measurements.

  7. MAD (Multi-Agent-Delivery) Nanolayer: Delivering Multiple Therapeutics from Hierarchical Assembled Surface Coatings

    PubMed Central

    Kim, Byeong-Su; Smith, Renée C.; Poon, Zhiyong; Hammond, Paula T.

    2014-01-01

    We present the hydrolytically degradable polymeric multilayer films that can co-deliver multiple therapeutics of differing chemical characteristics (charged biomacromolecules and neutral hydrophobic small molecules) from a surface. This multi-agent-delivery (MAD) nanolayer system integrates the hydrolytically degradable poly(β-amino ester) as a structural component to control the degradation of the multilayers to release active therapeutic macromolecules, as well as hydrophobic drugs imbedded within amphiphilic block copolymer micellar carriers within layer-by-layer (LbL) films, which would otherwise be difficult to include within the multilayers. By varying the anionic therapeutic agents (heparin and dextran sulfate) within the multilayer, we examine how different structural components can be used to control the release kinetics of multiple therapeutics from MAD nanolayers. Controlled release profiles and the in vitro efficacy of the MAD nanolayers in suppressing the growth of human smooth muscle cell lines were evaluated. The dual delivery of a charged macromolecular heparin and a small hydrophobic drug, paclitaxel, is found to be synergistic and beneficial toward effective therapeutic activity. Furthermore, we compared the classical dipping method we employed here with an automated spray-LbL technique. Spray-LbL significantly facilitates film processing time while preserving the characteristic release profiles of the MAD nanolayers. With the highly versatile and tunable nature of LbL assembly, we anticipate that MAD nanolayers can provide a unique platform for delivering multiple therapeutics from macromolecular to small molecules with distinct release profiles for applications in biological and biomedical surface coatings. PMID:19630389

  8. From crystallography to life

    NASA Astrophysics Data System (ADS)

    Allen, Roland E.

    2014-06-01

    2014 is the International Year of Crystallography, an extremely broad field which has had enormous impact in biology and materials science. Both experimental facilities and methods for interpreting the data have become increasingly sophisticated during the past century, and many highly complex systems have now been characterized, including large proteins and other biological macromolecules. A very few representative examples are mentioned here, including crystallographic studies of proteins that regulate programmed cell death (apoptosis), and structure determinations of G-protein coupled receptors (GPCRs), respectively the subjects of the 2014 Aminoff Prize and the 2012 Nobel Prize in chemistry. Normal apoptosis is essential for human embryonic development, prevention of cancer, and other processes within multicellular organisms. GPCRs are the targets of about half of all modern medicinal drugs, since they are responsible for the majority of cellular responses to hormones and neurotransmitters, as well as the senses of sight, taste, and smell. In materials, the behavior of electrons (both ordinary and exotic) is largely determined by the arrangement of the atoms. As examples, we mention carbon-based materials (diamond, buckyballs, nanotubes, and graphene) and high-temperature superconductors (cuprate and iron-based).

  9. Infrared Protein Crystallography

    SciTech Connect

    J Sage; Y Zhang; J McGeehan; R Ravelli; M Weik; J van Thor

    2011-12-31

    We consider the application of infrared spectroscopy to protein crystals, with particular emphasis on exploiting molecular orientation through polarization measurements on oriented single crystals. Infrared microscopes enable transmission measurements on individual crystals using either thermal or nonthermal sources, and can accommodate flow cells, used to measure spectral changes induced by exposure to soluble ligands, and cryostreams, used for measurements of flash-cooled crystals. Comparison of unpolarized infrared measurements on crystals and solutions probes the effects of crystallization and can enhance the value of the structural models refined from X-ray diffraction data by establishing solution conditions under which they are most relevant. Results on several proteins are consistent with similar equilibrium conformational distributions in crystal and solutions. However, the rates of conformational change are often perturbed. Infrared measurements also detect products generated by X-ray exposure, including CO{sub 2}. Crystals with favorable symmetry exhibit infrared dichroism that enhances the synergy with X-ray crystallography. Polarized infrared measurements on crystals can distinguish spectral contributions from chemically similar sites, identify hydrogen bonding partners, and, in opportune situations, determine three-dimensional orientations of molecular groups. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.

  10. Function and dynamics of macromolecular complexes explored by integrative structural and computational biology.

    PubMed

    Purdy, Michael D; Bennett, Brad C; McIntire, William E; Khan, Ali K; Kasson, Peter M; Yeager, Mark

    2014-08-01

    Three vignettes exemplify the potential of combining EM and X-ray crystallographic data with molecular dynamics (MD) simulation to explore the architecture, dynamics and functional properties of multicomponent, macromolecular complexes. The first two describe how EM and X-ray crystallography were used to solve structures of the ribosome and the Arp2/3-actin complex, which enabled MD simulations that elucidated functional dynamics. The third describes how EM, X-ray crystallography, and microsecond MD simulations of a GPCR:G protein complex were used to explore transmembrane signaling by the β-adrenergic receptor. Recent technical advancements in EM, X-ray crystallography and computational simulation create unprecedented synergies for integrative structural biology to reveal new insights into heretofore intractable biological systems.

  11. ["Imperial madness" - truth or legend?].

    PubMed

    V Zerssen, D

    2011-03-01

    The notion of "imperial madness" was coined in the historical literature and belles-lettres of the 19th century. Around that time up to the first quarter of the 20th century, it was adopted by a few German psychiatrists. Two of them viewed "imperial madness" as ordinary forms of insanity which became excessive only due to reactions of the social environment. Another one, however, classified it as one of "mental borderland states" in between insanity and normality, although he conceived the final stage of the disorder as a paranoid one. In agreement with the historians he postulated that "imperial madness" resulted from unlimited power of predisposed rulers. In recent times the whole concept of "imperial madness" was referred to the realm of legends by historians of antiquity and other historically interested authors. Yet the existence of the phenomenon cannot be denied. Despite its rarity it has played and is still playing an important role with often catastrophic consequences in various cultures all over the world. Therefore, psychiatrists and other physicians as well as clinical psychologists should be acquainted with it. From a modern point of view, it is not a paranoid disorder but rather a syndrome of addiction-like behavioural excesses representing an intensification of a Hybris syndrome as described by Anglo-Saxon psychiatrists. According to the present authors' view, it should be classified nosologically as a chronic adjustment disorder. In this case, the underlying stresses need to be extended to situations of temptation (here: the temptation to abuse almost unlimited power).

  12. The Neurobiologist's Guide to Structural Biology: A Primer on Why Macromolecular Structure Matters and How to Evaluate Structural Data

    PubMed Central

    Minor, Daniel L.

    2010-01-01

    Structural biology now plays a prominent role in addressing questions central to understanding how excitable cells function. Although interest in the insights gained from the definition and dissection of macromolecular anatomy is high, many neurobiologists remain unfamiliar with the methods employed. This primer aims to help neurobiologists understand approaches for probing macromolecular structure and where the limits and challenges remain. Using examples of macromolecules with neurobiological importance, the review covers X-ray crystallography, electron microscopy (EM), small-angle X-ray scattering (SAXS), and nuclear magnetic resonance (NMR) and biophysical methods with which these approaches are often paired: isothermal titration calorimetry (ITC), equilibrium analytical ultracentifugation, and molecular dynamics (MD). PMID:17521566

  13. The neurobiologist's guide to structural biology: a primer on why macromolecular structure matters and how to evaluate structural data.

    PubMed

    Minor, Daniel L

    2007-05-24

    Structural biology now plays a prominent role in addressing questions central to understanding how excitable cells function. Although interest in the insights gained from the definition and dissection of macromolecular anatomy is high, many neurobiologists remain unfamiliar with the methods employed. This primer aims to help neurobiologists understand approaches for probing macromolecular structure and where the limits and challenges remain. Using examples of macromolecules with neurobiological importance, the review covers X-ray crystallography, electron microscopy (EM), small-angle X-ray scattering (SAXS), and nuclear magnetic resonance (NMR) and biophysical methods with which these approaches are often paired: isothermal titration calorimetry (ITC), equilibrium analytical ultracentifugation, and molecular dynamics (MD).

  14. Cryogenic Neutron Protein Crystallography: routine methods and potential benefits

    SciTech Connect

    Weiss, Kevin L; Tomanicek, Stephen J; NG, Joseph D

    2014-01-01

    The use of cryocooling in neutron diffraction has been hampered by several technical challenges such as the need for specialized equipment and techniques. Recently we have developed and deployed equipment and strategies that allow for routine neutron data collection on cryocooled crystals using off the shelf components. This system has several advantages, compared to a closed displex cooling system such as fast cooling coupled with easier crystal mounting and centering. The ability to routinely collect cryogenic neutron data for analysis will significantly broaden the range of scientific questions that can be examined by neutron protein crystallography. Cryogenic neutron data collection for macromolecules has recently become available at the new Biological Diffractometer BIODIFF at FRM II and the Macromolecular Diffractometer (MaNDi) at the Spallation Neutron Source, Oak Ridge National Laboratory. To evaluate the benefits of a cryocooled neutron structure we collected a full neutron data set on the BIODIFF instrument on a Toho-1 lactamase structure at 100K.

  15. MAD for visual tracker fusion

    NASA Astrophysics Data System (ADS)

    Becker, Stefan; Krah, Sebastian B.; Hübner, Wolfgang; Arens, Michael

    2016-10-01

    Existing tracking methods vary strongly in their approach and therefore have different strengths and weaknesses. For example, a single tracking algorithm may be good at handling variations in illumination, but does not cope well with deformation. Hence, their failures can occur on entirely different time intervals on the same sequence. One possible solution for overcoming limitations of a single tracker and for benefitting from individual strengths, is to run a set of tracking algorithms in parallel and fuse their outputs. But in general, tracking algorithms are not designed to receive feedback from a higher level fusion strategy or require a high degree of integration between individual levels. Towards this end, we introduce a fusion strategy serving the purpose of online single object tracking, for which no knowledge about individual tracker characteristics is needed. The key idea is to combine several independent and heterogeneous tracking approaches and to robustly identify an outlier subset based on the "Median Absolute Deviations" (MAD) measure. The MAD fusion strategy is very generic and only requires frame-based object bounding boxes as input. Thus, it can work with arbitrary tracking algorithms. Furthermore, the MAD fusion strategy can also be applied for combining several instances of the same tracker to form a more robust ensemble for tracking an object. The evaluation is done on public available datasets. With a set of heterogeneous, commonly used trackers we show that the proposed MAD fusion strategy improves the tracking results in comparison to a classical combination of parallel trackers and that the tracker ensemble helps to deal with the initialization uncertainty of a single tracker.

  16. Development of a dose-limiting data collection strategy for serial synchrotron rotation crystallography

    PubMed Central

    Hasegawa, Kazuya; Yamashita, Keitaro; Murai, Tomohiro; Nuemket, Nipawan; Hirata, Kunio; Ueno, Go; Ago, Hideo; Nakatsu, Toru; Kumasaka, Takashi; Yamamoto, Masaki

    2017-01-01

    Serial crystallography, in which single-shot diffraction images are collected, has great potential for protein microcrystallography. Although serial femtosecond crystallography (SFX) has been successfully demonstrated, limited beam time prevents its routine use. Inspired by SFX, serial synchrotron crystallography (SSX) has been investigated at synchrotron macromolecular crystallography beamlines. Unlike SFX, the longer exposure time of milliseconds to seconds commonly used in SSX causes radiation damage. However, in SSX, crystals can be rotated during the exposure, which can achieve efficient coverage of the reciprocal space. In this study, mercury single-wavelength anomalous diffraction (Hg-SAD) phasing of the luciferin regenerating enzyme (LRE) was performed using serial synchrotron rotation crystallography. The advantages of rotation and influence of dose on the data collected were evaluated. The results showed that sample rotation was effective for accurate data collection, and the optimum helical rotation step depended on multiple factors such as multiplicity and partiality of reflections, exposure time per rotation angle and the contribution from background scattering. For the LRE microcrystals, 0.25° was the best rotation step for the achievable resolution limit, whereas a rotation step larger than or equal to 1° was favorable for Hg-SAD phasing. Although an accumulated dose beyond 1.1 MGy caused specific damage at the Hg site, increases in resolution and anomalous signal were observed up to 3.4 MGy because of a higher signal-to-noise ratio. PMID:28009544

  17. On macromolecular refinement at subatomic resolution withinteratomic scatterers

    SciTech Connect

    Afonine, Pavel V.; Grosse-Kunstleve, Ralf W.; Adams, Paul D.; Lunin, Vladimir Y.; Urzhumtsev, Alexandre

    2007-11-09

    A study of the accurate electron density distribution in molecular crystals at subatomic resolution, better than {approx} 1.0 {angstrom}, requires more detailed models than those based on independent spherical atoms. A tool conventionally used in small-molecule crystallography is the multipolar model. Even at upper resolution limits of 0.8-1.0 {angstrom}, the number of experimental data is insufficient for the full multipolar model refinement. As an alternative, a simpler model composed of conventional independent spherical atoms augmented by additional scatterers to model bonding effects has been proposed. Refinement of these mixed models for several benchmark datasets gave results comparable in quality with results of multipolar refinement and superior of those for conventional models. Applications to several datasets of both small- and macro-molecules are shown. These refinements were performed using the general-purpose macromolecular refinement module phenix.refine of the PHENIX package.

  18. Abject Magic: Reasoning Madness in Justine Larbalestier's "Magic or Madness" Trilogy

    ERIC Educational Resources Information Center

    Potter, Troy

    2013-01-01

    This paper explores the representation of magic and madness in Justine Larbalestier's "Magic or Madness" trilogy (2005-2007). Throughout the series, magic is constructed as an abject and disabling force that threatens to disable magic-wielders, either through madness or death. Despite being represented as a ubiquitous force, the…

  19. Transmucosal macromolecular drug delivery.

    PubMed

    Prego, C; García, M; Torres, D; Alonso, M J

    2005-01-03

    Mucosal surfaces are the most common and convenient routes for delivering drugs to the body. However, macromolecular drugs such as peptides and proteins are unable to overcome the mucosal barriers and/or are degraded before reaching the blood stream. Among the approaches explored so far in order to optimize the transport of these macromolecules across mucosal barriers, the use of nanoparticulate carriers represents a challenging but promising strategy. The present paper aims to compare the characteristics and potential of nanostructures based on the mucoadhesive polysaccharide chitosan (CS). These are CS nanoparticles, CS-coated oil nanodroplets (nanocapsules) and CS-coated lipid nanoparticles. The characteristics and behavior of CS nanoparticles and CS-coated lipid nanoparticles already reported [A. Vila, A. Sanchez, M. Tobio, P. Calvo, M.J. Alonso, Design of biodegradable particles for protein delivery, J. Control. Rel. 78 (2002) 15-24; R. Fernandez-Urrusuno, P. Calvo, C. Remunan-Lopez, J.L. Vila-Jato, M.J. Alonso, Enhancement of nasal absorption of insulin using chitosan nanoparticles, Pharm. Res. 16 (1999) 1576-1581; M. Garcia-Fuentes, D. Torres, M.J. Alonso, New surface-modified lipid nanoparticles as delivery vehicles for salmon calcitonin (submitted for publication).] are compared with those of CS nanocapsules originally reported here. The three types of systems have a size in the nanometer range and a positive zeta potential that was attributed to the presence of CS on their surface. They showed an important capacity for the association of peptides such as insulin, salmon calcitonin and proteins, such as tetanus toxoid. Their mechanism of interaction with epithelia was investigated using the Caco-2 model cell line. The results showed that CS-coated systems caused a concentration-dependent reduction in the transepithelial resistance of the cell monolayer. Moreover, within the range of concentrations investigated, these systems were internalized in the

  20. Protein energy landscapes determined by five-dimensional crystallography

    SciTech Connect

    Schmidt, Marius; Srajer, Vukica; Henning, Robert; Ihee, Hyotcherl; Purwar, Namrta; Tenboer, Jason; Tripathi, Shailesh

    2013-12-01

    Barriers of activation within the photocycle of a photoactive protein were extracted from comprehensive time courses of time resolved crystallographic data collected at multiple temperature settings. Free-energy landscapes decisively determine the progress of enzymatically catalyzed reactions [Cornish-Bowden (2012 ▶), Fundamentals of Enzyme Kinetics, 4th ed.]. Time-resolved macromolecular crystallography unifies transient-state kinetics with structure determination [Moffat (2001 ▶), Chem. Rev.101, 1569–1581; Schmidt et al. (2005 ▶), Methods Mol. Biol.305, 115–154; Schmidt (2008 ▶), Ultrashort Laser Pulses in Medicine and Biology] because both can be determined from the same set of X-ray data. Here, it is demonstrated how barriers of activation can be determined solely from five-dimensional crystallography, where in addition to space and time, temperature is a variable as well [Schmidt et al. (2010 ▶), Acta Cryst. A66, 198–206]. Directly linking molecular structures with barriers of activation between them allows insight into the structural nature of the barrier to be gained. Comprehensive time series of crystallographic data at 14 different temperature settings were analyzed and the entropy and enthalpy contributions to the barriers of activation were determined. One hundred years after the discovery of X-ray scattering, these results advance X-ray structure determination to a new frontier: the determination of energy landscapes.

  1. SIBYLS - A SAXS and protein crystallography beamline at the ALS

    SciTech Connect

    Trame, Christine; MacDowell, Alastair A.; Celestre, Richard S.; Padmore, Howard A.; Cambie, Daniella; Domning, Edward E.; Duarte, Robert M.; Kelez, Nicholas; Plate, David W.; Holton, James M.; Frankel, Kenneth; Tsutakawa, Susan; Tsuruta, Hiro; Tainer, John A.; Cooper, Priscilla K.

    2003-08-22

    The new Structurally Integrated BiologY for Life Sciences (SIBYLS) beamline at the Advanced Light Source will be dedicated to Macromolecular Crystallography (PX) and Small Angle X-ray Scattering (SAXS). SAXS will provide structural information of macromolecules in solutions and will complement high resolution PX studies on the same systems but in a crystalline state. The x-ray source is one of the 5 Tesla superbend dipoles recently installed at the ALS that allows for a hard x-ray program to be developed on the relatively low energy Advanced Light Source (ALS) ring (1.9 GeV). The beamline is equipped with fast interchangeable monochromator elements, consisting of either a pair of single Si(111) crystals for crystallography, or a pair of multilayers for the SAXS mode data collection (E/{Delta}E {approx} 1/110). Flux rates with Si(111) crystals for PX are measured as 2 x 10{sup 11} hv/sec/400 mA through a 100 {micro}m pinhole at 12.4 KeV. For SAXS the flux is up to 3 x 10{sup 13} photons/sec at 10 KeV with all apertures open when using the multilayer monochromator elements. The performance characteristics of this unique beamline will be described.

  2. Identifying and Visualizing Macromolecular Flexibility in Structural Biology

    PubMed Central

    Palamini, Martina; Canciani, Anselmo; Forneris, Federico

    2016-01-01

    Structural biology comprises a variety of tools to obtain atomic resolution data for the investigation of macromolecules. Conventional structural methodologies including crystallography, NMR and electron microscopy often do not provide sufficient details concerning flexibility and dynamics, even though these aspects are critical for the physiological functions of the systems under investigation. However, the increasing complexity of the molecules studied by structural biology (including large macromolecular assemblies, integral membrane proteins, intrinsically disordered systems, and folding intermediates) continuously demands in-depth analyses of the roles of flexibility and conformational specificity involved in interactions with ligands and inhibitors. The intrinsic difficulties in capturing often subtle but critical molecular motions in biological systems have restrained the investigation of flexible molecules into a small niche of structural biology. Introduction of massive technological developments over the recent years, which include time-resolved studies, solution X-ray scattering, and new detectors for cryo-electron microscopy, have pushed the limits of structural investigation of flexible systems far beyond traditional approaches of NMR analysis. By integrating these modern methods with powerful biophysical and computational approaches such as generation of ensembles of molecular models and selective particle picking in electron microscopy, more feasible investigations of dynamic systems are now possible. Using some prominent examples from recent literature, we review how current structural biology methods can contribute useful data to accurately visualize flexibility in macromolecular structures and understand its important roles in regulation of biological processes. PMID:27668215

  3. The Phenix software for automated determination of macromolecular structures.

    PubMed

    Adams, Paul D; Afonine, Pavel V; Bunkóczi, Gábor; Chen, Vincent B; Echols, Nathaniel; Headd, Jeffrey J; Hung, Li-Wei; Jain, Swati; Kapral, Gary J; Grosse Kunstleve, Ralf W; McCoy, Airlie J; Moriarty, Nigel W; Oeffner, Robert D; Read, Randy J; Richardson, David C; Richardson, Jane S; Terwilliger, Thomas C; Zwart, Peter H

    2011-09-01

    X-ray crystallography is a critical tool in the study of biological systems. It is able to provide information that has been a prerequisite to understanding the fundamentals of life. It is also a method that is central to the development of new therapeutics for human disease. Significant time and effort are required to determine and optimize many macromolecular structures because of the need for manual interpretation of complex numerical data, often using many different software packages, and the repeated use of interactive three-dimensional graphics. The Phenix software package has been developed to provide a comprehensive system for macromolecular crystallographic structure solution with an emphasis on automation. This has required the development of new algorithms that minimize or eliminate subjective input in favor of built-in expert-systems knowledge, the automation of procedures that are traditionally performed by hand, and the development of a computational framework that allows a tight integration between the algorithms. The application of automated methods is particularly appropriate in the field of structural proteomics, where high throughput is desired. Features in Phenix for the automation of experimental phasing with subsequent model building, molecular replacement, structure refinement and validation are described and examples given of running Phenix from both the command line and graphical user interface.

  4. The madness of Gerard de Nerval.

    PubMed

    Beveridge, Allan

    2014-06-01

    This paper examines the madness of Gerard de Nerval, the nineteenth-century French writer. It looks at his account of mental disturbance, how he responded to the psychiatric profession and how he reacted to being diagnosed as insane. It considers his autobiographical novella of madness, Aurelia, which he began at the suggestion of his alienist, Dr Emile Blanche, and while he was still an asylum inmate. Nerval's story raises important questions about the nature of madness. Is it, as he contended, a mystical experience revealing truths about spiritual worlds inaccessible to the 'sane'? Does psychiatry fail to understand it and inappropriately reduce it to the categories of scientific reason? Or are such notions of the spiritual value of madness guilty of the charge that they romanticise insanity? Do they make extravagant claims for an experience that is often disturbing and debilitating? What is the relationship between madness and recovery? Should an individual try to forget their experience of mental disturbance once they recover, or should they examine what the event reveals about themselves? Can the language of madness be decoded to unveil profound truths as Carl Jung and R.D. Laing have suggested, or is it, as the psychiatrist German Berrios maintains, merely a series of 'empty speech acts', signifying nothing? And finally, how does one avoid writing about madness, and instead write madness?

  5. [Women and madness in the Eneid].

    PubMed

    Totola, Giorgia

    2012-01-01

    The article presents female cases of madness in Latin Vergilian Literature, comparing the Greek Dyonisian divine possession of the Maenads and Bacchae with the madness of Dido and Amata. Transcultural psychiatry is here proposed as a useful tool for reading the descriptions of the Aeneid - to try to understand every kind of world where barriers disappear between visible and invisible.

  6. Elizabethan madness: on London's stage.

    PubMed

    Dalby, J T

    1997-12-01

    During the reign of Elizabeth I (1558-1603) a renaissance of both literary and political history occurred. The stage was transformed from primitive echoes of the morality plays to a vibrant and diverse exploration of human endeavor and man's place in the universe. The titanic literary figure of Shakespeare today veils a group of friends and challengers whose pens strove for the same goal. The depiction of madness was ubiquitous during plays of this time and reflection on the views of this group of men gives us a more reliable insight into mental illness then and today.

  7. Radiation damage in macromolecular cryocrystallography.

    PubMed

    Ravelli, Raimond B G; Garman, Elspeth F

    2006-10-01

    X-ray radiation damage to cryocooled ( approximately 100 K) macromolecular crystals has emerged as a general problem, especially since the advent of third generation synchrotron undulator sources. Interest in understanding the physical and chemical phenomena behind the observed effects is growing rapidly. The specific structural damage seen in electron density maps has to be accounted for when studying intermediates, and can sometimes be related to biological function. Radiation damage induces non-isomorphism, thus hampering traditional phasing methods. However, specific damage can also be used to obtain phases. With an increased knowledge of expected crystal lifetime, beamline characteristics and types of damage, macromolecular crystallographers might soon be able to account for radiation damage in data collection, processing and phasing.

  8. Madness Decolonized?: Madness as Transnational Identity in Gail Hornstein's Agnes's Jacket.

    PubMed

    Miller, Gavin

    2017-02-13

    The US psychologist Gail Hornstein's monograph, Agnes's Jacket: A Psychologist's Search for the Meanings of Madness (2009), is an important intervention in the identity politics of the mad movement. Hornstein offers a resignified vision of mad identity that embroiders the central trope of an "anti-colonial" struggle to reclaim the experiential world "colonized" by psychiatry. A series of literal and figurative appeals makes recourse to the inner world and (corresponding) cultural world of the mad as well as to the ethno-symbolic cultural materials of dormant nationhood. This rhetoric is augmented by a model in which the mad comprise a diaspora without an origin, coalescing into a single transnational community. The mad are also depicted as persons displaced from their metaphorical homeland, the "inner" world "colonized" by the psychiatric regime. There are a number of difficulties with Hornstein's rhetoric, however. Her "ethnicity-and-rights" response to the oppression of the mad is symptomatic of Western parochialism, while her proposed transmutation of putative psychopathology from limit upon identity to parameter of successful identity is open to contestation. Moreover, unless one accepts Hornstein's porous vision of mad identity, her self-ascribed insider status in relation to the mad community may present a problematic "re-colonization" of mad experience.

  9. Mad2 phosphorylation regulates its association with Mad1 and the APC/C

    PubMed Central

    Wassmann, Katja; Liberal, Vasco; Benezra, Robert

    2003-01-01

    Improper attachment of the mitotic spindle to the kinetochores of paired sister chromatids in mitosis is monitored by a checkpoint that leads to an arrest in early metaphase. This arrest requires the inhibitory association of Mad2 with the anaphase promoting complex/cyclosome (APC/C). It is not known how the association of Mad2 with the kinetochore and the APC/C is regulated in mitosis. Here, we demonstrate that human Mad2 is modified through phosphorylation on multiple serine residues in vivo in a cell cycle dependent manner and that only unphosphorylated Mad2 interacts with Mad1 or the APC/C in vivo. A Mad2 mutant containing serine to aspartic acid mutations mimicking the C-terminal phosphorylation events fails to interact with Mad1 or the APC/C and acts as a dominant-negative antagonist of wild-type Mad2. These data suggest that the phosphorylation state of Mad2 regulates its checkpoint activity by modulating its association with Mad1 and the APC/C. PMID:12574116

  10. Operational experience of a large area x-ray camera for protein crystallography.

    SciTech Connect

    Joachimiak, A.; Jorden, A. R.; Loeffen, P. W.; Naday, I.; Sanishvili, R.; Westbrook, E. M.

    1999-07-13

    After 3 years experience of operating very large area (210mm x 210mm) CCD-based detectors at the Advanced Photon Source, operational experience is reported. Four such detectors have been built, two for Structural Biology Center (APS-1 and SBC-2), one for Basic Energy Sciences Synchrotrons Radiation Center (Gold-2) at Argonne National Laboratory's Advanced Photon Source and one for Osaka University by Oxford Instruments, for use at Spring 8 (PX-21O). The detector is specifically designed as a high resolution and fast readout camera for macromolecular crystallography. Design trade-offs for speed and size are reviewed in light of operational experience and future requirements are considered. Operational data and examples of crystallography data are presented, together with plans for more development.

  11. PHENIX: a comprehensive Python-based system for macromolecular structure solution

    PubMed Central

    Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor; Chen, Vincent B.; Davis, Ian W.; Echols, Nathaniel; Headd, Jeffrey J.; Hung, Li-Wei; Kapral, Gary J.; Grosse-Kunstleve, Ralf W.; McCoy, Airlie J.; Moriarty, Nigel W.; Oeffner, Robert; Read, Randy J.; Richardson, David C.; Richardson, Jane S.; Terwilliger, Thomas C.; Zwart, Peter H.

    2010-01-01

    Macromolecular X-ray crystallography is routinely applied to understand biological processes at a molecular level. How­ever, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromolecular crystallo­graphic structure solution with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms. PMID:20124702

  12. New computational tools for H/D determination in macromolecular structures from neutron data.

    PubMed

    Siliqi, Dritan; Caliandro, Rocco; Carrozzini, Benedetta; Cascarano, Giovanni Luca; Mazzone, Annamaria

    2010-11-01

    Two new computational methods dedicated to neutron crystallography, called n-FreeLunch and DNDM-NDM, have been developed and successfully tested. The aim in developing these methods is to determine hydrogen and deuterium positions in macromolecular structures by using information from neutron density maps. Of particular interest is resolving cases in which the geometrically predicted hydrogen or deuterium positions are ambiguous. The methods are an evolution of approaches that are already applied in X-ray crystallography: extrapolation beyond the observed resolution (known as the FreeLunch procedure) and a difference electron-density modification (DEDM) technique combined with the electron-density modification (EDM) tool (known as DEDM-EDM). It is shown that the two methods are complementary to each other and are effective in finding the positions of H and D atoms in neutron density maps.

  13. Selenium-Assisted Nucleic Acid Crystallography: Use of DNA Phosphoroselenoates for MAD Phasing

    SciTech Connect

    Wilds, C.J.; Pattanayek, R.; Pan, C.; Wawrzak, Z.; Egli, M.

    2010-03-08

    The combination of synchrotron radiation and a variety of atoms or ions (either covalently attached to the biomolecule prior to crystallization or soaked into crystals) that serve as anomalous scatterers constitutes a powerful tool in the X-ray crystallographer's repertoire of structure determination techniques. Phosphoroselenoates in which one of the nonbridging phosphate oxygens in the backbone is replaced by selenium offer a simplified means for introducing an anomalous scatterer into oligonucleotides by conventional solid-phase synthesis. Unlike other methods that are used to derivatize DNA or RNA by covalent attachment of a heavy atom (i.e., bromine at the C5 position of pyrimidines), tedious synthesis of specialized nucleosides is not required. Introduction of selenium is readily accomplished in solid-phase oligonucleotide synthesis by replacing the standard oxidation agent with a solution of potassium selenocyanide. This results in a diastereomeric mixture of phosphoroselenoates that can be separated by strong anion-exchange HPLC. As a test case, all 10 DNA hexamers of the sequence CGCGCG containing a single phosphoroselenoate linkage (PSe) were prepared. Crystals were grown for a subset of them, and the structure of [d(C{sub PSe}GCGCG)]{sub 2} was determined by the multiwavelength anomalous dispersion technique and refined to 1.1 {angstrom} resolution.

  14. Closed MAD2 (C-MAD2) is selectively incorporated into the mitotic checkpoint complex (MCC)

    PubMed Central

    Tipton, Aaron R; Tipton, Michael; Yen, Tim

    2011-01-01

    The mitotic checkpoint is a specialized signal transduction pathway that monitors kinetochore-microtubule attachment to achieve faithful chromosome segregation. MAD2 is an evolutionarily conserved mitotic checkpoint protein that exists in open (O) and closed (C) conformations. The increase of intracellular C-MAD2 level during mitosis, through O→C-MAD2 conversion as catalyzed by unattached kinetochores, is a critical signaling event for the mitotic checkpoint. However, it remains controversial whether MAD2 is an integral component of the effector of the mitotic checkpoint—the mitotic checkpoint complex (MCC). We show here that endogenous human MCC is assembled by first forming a BUBR1:BUB3:CDC20 complex in G2 and then selectively incorporating C-MAD2 during mitosis. Nevertheless, MCC can be induced to form in G1/S cells by expressing a C-conformation locked MAD2 mutant, indicating intracellular level of C-MAD2 as a major limiting factor for MCC assembly. In addition, a recombinant MCC containing C-MAD2 exhibits effective inhibitory activity toward APC/C isolated from mitotic HeLa cells, while a recombinant BUBR1:BUB3:CDC20 ternary complex is ineffective at comparable concentrations despite association with APC/C. These results help establish a direct connection between a major signal transducer (C-MAD2) and the potent effector (MCC) of the mitotic checkpoint, and provide novel insights into protein-protein interactions during assembly of a functional MCC. PMID:22037211

  15. Protein crystallography with spallation neutrons

    SciTech Connect

    Langan, P.; Schoenborn, Benno P.

    2003-01-01

    proteins and oriented molecular complexes. With spallation neutrons and their time dependent wavelength structure, one can select data with an optimal wavelength bandwidth and cover the whole Laue spectrum as time (wavelength) resolved diffraction data. This optimizes data quality with best peak to background ratios and provides spatial and energy resolution to eliminate peak overlaps. Such a Protein Crystallography Station (PCS) has been built and tested at Los Alamos Neutron Science Center. A partially coupled moderator is used to increase flux and data are collected by a Cylindrical He3 detector covering 120' with 200mm height. The PCS is described along with examples of data collected from a number of proteins.

  16. Mad honey intoxication mimicking acute coronary syndrome.

    PubMed

    Dur, Ali; Sonmez, Ertan; Civelek, Cemil; AhmetTurkdogan, Kenan; AkifVatankulu, Mehmet; Sogut, Ozgur

    2014-09-01

    Mad honey intoxication or grayanotoxin poisoning is caused by consumption of grayanotoxin-containing toxic honey produced from leaves and flowers of the Rhododendron family. Despite the rarity of intoxication cases, the correct diagnosis and treatment are required because of the significance of haemodynamic disturbance and confounding of symptoms for disease identification. We report herein a case of a patient with mad honey intoxication mimicking acute non-ST segment elevation myocardial infarction and review the pathophysiology and diagnostic considerations.

  17. Performance of PILATUS detector technology for long-wavelength macromolecular crystallography

    NASA Astrophysics Data System (ADS)

    Marchal, J.; Wagner, A.

    2011-05-01

    The long-wavelength MX beamline I23 currently under design at Diamond Light Source will be optimized in the X-ray energy range between 3 and 5 keV. At the moment no commercial off-the-shelf detector with high quantum efficiency and dynamic range is available to cover the large area required for diffraction experiments in this energy range. The hybrid pixel detector technology used in PILATUS detectors could overcome these limitations as the modular design could allow a large coverage in reciprocal space and high detection efficiency. Experiments were carried out on the Microfocus Spectroscopy beamline I18 at Diamond Light Source to test the performance of a 100K PILATUS module in the low-energy range from 2.3 to 3.7 keV.

  18. Macromolecular Diffusion in Polymer Nanocomposites

    NASA Astrophysics Data System (ADS)

    Gam, Sangah; Meth, Jeff; Zane, Steve; Winey, Karen; Clarke, Nigel; Composto, Russell

    2011-03-01

    Macromolecular diffusion in crowded systems is important in biological and engineered systems. We have studied macromolecular diffusion through a model polymer nanocomposite (PNC) containing phenyl grafted silica nanoparticles (NPs), randomly distributed in a polystyrene matrix. Over a wide range of NP loading and tracer molecular weight (M), the scaling of the diffusion coefficient with M is in excellent agreement with the entropic barrier model (EBM) previously used to describe diffusion of DNA through confined media (e.g., gels and nanopores). To investigate the effect of NP size, diffusion was measured in PNC's with silica NPs having diameters of 28 and 12 nm. The normalized diffusion coefficients (D / D0) plotted against the interparticle separation relative to probe size (i.e., ID/ 2 Rg) collapse on a master curve. Diffusion in a poly(methyl methacrylate):silica NP system was also investigated to understand how attractive interactions (i.e., enthalpy) perturb motion relative to the polystyrene and phenyl-silica NP system which is athermal. Finally, a flux-based model is proposed and compared with experimental results.

  19. Fluid Physics and Macromolecular Crystal Growth in Microgravity

    NASA Technical Reports Server (NTRS)

    Helliwell, John R.; Snell, Edward H.; Chayen, Naomi E.; Judge, Russell A.; Boggon, Titus J.; Pusey, M. L.; Rose, M. Franklin (Technical Monitor)

    2000-01-01

    The first protein crystallization experiment in microgravity was launched in April, 1981 and used Germany's Technologische Experimente unter Schwerelosigkeit (TEXUS 3) sounding rocket. The protein P-galactosidase (molecular weight 465Kda) was chosen as the sample with a liquid-liquid diffusion growth method. A sliding device brought the protein, buffer and salt solution into contact when microgravity was reached. The sounding rocket gave six minutes of microgravity time with a cine camera and schlieren optics used to monitor the experiment, a single growth cell. In microgravity a strictly laminar diffusion process was observed in contrast to the turbulent convection seen on the ground. Several single crystals, approx 100micron in length, were formed in the flight which were of inferior but of comparable visual quality to those grown on the ground over several days. A second experiment using the same protocol but with solutions cooled to -8C (kept liquid with glycerol antifreeze) again showed laminar diffusion. The science of macromolecular structural crystallography involves crystallization of the macromolecule followed by use of the crystal for X-ray diffraction experiments to determine the three dimensional structure of the macromolecule. Neutron protein crystallography is employed for elucidation of H/D exchange and for improved definition of the bound solvent (D20). The structural information enables an understanding of how the molecule functions with important potential for rational drug design, improved efficiency of industrial enzymes and agricultural chemical development. The removal of turbulent convection and sedimentation in microgravity, and the assumption that higher quality crystals will be produced, has given rise to the growing number of crystallization experiments now flown. Many experiments can be flown in a small volume with simple, largely automated, equipment - an ideal combination for a microgravity experiment. The term "protein crystal growth

  20. Microfluidic Tools for Protein Crystallography

    NASA Astrophysics Data System (ADS)

    Abdallah, Bahige G.

    X-ray crystallography is the most widely used method to determine the structure of proteins, providing an understanding of their functions in all aspects of life to advance applications in fields such as drug development and renewable energy. New techniques, namely serial femtosecond crystallography (SFX), have unlocked the ability to unravel the structures of complex proteins with vital biological functions. A key step and major bottleneck of structure determination is protein crystallization, which is very arduous due to the complexity of proteins and their natural environments. Furthermore, crystal characteristics govern data quality, thus need to be optimized to attain the most accurate reconstruction of the protein structure. Crystal size is one such characteristic in which narrowed distributions with a small modal size can significantly reduce the amount of protein needed for SFX. A novel microfluidic sorting platform was developed to isolate viable ~200 nm -- ~600 nm photosystem I (PSI) membrane protein crystals from ~200 nm -- ~20 ?m crystal samples using dielectrophoresis, as confirmed by fluorescence microscopy, second-order nonlinear imaging of chiral crystals (SONICC), and dynamic light scattering. The platform was scaled-up to rapidly provide 100s of microliters of sorted crystals necessary for SFX, in which similar crystal size distributions were attained. Transmission electron microscopy was used to view the PSI crystal lattice, which remained well-ordered postsorting, and SFX diffraction data was obtained, confirming a high-quality, viable crystal sample. Simulations indicated sorted samples provided accurate, complete SFX datasets with 3500-fold less protein than unsorted samples. Microfluidic devices were also developed for versatile, rapid protein crystallization screening using nanovolumes of sample. Concentration gradients of protein and precipitant were generated to crystallize PSI, phycocyanin, and lysozyme using modified counterdiffusion

  1. Phase retrieval in protein crystallography.

    PubMed

    Liu, Zhong Chuan; Xu, Rui; Dong, Yu Hui

    2012-03-01

    Solution of the phase problem is central to crystallographic structure determination. An oversampling method is proposed, based on the hybrid input-output algorithm (HIO) [Fienup (1982). Appl. Opt. 21, 2758-2769], to retrieve the phases of reflections in crystallography. This method can extend low-resolution structures to higher resolution for structure determination of proteins without additional sample preparation. The method requires an envelope of the protein which divides a unit cell into the density region where the proteins are located and the non-density region occupied by solvents. After a few hundred to a few thousand iterations, the correct phases and density maps are recovered. The method has been used successfully in several cases to retrieve the phases from the experimental X-ray diffraction data and the envelopes of proteins constructed from structure files downloaded from the Protein Data Bank. It is hoped that this method will greatly facilitate the ab initio structure determination of proteins.

  2. Sample mounts for microcrystal crystallography

    NASA Technical Reports Server (NTRS)

    Thorne, Robert E. (Inventor); Stum, Zachary (Inventor); O'Neill, Kevin (Inventor); Kmetko, Jan (Inventor)

    2007-01-01

    Sample mounts (10) for mounting microcrystals of biological macromolecules for X-ray crystallography are prepared by using patterned thin polyimide films (12) that have curvature imparted thereto, for example, by being attached to a curved outer surface of a small metal rod (16). The patterned film (12) preferably includes a tapered tip end (24) for holding a crystal. Preferably, a small sample aperture is disposed in the film for reception of the crystal. A second, larger aperture can also be provided that is connected to the sample aperture by a drainage channel, allowing removal of excess liquid and easier manipulation in viscous solutions. The curvature imparted to the film (12) increases the film's rigidity and allows a convenient scoop-like action for retrieving crystals. The polyimide contributes minimally to background and absorption, and can be treated to obtain desired hydrophobicity or hydrophilicity.

  3. Sample mounts for microcrystal crystallography

    NASA Technical Reports Server (NTRS)

    Thorne, Robert E. (Inventor); Stum, Zachary (Inventor); O'Neill, Kevin (Inventor); Kmetko, Jan (Inventor)

    2009-01-01

    Sample mounts (10) for mounting microcrystals of biological macromolecules for X-ray crystallography are prepared by using patterned thin polyimide films (12) that have curvature imparted thereto, for example, by being attached to a curved outer surface of a small metal rod (16). The patterned film (12) preferably includes a tip end (24) for holding a crystal. Preferably, a small sample aperture is disposed in the film for reception of the crystal. A second, larger aperture can also be provided that is connected to the sample aperture by a drainage channel, allowing removal of excess liquid and easier manipulation in viscous solutions. The curvature imparted to the film (12) increases the film's rigidity and allows a convenient scoop-like action for retrieving crystals. The polyimide contributes minimally to background and absorption, and can be treated to obtain desired hydrophobicity or hydrophilicity.

  4. Wrinkling crystallography on spherical surfaces

    PubMed Central

    Brojan, Miha; Terwagne, Denis; Lagrange, Romain; Reis, Pedro M.

    2015-01-01

    We present the results of an experimental investigation on the crystallography of the dimpled patterns obtained through wrinkling of a curved elastic system. Our macroscopic samples comprise a thin hemispherical shell bound to an equally curved compliant substrate. Under compression, a crystalline pattern of dimples self-organizes on the surface of the shell. Stresses are relaxed by both out-of-surface buckling and the emergence of defects in the quasi-hexagonal pattern. Three-dimensional scanning is used to digitize the topography. Regarding the dimples as point-like packing units produces spherical Voronoi tessellations with cells that are polydisperse and distorted, away from their regular shapes. We analyze the structure of crystalline defects, as a function of system size. Disclinations are observed and, above a threshold value, dislocations proliferate rapidly with system size. Our samples exhibit striking similarities with other curved crystals of charged particles and colloids. Differences are also found and attributed to the far-from-equilibrium nature of our patterns due to the random and initially frozen material imperfections which act as nucleation points, the presence of a physical boundary which represents an additional source of stress, and the inability of dimples to rearrange during crystallization. Even if we do not have access to the exact form of the interdimple interaction, our experiments suggest a broader generality of previous results of curved crystallography and their robustness on the details of the interaction potential. Furthermore, our findings open the door to future studies on curved crystals far from equilibrium. PMID:25535355

  5. Visual automated macromolecular model building.

    PubMed

    Langer, Gerrit G; Hazledine, Saul; Wiegels, Tim; Carolan, Ciaran; Lamzin, Victor S

    2013-04-01

    Automated model-building software aims at the objective interpretation of crystallographic diffraction data by means of the construction or completion of macromolecular models. Automated methods have rapidly gained in popularity as they are easy to use and generate reproducible and consistent results. However, the process of model building has become increasingly hidden and the user is often left to decide on how to proceed further with little feedback on what has preceded the output of the built model. Here, ArpNavigator, a molecular viewer tightly integrated into the ARP/wARP automated model-building package, is presented that directly controls model building and displays the evolving output in real time in order to make the procedure transparent to the user.

  6. Kernel MAD Algorithm for Relative Radiometric Normalization

    NASA Astrophysics Data System (ADS)

    Bai, Yang; Tang, Ping; Hu, Changmiao

    2016-06-01

    The multivariate alteration detection (MAD) algorithm is commonly used in relative radiometric normalization. This algorithm is based on linear canonical correlation analysis (CCA) which can analyze only linear relationships among bands. Therefore, we first introduce a new version of MAD in this study based on the established method known as kernel canonical correlation analysis (KCCA). The proposed method effectively extracts the non-linear and complex relationships among variables. We then conduct relative radiometric normalization experiments on both the linear CCA and KCCA version of the MAD algorithm with the use of Landsat-8 data of Beijing, China, and Gaofen-1(GF-1) data derived from South China. Finally, we analyze the difference between the two methods. Results show that the KCCA-based MAD can be satisfactorily applied to relative radiometric normalization, this algorithm can well describe the nonlinear relationship between multi-temporal images. This work is the first attempt to apply a KCCA-based MAD algorithm to relative radiometric normalization.

  7. Mad2 and Mad3 Cooperate to Arrest Budding Yeast in Mitosis

    PubMed Central

    Lau, Derek T. C.; Murray, Andrew W.

    2012-01-01

    Summary Background The spindle checkpoint ensures accurate chromosome transmission by delaying chromosome segregation until all chromosomes are correctly aligned on the mitotic spindle. The checkpoint is activated by kinetochores that are not attached to microtubules or are attached but not under tension and arrests cells at metaphase by inhibiting the anaphase-promoting complex (APC) and its co-activator Cdc20. Despite numerous studies, we still do not understand how the checkpoint proteins coordinate with each other to inhibit APCCdc20 activity. Results To ask how the checkpoint components induce metaphase arrest, we constructed fusions of checkpoint proteins and expressed them in the budding yeast, Saccharomyces cerevisiae, to mimic possible protein interactions during checkpoint activation. We found that expression of a Mad2-Mad3 protein fusion or non-covalently linked Mad2 and Mad3, but not the overexpression of the two separate proteins, induces metaphase arrest that is independent of functional kinetochores or other checkpoint proteins. We further showed that artificially tethering Mad2 to Cdc20 also arrests cells in metaphase independently of other checkpoint components. Conclusion Our results suggest that Mad3 is required for the stable binding of Mad2 to Cdc20 in vivo, which is sufficient to inhibit APC activity and is the most downstream event in spindle checkpoint activation. PMID:22209528

  8. Protein energy landscapes determined by five-dimensional crystallography

    PubMed Central

    Schmidt, Marius; Srajer, Vukica; Henning, Robert; Ihee, Hyotcherl; Purwar, Namrta; Tenboer, Jason; Tripathi, Shailesh

    2013-01-01

    Free-energy landscapes decisively determine the progress of enzymatically catalyzed reactions [Cornish-Bowden (2012 ▶), Fundamentals of Enzyme Kinetics, 4th ed.]. Time-resolved macromolecular crystallography unifies transient-state kinetics with structure determination [Moffat (2001 ▶), Chem. Rev. 101, 1569–1581; Schmidt et al. (2005 ▶), Methods Mol. Biol. 305, 115–154; Schmidt (2008 ▶), Ultrashort Laser Pulses in Medicine and Biology] because both can be determined from the same set of X-ray data. Here, it is demonstrated how barriers of activation can be determined solely from five-dimensional crystallo­graphy, where in addition to space and time, temperature is a variable as well [Schmidt et al. (2010 ▶), Acta Cryst. A66, 198–206]. Directly linking molecular structures with barriers of activation between them allows insight into the structural nature of the barrier to be gained. Comprehensive time series of crystallo­graphic data at 14 different temperature settings were analyzed and the entropy and enthalpy contributions to the barriers of activation were determined. One hundred years after the discovery of X-ray scattering, these results advance X-ray structure determination to a new frontier: the determination of energy landscapes. PMID:24311594

  9. Electron crystallography of ultrathin 3D protein crystals: atomic model with charges.

    PubMed

    Yonekura, Koji; Kato, Kazuyuki; Ogasawara, Mitsuo; Tomita, Masahiro; Toyoshima, Chikashi

    2015-03-17

    Membrane proteins and macromolecular complexes often yield crystals too small or too thin for even the modern synchrotron X-ray beam. Electron crystallography could provide a powerful means for structure determination with such undersized crystals, as protein atoms diffract electrons four to five orders of magnitude more strongly than they do X-rays. Furthermore, as electron crystallography yields Coulomb potential maps rather than electron density maps, it could provide a unique method to visualize the charged states of amino acid residues and metals. Here we describe an attempt to develop a methodology for electron crystallography of ultrathin (only a few layers thick) 3D protein crystals and present the Coulomb potential maps at 3.4-Å and 3.2-Å resolution, respectively, obtained from Ca(2+)-ATPase and catalase crystals. These maps demonstrate that it is indeed possible to build atomic models from such crystals and even to determine the charged states of amino acid residues in the Ca(2+)-binding sites of Ca(2+)-ATPase and that of the iron atom in the heme in catalase.

  10. 10 years of protein crystallography at AR-NW12A beamline

    NASA Astrophysics Data System (ADS)

    Chavas, L. M. G.; Yamada, Y.; Hiraki, M.; Igarashi, N.; Matsugaki, N.; Wakatsuki, S.

    2013-03-01

    The exponential growth of protein crystallography can be observed in the continuously increasing demand for synchrotron beam time, both from academic and industrial users. Nowadays, the screening of a profusion of sample crystals for more and more projects is being implemented by taking advantage of fully automated procedures at every level of the experiments. The insertion device AR-NW12A beamline is one of the five macromolecular crystallography (MX) beamlines at the Photon Factory (PF). Currently the oldest MX beamline operational at the High Energy Accelerator Research Organization (KEK), the end-station was launched in 2001 as part of an upgrade of the PF Advanced Ring. Since its commissioning, AR-NW12A has been operating as a high-throughput beamline, slowly evolving to a multipurpose end-station for MX experiments. The development of the beamline took place about a decade ago, in parallel with a drastic development of protein crystallography and more general synchrotron technology. To keep the beamline up-to-date and competitive with other MX stations in Japan and worldwide, new features have been constantly added, with the goal of user friendliness of the various beamline optics and other instruments. Here we describe the evolution of AR-NW12A for its tenth anniversary. We also discuss the plans for upgrades for AR-NW12A, the future objectives in terms of the beamline developments, and especially the strong desire to open the beamline to a larger user community.

  11. [In search of puerperal madness in Argentina].

    PubMed

    Vaschetto, Emilio

    2008-01-01

    The aim of this paper is to identify cases and scientific works about "puerperal madness" between 1850 and 1940 in Buenos Aires. The gradual installation of the device of maternities in the Argentina, and especially in the city of Buenos Aires, was not exempt from difficulties, in the medical boarding of the pregnant woman and parturient as well as in the cultural conception of the mothers. This fact presupposes an increase of the phenomenon called "puerperal madness" in the moment where the displacement of the act of giving birth in the house to the hospital takes place. Certainly, the clinical fact of the "puerperal madness" exceeds to the historical social context, and in this way appear all the psychopathologies related to the pregnancy, childbirth and the puerperal fevers. We also must take in account the influence of the medical speech about the feminine body and his translation to a scientific and institutional speech never before experienced in Argentina.

  12. Gorgon and Pathwalking: Macromolecular Modeling Tools for Subnanometer Resolution Density Maps

    PubMed Central

    Baker, Matthew L.; Baker, Mariah R.; Hryc, Corey F.; Ju, Tao; Chiu, Wah

    2013-01-01

    The complex interplay of proteins and other molecules, often in the form of large transitory assemblies, are critical to cellular function. Today, X-ray crystallography and electron cryo-microscopy (cryo-EM) are routinely used to image these macromolecular complexes, though often at limited resolutions. Despite the rapidly growing number of macromolecular structures, few tools exist for modeling and annotating structures in the range of 3-10Å resolution. To address this need, we have developed a number of utilities specifically targeting subnanometer resolution density maps. As part of the 2010 Cryo-EM Modeling Challenge, we demonstrated two of our latest de novo modeling tools, Pathwalking and Gorgon, as well as a tool for secondary structure identification (SSEHunter) and a new rigid-body/flexible fitting tool in Gorgon. In total, we submitted 30 structural models from ten different subnanometer resolution data sets in four of the six challenge categories. Each of our utlities produced accurate structural models and annotations across the various density maps. In the end, the utilities that we present here offer users a robust toolkit for analyzing and modeling protein structure in macromolecular assemblies at non-atomic resolutions. PMID:22696403

  13. Inhibition of cell proliferation by the Mad1 transcriptional repressor.

    PubMed Central

    Roussel, M F; Ashmun, R A; Sherr, C J; Eisenman, R N; Ayer, D E

    1996-01-01

    Mad1 is a basic helix-loop-helix-leucine zipper protein that is induced upon differentiation of a number of distinct cell types. Mad1 dimerizes with Max and recognizes the same DNA sequences as do Myc:Max dimers. However, Mad1 and Myc appear to have opposing functions. Myc:Max heterodimers activate transcription while Mad:Max heterodimers repress transcription from the same promoter. In addition Mad1 has been shown to block the oncogenic activity of Myc. Here we show that ectopic expression of Mad1 inhibits the proliferative response of 3T3 cells to signaling through the colony-stimulating factor-1 (CSF-1) receptor. The ability of over-expressed Myc and cyclin D1 to complement the mutant CSF-1 receptor Y809F (containing a Y-to-F mutation at position 809) is also inhibited by Mad1. Cell cycle analysis of proliferating 3T3 cells transfected with Mad1 demonstrates a significant decrease in the fraction of cells in the S and G2/M phases and a concomitant increase in the fraction of G1 phase cells, indicating that Mad1 negatively influences cell cycle progression from the G1 to the S phase. Mutations in Mad1 which inhibit its activity as a transcription repressor also result in loss of Mad1 cell cycle inhibitory activity. Thus, the ability of Mad1 to inhibit cell cycle progression is tightly coupled to its function as a transcriptional repressor. PMID:8649388

  14. Timely deposition of macromolecular structures is necessary for peer review

    SciTech Connect

    Joosten, Robbie P.; Soueidan, Hayssam; Wessels, Lodewyk F. A.; Perrakis, Anastassis

    2013-12-01

    Deposition of crystallographic structures should be concurrent with or prior to manuscript submission for peer review, enabling validation and increasing reliability of the PDB. Most of the macromolecular structures in the Protein Data Bank (PDB), which are used daily by thousands of educators and scientists alike, are determined by X-ray crystallography. It was examined whether the crystallographic models and data were deposited to the PDB at the same time as the publications that describe them were submitted for peer review. This condition is necessary to ensure pre-publication validation and the quality of the PDB public archive. It was found that a significant proportion of PDB entries were submitted to the PDB after peer review of the corresponding publication started, and many were only submitted after peer review had ended. It is argued that clear description of journal policies and effective policing is important for pre-publication validation, which is key in ensuring the quality of the PDB and of peer-reviewed literature.

  15. Ordered macromolecular structures in ferrofluid mixtures

    SciTech Connect

    Hayter, J.B.; Pynn, R.; Charles, S.; Skjeltorp, A.T.; Trewhella, J.; Stubbs, G.; Timmins, P.

    1989-04-03

    We have observed ordering of dilute dispersions of spherical and cylindrical macromolecules in magnetized ferrofluids. The order results from structural correlations between macromolecular and ferrofluid particles rather than from macroscopic magnetostatic effects. We have aligned elongated macromolecules by this technique and have obtained anisotropic neutron-diffraction patterns, which reflect the internal structure of the macromolecules. The method provides a tool for orienting suspended macromolecular assemblies which are not amenable to conventional alignment techniques.

  16. Reduced Mad2 expression keeps relaxed kinetochores from arresting budding yeast in mitosis

    PubMed Central

    Barnhart, Erin L.; Dorer, Russell K.; Murray, Andrew W.; Schuyler, Scott C.

    2011-01-01

    Chromosome segregation depends on the spindle checkpoint, which delays anaphase until all chromosomes have bound microtubules and have been placed under tension. The Mad1–Mad2 complex is an essential component of the checkpoint. We studied the consequences of removing one copy of MAD2 in diploid cells of the budding yeast, Saccharomyces cerevisiae. Compared to MAD2/MAD2 cells, MAD2/mad2Δ heterozygotes show increased chromosome loss and have different responses to two insults that activate the spindle checkpoint: MAD2/mad2Δ cells respond normally to antimicrotubule drugs but cannot respond to chromosomes that lack tension between sister chromatids. In MAD2/mad2Δ cells with normal sister chromatid cohesion, removing one copy of MAD1 restores the checkpoint and returns chromosome loss to wild-type levels. We conclude that cells need the normal Mad2:Mad1 ratio to respond to chromosomes that are not under tension. PMID:21593209

  17. The use of polyoxometalates in protein crystallography – An attempt to widen a well-known bottleneck

    PubMed Central

    Bijelic, Aleksandar; Rompel, Annette

    2015-01-01

    Polyoxometalates (POMs) are discrete polynuclear metal-oxo anions with a fascinating variety of structures and unique chemical and physical properties. Their application in various fields is well covered in the literature, however little information about their usage in protein crystallization is available. This review summarizes the impact of the vast class of POMs on the formation of protein crystals, a well-known (frustrating) bottleneck in macromolecular crystallography, with the associated structure elucidation and a particular emphasis focused on POM's potential as a powerful crystallization additive for future research. The Protein Data Bank (PDB) was scanned for protein structures with incorporated POMs which were assigned a PDB ligand ID resulting in 30 PDB entries. These structures have been analyzed with regard to (i) the structure of POM itself in the immediate protein environment, (ii) the kind of interaction and position of the POM within the protein structure and (iii) the beneficial effects of POM on protein crystallography apparent so far. PMID:26339074

  18. The magic triangle goes MAD: experimental phasing with a bromine derivative

    SciTech Connect

    Beck, Tobias Gruene, Tim; Sheldrick, George M.

    2010-04-01

    5-Amino-2, 4, 6-tribromoisophthalic acid is used as a phasing tool for protein structure determination by MAD phasing. It is the second representative of a novel class of compounds for heavy-atom derivatization that combine heavy atoms with amino and carboxyl groups for binding to proteins. Experimental phasing is an essential technique for the solution of macromolecular structures. Since many heavy-atom ion soaks suffer from nonspecific binding, a novel class of compounds has been developed that combines heavy atoms with functional groups for binding to proteins. The phasing tool 5-amino-2, 4, 6-tribromoisophthalic acid (B3C) contains three functional groups (two carboxylate groups and one amino group) that interact with proteins via hydrogen bonds. Three Br atoms suitable for anomalous dispersion phasing are arranged in an equilateral triangle and are thus readily identified in the heavy-atom substructure. B3C was incorporated into proteinase K and a multiwavelength anomalous dispersion (MAD) experiment at the Br K edge was successfully carried out. Radiation damage to the bromine–carbon bond was investigated. A comparison with the phasing tool I3C that contains three I atoms for single-wavelength anomalous dispersion (SAD) phasing was also carried out.

  19. America's Descent into Madness

    ERIC Educational Resources Information Center

    Giroux, Henry A.

    2014-01-01

    This article describes America's descent into madness under the regime of neoliberalism that has emerged in the United States since the late 1970s. In part, this is due to the emergence of a public pedagogy produced by the corporate-owned media that now saturates Americans with a market-driven value system that undermines those formative…

  20. Phosphorylation of Mad controls competition between wingless and BMP signaling.

    PubMed

    Eivers, Edward; Demagny, Hadrien; Choi, Renee H; De Robertis, Edward M

    2011-10-11

    Bone morphogenetic proteins (BMPs) and Wnts are growth factors that provide essential patterning signals for cell proliferation and differentiation. Here, we describe a molecular mechanism by which the phosphorylation state of the Drosophila transcription factor Mad determines its ability to transduce either BMP or Wingless (Wg) signals. Previously, Mad was thought to function in gene transcription only when phosphorylated by BMP receptors. We found that the unphosphorylated form of Mad was required for canonical Wg signaling by interacting with the Pangolin-Armadillo transcriptional complex. Phosphorylation of the carboxyl terminus of Mad by BMP receptor directed Mad toward BMP signaling, thereby preventing Mad from functioning in the Wg pathway. The results show that Mad has distinct signal transduction roles in the BMP and Wnt pathways depending on its phosphorylation state.

  1. MADS goes genomic in conifers: towards determining the ancestral set of MADS-box genes in seed plants

    PubMed Central

    Gramzow, Lydia; Weilandt, Lisa; Theißen, Günter

    2014-01-01

    Background and Aims MADS-box genes comprise a gene family coding for transcription factors. This gene family expanded greatly during land plant evolution such that the number of MADS-box genes ranges from one or two in green algae to around 100 in angiosperms. Given the crucial functions of MADS-box genes for nearly all aspects of plant development, the expansion of this gene family probably contributed to the increasing complexity of plants. However, the expansion of MADS-box genes during one important step of land plant evolution, namely the origin of seed plants, remains poorly understood due to the previous lack of whole-genome data for gymnosperms. Methods The newly available genome sequences of Picea abies, Picea glauca and Pinus taeda were used to identify the complete set of MADS-box genes in these conifers. In addition, MADS-box genes were identified in the growing number of transcriptomes available for gymnosperms. With these datasets, phylogenies were constructed to determine the ancestral set of MADS-box genes of seed plants and to infer the ancestral functions of these genes. Key Results Type I MADS-box genes are under-represented in gymnosperms and only a minimum of two Type I MADS-box genes have been present in the most recent common ancestor (MRCA) of seed plants. In contrast, a large number of Type II MADS-box genes were found in gymnosperms. The MRCA of extant seed plants probably possessed at least 11–14 Type II MADS-box genes. In gymnosperms two duplications of Type II MADS-box genes were found, such that the MRCA of extant gymnosperms had at least 14–16 Type II MADS-box genes. Conclusions The implied ancestral set of MADS-box genes for seed plants shows simplicity for Type I MADS-box genes and remarkable complexity for Type II MADS-box genes in terms of phylogeny and putative functions. The analysis of transcriptome data reveals that gymnosperm MADS-box genes are expressed in a great variety of tissues, indicating diverse roles of MADS

  2. A database of macromolecular motions.

    PubMed Central

    Gerstein, M; Krebs, W

    1998-01-01

    We describe a database of macromolecular motions meant to be of general use to the structural community. The database, which is accessible on the World Wide Web with an entry point at http://bioinfo.mbb.yale.edu/MolMovDB , attempts to systematize all instances of protein and nucleic acid movement for which there is at least some structural information. At present it contains >120 motions, most of which are of proteins. Protein motions are further classified hierarchically into a limited number of categories, first on the basis of size (distinguishing between fragment, domain and subunit motions) and then on the basis of packing. Our packing classification divides motions into various categories (shear, hinge, other) depending on whether or not they involve sliding over a continuously maintained and tightly packed interface. In addition, the database provides some indication about the evidence behind each motion (i.e. the type of experimental information or whether the motion is inferred based on structural similarity) and attempts to describe many aspects of a motion in terms of a standardized nomenclature (e.g. the maximum rotation, the residue selection of a fixed core, etc.). Currently, we use a standard relational design to implement the database. However, the complexity and heterogeneity of the information kept in the database makes it an ideal application for an object-relational approach, and we are moving it in this direction. Specifically, in terms of storing complex information, the database contains plausible representations for motion pathways, derived from restrained 3D interpolation between known endpoint conformations. These pathways can be viewed in a variety of movie formats, and the database is associated with a server that can automatically generate these movies from submitted coordinates. PMID:9722650

  3. Molecular Control of Macromolecular Properties

    NASA Astrophysics Data System (ADS)

    Holcombe, Thomas Wesley, III

    Molecular level control over macromolecules has been at the heart of human advancement, long before Hermann Staudinger coined the term Makromolekule. From the development of primitive pharmaceuticals to the advanced materials that sent Man into outer-space, We have been tinkering with God's paint since our inception. The work described herein primarily involves advances concerning poly-aromatic macromolecules for use in future electronic applications, particularly that of organic photovoltaics. There is a final chapter, however, that gives the reader a taste of how some molecular level changes can be directly visualized with modern microscopy techniques. Chapter 1 provides a very brief introduction to conjugated polymers and molecular level control over macromolecular properties. Chapters 2--4 introduces the concept of polymer substitution as a means by which to control and improve charge generation in organic photovoltaic devices. Chapters 5 and 6 show how these polymers can take on larger, defined structures, yet are still beholden to intrinsic molecular properties---such as regioregularity, a fancy word for the regularity of the position in which two aromatic rings are joined together. Chapter 7 re-examines the role of polymer substitution on photovoltaic performance, this time with an emphasis on homo-polymer packing rather than electron transfer at the donor/acceptor interface. Finally, Chapter 8 visualizes how controlling the environment about a single metal atom can lead directly to a cyclic polyolefin. Individually, these advances do not yield any breakthroughs noticeable to a general audience; collectively, they sit atop a mountain of human knowledge, waiting to provide a stepping stone for the next generation.

  4. Asymmetry in serial femtosecond crystallography data

    PubMed Central

    Sharma, Amit; Johansson, Linda; Dunevall, Elin; Wahlgren, Weixiao Y.; Neutze, Richard

    2017-01-01

    Serial crystallography is an increasingly important approach to protein crystallography that exploits both X-ray free-electron laser (XFEL) and synchrotron radiation. Serial crystallography recovers complete X-ray diffraction data by processing and merging diffraction images from thousands of randomly oriented non-uniform microcrystals, of which all observations are partial Bragg reflections. Random fluctuations in the XFEL pulse energy spectrum, variations in the size and shape of microcrystals, integrating over millions of weak partial observations and instabilities in the XFEL beam position lead to new types of experimental errors. The quality of Bragg intensity estimates deriving from serial crystallography is therefore contingent upon assumptions made while modeling these data. Here it is observed that serial femtosecond crystallography (SFX) Bragg reflections do not follow a unimodal Gaussian distribution and it is recommended that an idealized assumption of single Gaussian peak profiles be relaxed to incorporate apparent asymmetries when processing SFX data. The phenomenon is illustrated by re-analyzing data collected from microcrystals of the Blastochloris viridis photosynthetic reaction center and comparing these intensity observations with conventional synchrotron data. The results show that skewness in the SFX observations captures the essence of the Wilson plot and an empirical treatment is suggested that can help to separate the diffraction Bragg intensity from the background. PMID:28248658

  5. Fragment screening using X-ray crystallography.

    PubMed

    Davies, Thomas G; Tickle, Ian J

    2012-01-01

    The fragment-based approach is now well established as an important component of modern drug discovery. A key part in establishing its position as a viable technique has been the development of a range of biophysical methodologies with sufficient sensitivity to detect the binding of very weakly binding molecules. X-ray crystallography was one of the first techniques demonstrated to be capable of detecting such weak binding, but historically its potential for screening was under-appreciated and impractical due to its relatively low throughput. In this chapter we discuss the various benefits associated with fragment-screening by X-ray crystallography, and describe the technical developments we have implemented to allow its routine use in drug discovery. We emphasize how this approach has allowed a much greater exploitation of crystallography than has traditionally been the case within the pharmaceutical industry, with the rapid and timely provision of structural information having maximum impact on project direction.

  6. The pineapple AcMADS1 promoter confers high level expression in tomato and arabidopsis flowering and fruiting tissues, but AcMADS1 does not complement the tomato LeMADS-RIN (rin) mutant

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A previous EST study identified a MADS box transcription factor coding sequence, AcMADS1, that is strongly induced during non-climacteric pineapple fruit ripening. Phylogenetic analyses place the AcMADS1 protein in the same superclade as LeMADS-RIN, a master regulator of fruit ripening upstream of e...

  7. Effects of macromolecular crowding on genetic networks.

    PubMed

    Morelli, Marco J; Allen, Rosalind J; Wolde, Pieter Rein ten

    2011-12-21

    The intracellular environment is crowded with proteins, DNA, and other macromolecules. Under physiological conditions, macromolecular crowding can alter both molecular diffusion and the equilibria of bimolecular reactions and therefore is likely to have a significant effect on the function of biochemical networks. We propose a simple way to model the effects of macromolecular crowding on biochemical networks via an appropriate scaling of bimolecular association and dissociation rates. We use this approach, in combination with kinetic Monte Carlo simulations, to analyze the effects of crowding on a constitutively expressed gene, a repressed gene, and a model for the bacteriophage λ genetic switch, in the presence and absence of nonspecific binding of transcription factors to genomic DNA. Our results show that the effects of crowding are mainly caused by the shift of association-dissociation equilibria rather than the slowing down of protein diffusion, and that macromolecular crowding can have relevant and counterintuitive effects on biochemical network performance.

  8. MADS-box transcription factor OsMADS25 regulates root development through affection of nitrate accumulation in rice.

    PubMed

    Yu, Chunyan; Liu, Yihua; Zhang, Aidong; Su, Sha; Yan, An; Huang, Linli; Ali, Imran; Liu, Yu; Forde, Brian G; Gan, Yinbo

    2015-01-01

    MADS-box transcription factors are vital regulators participating in plant growth and development process and the functions of most of them are still unknown. ANR1 was reported to play a key role in controlling lateral root development through nitrate signal in Arabidopsis. OsMADS25 is one of five ANR1-like genes in Oryza Sativa and belongs to the ANR1 clade. Here we have investigated the role of OsMADS25 in the plant's responses to external nitrate in Oryza Sativa. Our results showed that OsMADS25 protein was found in the nucleus as well as in the cytoplasm. Over-expression of OsMADS25 significantly promoted lateral and primary root growth as well as shoot growth in a nitrate-dependent manner in Arabidopsis. OsMADS25 overexpression in transgenic rice resulted in significantly increased primary root length, lateral root number, lateral root length and shoot fresh weight in the presence of nitrate. Down-regulation of OsMADS25 in transgenic rice exhibited significantly reduced shoot and root growth in the presence of nitrate. Furthermore, over-expression of OsMADS25 in transgenic rice promoted nitrate accumulation and significantly increased the expressions of nitrate transporter genes at high rates of nitrate supply while down-regulation of OsMADS25 produced the opposite effect. Taken together, our findings suggest that OsMADS25 is a positive regulator control lateral and primary root development in rice.

  9. A tomato MADS-box transcription factor, SlMADS1, acts as a negative regulator of fruit ripening.

    PubMed

    Dong, Tingting; Hu, Zongli; Deng, Lei; Wang, Yi; Zhu, Mingku; Zhang, Jianling; Chen, Guoping

    2013-10-01

    MADS-box genes encode a highly conserved gene family of transcriptional factors that regulate numerous developmental processes in plants. In this study, a tomato (Solanum lycopersicum) MADS-box gene, SlMADS1, was cloned and its tissue-specific expression profile was analyzed. The real-time polymerase chain reaction results showed that SlMADS1 was highly expressed in sepals and fruits; its expression level was increased with the development of sepals, while the transcript of SlMADS1 decreased significantly in accordance with fruit ripening. To further explore the function of SlMADS1, an RNA interference (RNAi) expression vector targeting SlMADS1 was constructed and transformed into tomato plants. Shorter ripening time of fruit was observed in SlMADS1-silenced tomatoes. The accumulation of carotenoid and the expression of PHYTOENE SYNTHETASE1 were enhanced in RNAi fruits. Besides, ethylene biosynthetic genes, including 1-AMINOCYCLOPROPANE-1-CARBOXYLATE SYNTHASE1A, 1-AMINOCYCLOPROPANE-1-CARBOXYLATE SYNTHASE6, 1-AMINOCYCLOPROPANE-1-CARBOXYLATE OXIDASE1, and 1-AMINOCYCLOPROPANE-1-CARBOXYLATE OXIDASE3, and the ethylene-responsive genes E4 and E8, which were involved in fruit ripening, were also up-regulated in silenced plants. SlMADS1 RNAi fruits showed approximately 2- to 4-fold increases in ethylene production compared with the wild type. Furthermore, SlMADS1-silenced seedlings displayed shorter hypocotyls and were more sensitive to 1-aminocyclopropane-1-carboxylate than the wild type. Additionally, a yeast two-hybrid assay revealed a clear interaction between SlMADS1 and SlMADS-RIN. These results suggest that SlMADS1 plays an important role in fruit ripening as a repressive modulator.

  10. Ab-initio phasing in protein crystallography

    NASA Astrophysics Data System (ADS)

    van der Plas, J. L.; Millane, Rick P.

    2000-11-01

    The central problem in the determination of protein structures form x-ray diffraction dada (x-ray crystallography) corresponds to a phase retrieval problem with undersampled amplitude data. Algorithms for this problem that have an increased radius of convergence have the potential for reducing the amount of experimental work, and cost, involved in determining protein structures. We describe such an algorithm. Application of the algorithm to a simulated crystallographic problem shows that it converges to the correct solution, with no initial phase information, where currently used algorithms fail. The results lend support to the possibility of ab initio phasing in protein crystallography.

  11. Towards long-wavelength protein crystallography: keeping a protein crystal frozen in vacuum

    NASA Astrophysics Data System (ADS)

    Mykhaylyk, Vitaliy; Wagner, Armin

    2013-03-01

    There is growing interest to explore the long-wavelength X-ray domain for macromolecular crystallography (MX) experiments but there are a number of practical issues that make these experiments difficult to perform. In this article we study several aspects related to cooling a protein crystal in a vacuum environment. We investigated thermal contact conductance (TCC) of copper-copper joints and designed a demountable sample holder assembly with a magnetic joint that facilitates good thermal conductivity and reliability over a long time period. The thermal conductivity of amorphous ice formed by a 20% solution of ethylene glycol was studied. It is concluded that the ice thickness is the factor that can compromise the cooling of protein crystals and therefore it should be carefully controlled.

  12. Phenix - a comprehensive python-based system for macromolecular structure solution

    SciTech Connect

    Terwilliger, Thomas C; Hung, Li - Wei; Adams, Paul D; Afonine, Pavel V; Bunkoczi, Gabor; Chen, Vincent B; Davis, Ian; Echols, Nathaniel; Headd, Jeffrey J; Grosse Kunstleve, Ralf W; Mccoy, Airlie J; Moriarty, Nigel W; Oeffner, Robert; Read, Randy J; Richardson, David C; Richardson, Jane S; Zwarta, Peter H

    2009-01-01

    Macromolecular X-ray crystallography is routinely applied to understand biological processes at a molecular level. However, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages, and the repeated use of interactive three-dimensional graphics. Phenix has been developed to provide a comprehensive system for crystallographic structure solution with an emphasis on automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand, and finally the development of a framework that allows a tight integration between the algorithms.

  13. A lex-based mad parser and its applications

    SciTech Connect

    Oleg Krivosheev et al.

    2001-07-03

    An embeddable and portable Lex-based MAD language parser has been developed. The parser consists of a front-end which reads a MAD file and keeps beam elements, beam line data and algebraic expressions in tree-like structures, and a back-end, which processes the front-end data to generate an input file or data structures compatible with user applications. Three working programs are described, namely, a MAD to C++ converter, a dynamic C++ object factory and a MAD-MARS beam line builder. Design and implementation issues are discussed.

  14. RZZ and Mad1 dynamics in Drosophila mitosis.

    PubMed

    Défachelles, Lénaïg; Raich, Natacha; Terracol, Régine; Baudin, Xavier; Williams, Byron; Goldberg, Michael; Karess, Roger E

    2015-06-01

    The presence or absence of Mad1 at kinetochores is a major determinant of spindle assembly checkpoint (SAC) activity, the surveillance mechanism that delays anaphase onset if one or more kinetochores remain unattached to spindle fibers. Among the factors regulating the levels of Mad1 at kinetochores is the Rod, Zw10, and Zwilch (RZZ) complex, which is required for Mad1 recruitment through a mechanism that remains unknown. The relative dynamics and interactions of Mad1 and RZZ at kinetochores have not been extensively investigated, although Mad1 has been reported to be stably recruited to unattached kinetochores. In this study, we directly compare Mad1-green fluorescent protein (GFP) turnover dynamics on unattached Drosophila kinetochores with that of RZZ, tagged either with GFP-Rod or GFP-Zw10. We find that nearly 40 % of kinetochore-bound Mad1 has a significant dynamic component, turning over with a half-life of 12 s. RZZ in contrast is essentially stable on unattached kinetochores. In addition, we report that a fraction of RZZ and Mad1 can co-immunoprecipitate, indicating that the genetically determined recruitment hierarchy (in which Mad1 depends on RZZ) may reflect a physical association of the two complexes.

  15. Protein Crystallography in Vaccine Research and Development

    PubMed Central

    Malito, Enrico; Carfi, Andrea; Bottomley, Matthew J.

    2015-01-01

    The use of protein X-ray crystallography for structure-based design of small-molecule drugs is well-documented and includes several notable success stories. However, it is less well-known that structural biology has emerged as a major tool for the design of novel vaccine antigens. Here, we review the important contributions that protein crystallography has made so far to vaccine research and development. We discuss several examples of the crystallographic characterization of vaccine antigen structures, alone or in complexes with ligands or receptors. We cover the critical role of high-resolution epitope mapping by reviewing structures of complexes between antigens and their cognate neutralizing, or protective, antibody fragments. Most importantly, we provide recent examples where structural insights obtained via protein crystallography have been used to design novel optimized vaccine antigens. This review aims to illustrate the value of protein crystallography in the emerging discipline of structural vaccinology and its impact on the rational design of vaccines. PMID:26068237

  16. Structure of an intermediate conformer of the spindle checkpoint protein Mad2

    PubMed Central

    Hara, Mayuko; Özkan, Engin; Sun, Hongbin; Yu, Hongtao; Luo, Xuelian

    2015-01-01

    The spindle checkpoint senses unattached kinetochores during prometaphase and inhibits the anaphase-promoting complex or cyclosome (APC/C), thus ensuring accurate chromosome segregation. The checkpoint protein mitotic arrest deficient 2 (Mad2) is an unusual protein with multiple folded states. Mad2 adopts the closed conformation (C-Mad2) in a Mad1–Mad2 core complex. In mitosis, kinetochore-bound Mad1–C-Mad2 recruits latent, open Mad2 (O-Mad2) from the cytosol and converts it to an intermediate conformer (I-Mad2), which can then bind and inhibit the APC/C activator cell division cycle 20 (Cdc20) as C-Mad2. Here, we report the crystal structure and NMR analysis of I-Mad2 bound to C-Mad2. Although I-Mad2 retains the O-Mad2 fold in crystal and in solution, its core structural elements undergo discernible rigid-body movements and more closely resemble C-Mad2. Residues exhibiting methyl chemical shift changes in I-Mad2 form a contiguous, interior network that connects its C-Mad2–binding site to the conformationally malleable C-terminal region. Mutations of residues at the I-Mad2–C-Mad2 interface hinder I-Mad2 formation and impede the structural transition of Mad2. Our study provides insight into the conformational activation of Mad2 and establishes the basis of allosteric communication between two distal sites in Mad2. PMID:26305957

  17. Structure of an intermediate conformer of the spindle checkpoint protein Mad2

    SciTech Connect

    Hara, Mayuko; Özkan, Engin; Sun, Hongbin; Yu, Hongtao; Luo, Xuelian

    2015-08-24

    The spindle checkpoint senses unattached kinetochores during prometaphase and inhibits the anaphase-promoting complex or cyclosome (APC/C), thus ensuring accurate chromosome segregation. The checkpoint protein mitotic arrest deficient 2 (Mad2) is an unusual protein with multiple folded states. Mad2 adopts the closed conformation (C-Mad2) in a Mad1–Mad2 core complex. In mitosis, kinetochore-bound Mad1–C-Mad2 recruits latent, open Mad2 (O-Mad2) from the cytosol and converts it to an intermediate conformer (I-Mad2), which can then bind and inhibit the APC/C activator cell division cycle 20 (Cdc20) as C-Mad2. In this paper, we report the crystal structure and NMR analysis of I-Mad2 bound to C-Mad2. Although I-Mad2 retains the O-Mad2 fold in crystal and in solution, its core structural elements undergo discernible rigid-body movements and more closely resemble C-Mad2. Residues exhibiting methyl chemical shift changes in I-Mad2 form a contiguous, interior network that connects its C-Mad2–binding site to the conformationally malleable C-terminal region. Mutations of residues at the I-Mad2–C-Mad2 interface hinder I-Mad2 formation and impede the structural transition of Mad2. Finally, our study provides insight into the conformational activation of Mad2 and establishes the basis of allosteric communication between two distal sites in Mad2.

  18. Structure of an intermediate conformer of the spindle checkpoint protein Mad2

    DOE PAGES

    Hara, Mayuko; Özkan, Engin; Sun, Hongbin; ...

    2015-08-24

    The spindle checkpoint senses unattached kinetochores during prometaphase and inhibits the anaphase-promoting complex or cyclosome (APC/C), thus ensuring accurate chromosome segregation. The checkpoint protein mitotic arrest deficient 2 (Mad2) is an unusual protein with multiple folded states. Mad2 adopts the closed conformation (C-Mad2) in a Mad1–Mad2 core complex. In mitosis, kinetochore-bound Mad1–C-Mad2 recruits latent, open Mad2 (O-Mad2) from the cytosol and converts it to an intermediate conformer (I-Mad2), which can then bind and inhibit the APC/C activator cell division cycle 20 (Cdc20) as C-Mad2. In this paper, we report the crystal structure and NMR analysis of I-Mad2 bound to C-Mad2.more » Although I-Mad2 retains the O-Mad2 fold in crystal and in solution, its core structural elements undergo discernible rigid-body movements and more closely resemble C-Mad2. Residues exhibiting methyl chemical shift changes in I-Mad2 form a contiguous, interior network that connects its C-Mad2–binding site to the conformationally malleable C-terminal region. Mutations of residues at the I-Mad2–C-Mad2 interface hinder I-Mad2 formation and impede the structural transition of Mad2. Finally, our study provides insight into the conformational activation of Mad2 and establishes the basis of allosteric communication between two distal sites in Mad2.« less

  19. Current status and future prospects of an automated sample exchange system PAM for protein crystallography

    NASA Astrophysics Data System (ADS)

    Hiraki, M.; Yamada, Y.; Chavas, L. M. G.; Matsugaki, N.; Igarashi, N.; Wakatsuki, S.

    2013-03-01

    To achieve fully-automated and/or remote data collection in high-throughput X-ray experiments, the Structural Biology Research Centre at the Photon Factory (PF) has installed PF automated mounting system (PAM) for sample exchange robots at PF macromolecular crystallography beamlines BL-1A, BL-5A, BL-17A, AR-NW12A and AR-NE3A. We are upgrading the experimental systems, including the PAM for stable and efficient operation. To prevent human error in automated data collection, we installed a two-dimensional barcode reader for identification of the cassettes and sample pins. Because no liquid nitrogen pipeline in the PF experimental hutch is installed, the users commonly add liquid nitrogen using a small Dewar. To address this issue, an automated liquid nitrogen filling system that links a 100-liter tank to the robot Dewar has been installed on the PF macromolecular beamline. Here we describe this new implementation, as well as future prospects.

  20. HbMADS4, a MADS-box Transcription Factor from Hevea brasiliensis, Negatively Regulates HbSRPP

    PubMed Central

    Li, Hui-Liang; Wei, Li-Ran; Guo, Dong; Wang, Ying; Zhu, Jia-Hong; Chen, Xiong-Ting; Peng, Shi-Qing

    2016-01-01

    In plants MADS-box transcription factors (TFs) play important roles in growth and development. However, no plant MADS-box gene has been identified to have a function related to secondary metabolites regulation. Here, a MADS-box TF gene, designated as HbMADS4, was isolated from Hevea brasiliensis by the yeast one-hybrid experiment to screen the latex cDNA library using the promoter of the gene encoding H. brasiliensis small rubber particle protein (HbSRPP) as bait. HbMADS4 was 984-bp containing 633-bp open reading frame encoding a deduced protein of 230 amino acid residues with a typical conserved MADS-box motif at the N terminus. HbMADS4 was preferentially expressed in the latex, but little expression was detected in the leaves, flowers, and roots. Its expression was inducible by methyl jasmonate and ethylene. Furthermore, transient over-expression and over-expression of HbMADS4 in transgenic tobacco plants significantly suppressed the activity of the HbSRP promoter. Altogether, it is proposed that HbMADS4 is a negative regulator of HbSRPP which participates in the biosynthesis of natural rubber. PMID:27895659

  1. Ultrasonic acoustic levitation for fast frame rate X-ray protein crystallography at room temperature

    NASA Astrophysics Data System (ADS)

    Tsujino, Soichiro; Tomizaki, Takashi

    2016-05-01

    Increasing the data acquisition rate of X-ray diffraction images for macromolecular crystals at room temperature at synchrotrons has the potential to significantly accelerate both structural analysis of biomolecules and structure-based drug developments. Using lysozyme model crystals, we demonstrated the rapid acquisition of X-ray diffraction datasets by combining a high frame rate pixel array detector with ultrasonic acoustic levitation of protein crystals in liquid droplets. The rapid spinning of the crystal within a levitating droplet ensured an efficient sampling of the reciprocal space. The datasets were processed with a program suite developed for serial femtosecond crystallography (SFX). The structure, which was solved by molecular replacement, was found to be identical to the structure obtained by the conventional oscillation method for up to a 1.8-Å resolution limit. In particular, the absence of protein crystal damage resulting from the acoustic levitation was carefully established. These results represent a key step towards a fully automated sample handling and measurement pipeline, which has promising prospects for a high acquisition rate and high sample efficiency for room temperature X-ray crystallography.

  2. Ultrasonic acoustic levitation for fast frame rate X-ray protein crystallography at room temperature

    PubMed Central

    Tsujino, Soichiro; Tomizaki, Takashi

    2016-01-01

    Increasing the data acquisition rate of X-ray diffraction images for macromolecular crystals at room temperature at synchrotrons has the potential to significantly accelerate both structural analysis of biomolecules and structure-based drug developments. Using lysozyme model crystals, we demonstrated the rapid acquisition of X-ray diffraction datasets by combining a high frame rate pixel array detector with ultrasonic acoustic levitation of protein crystals in liquid droplets. The rapid spinning of the crystal within a levitating droplet ensured an efficient sampling of the reciprocal space. The datasets were processed with a program suite developed for serial femtosecond crystallography (SFX). The structure, which was solved by molecular replacement, was found to be identical to the structure obtained by the conventional oscillation method for up to a 1.8-Å resolution limit. In particular, the absence of protein crystal damage resulting from the acoustic levitation was carefully established. These results represent a key step towards a fully automated sample handling and measurement pipeline, which has promising prospects for a high acquisition rate and high sample efficiency for room temperature X-ray crystallography. PMID:27150272

  3. Ultrasonic acoustic levitation for fast frame rate X-ray protein crystallography at room temperature.

    PubMed

    Tsujino, Soichiro; Tomizaki, Takashi

    2016-05-06

    Increasing the data acquisition rate of X-ray diffraction images for macromolecular crystals at room temperature at synchrotrons has the potential to significantly accelerate both structural analysis of biomolecules and structure-based drug developments. Using lysozyme model crystals, we demonstrated the rapid acquisition of X-ray diffraction datasets by combining a high frame rate pixel array detector with ultrasonic acoustic levitation of protein crystals in liquid droplets. The rapid spinning of the crystal within a levitating droplet ensured an efficient sampling of the reciprocal space. The datasets were processed with a program suite developed for serial femtosecond crystallography (SFX). The structure, which was solved by molecular replacement, was found to be identical to the structure obtained by the conventional oscillation method for up to a 1.8-Å resolution limit. In particular, the absence of protein crystal damage resulting from the acoustic levitation was carefully established. These results represent a key step towards a fully automated sample handling and measurement pipeline, which has promising prospects for a high acquisition rate and high sample efficiency for room temperature X-ray crystallography.

  4. Quickly Getting the Best Data from Your Macromolecular Crystals with a New Generation of Beamline Instruments

    SciTech Connect

    Cipriani, Florent; Felisaz, Franck; Lavault, Bernard; Brockhauser, Sandor; Ravelli, Raimond; Launer, Ludovic

    2007-01-19

    While routine Macromolecular x-ray (MX) crystallography has relied on well established techniques for some years all the synchrotrons around the world are improving the throughput of their MX beamlines. Third generation synchrotrons provide small intense beams that make data collection of 5-10 microns sized crystals possible. The EMBL/ESRF MX Group in Grenoble has developed a new generation of instruments to easily collect data on 10 {mu}m size crystals in an automated environment. This work is part of the Grenoble automation program that enables FedEx like crystallography using fully automated data collection and web monitored experiments. Seven ESRF beamlines and the MRC BM14 ESRF/CRG beamline are currently equipped with these latest instruments. We describe here the main features of the MD2x diffractometer family and the SC3 sample changer robot. Although the SC3 was primarily designed to increase the throughput of MX beamlines, it has also been shown to be efficient in improving the quality of the data collected. Strategies in screening a large number of crystals, selecting the best, and collecting a full data set from several re-oriented micro-crystals can now be run with minimum time and effort. The MD2x and SC3 instruments are now commercialised by the company ACCEL GmbH.

  5. Interdisciplinary Critical Inquiry: Teaching about the Social Construction of Madness

    ERIC Educational Resources Information Center

    Connor-Greene, Patricia A.

    2006-01-01

    Theories and treatments of mental illness reflect the social, philosophical, and historical context in which they developed. This article describes ways to invite students to grapple with complex questions about "madness" from an interdisciplinary perspective. Looking at the construct of madness through multiple lenses (e.g., literature,…

  6. Web life: The Evil Mad Scientist Project

    NASA Astrophysics Data System (ADS)

    2009-04-01

    What is it? Have you ever tried to electrocute a hot dog? Wondered how to make a robot out of a toothbrush, watch battery and phone-pager motor? Seen a cantaloupe melon and thought, "Hmm, I could make this look like the Death Star from the original Star Wars films"? If you have not, but you would like to - preferably as soon as you can find a pager motor - then this is the site for you. The Evil Mad Scientist Project (EMSP) blog is packed full of ideas for unusual, silly and frequently physics-related creations that bring science out of the laboratory and into kitchens, backyards and tool sheds.

  7. Resolution of structural heterogeneity in dynamic crystallography

    PubMed Central

    Ren, Zhong; Chan, Peter W. Y.; Moffat, Keith; Pai, Emil F.; Royer, William E.; Šrajer, Vukica; Yang, Xiaojing

    2013-01-01

    Dynamic behavior of proteins is critical to their function. X-­ray crystallography, a powerful yet mostly static technique, faces inherent challenges in acquiring dynamic information despite decades of effort. Dynamic ‘structural changes’ are often indirectly inferred from ‘structural differences’ by comparing related static structures. In contrast, the direct observation of dynamic structural changes requires the initiation of a biochemical reaction or process in a crystal. Both the direct and the indirect approaches share a common challenge in analysis: how to interpret the structural heterogeneity intrinsic to all dynamic processes. This paper presents a real-space approach to this challenge, in which a suite of analytical methods and tools to identify and refine the mixed structural species present in multiple crystallographic data sets have been developed. These methods have been applied to representative scenarios in dynamic crystallography, and reveal structural information that is otherwise difficult to interpret or inaccessible using conventional methods. PMID:23695239

  8. Resolution of structural heterogeneity in dynamic crystallography.

    PubMed

    Ren, Zhong; Chan, Peter W Y; Moffat, Keith; Pai, Emil F; Royer, William E; Šrajer, Vukica; Yang, Xiaojing

    2013-06-01

    Dynamic behavior of proteins is critical to their function. X-ray crystallography, a powerful yet mostly static technique, faces inherent challenges in acquiring dynamic information despite decades of effort. Dynamic `structural changes' are often indirectly inferred from `structural differences' by comparing related static structures. In contrast, the direct observation of dynamic structural changes requires the initiation of a biochemical reaction or process in a crystal. Both the direct and the indirect approaches share a common challenge in analysis: how to interpret the structural heterogeneity intrinsic to all dynamic processes. This paper presents a real-space approach to this challenge, in which a suite of analytical methods and tools to identify and refine the mixed structural species present in multiple crystallographic data sets have been developed. These methods have been applied to representative scenarios in dynamic crystallography, and reveal structural information that is otherwise difficult to interpret or inaccessible using conventional methods.

  9. High-Throughput Methods for Electron Crystallography

    PubMed Central

    Stokes, David L.; Ubarretxena-Belandia, Iban; Gonen, Tamir; Engel, Andreas

    2013-01-01

    Membrane proteins play a tremendously important role in cell physiology and serve as a target for an increasing number of drugs. Structural information is key to understanding their function and for developing new strategies for combating disease. However, the complex physical chemistry associated with membrane proteins has made them more difficult to study than their soluble cousins. Electron crystallography has historically been a successful method for solving membrane protein structures and has the advantage of providing the natural environment of a lipid membrane. Specifically, when membrane proteins form two-dimensional arrays within a lipid bilayer, images and diffraction can be recorded by electron microscopy. The corresponding data can be combined to produce a three-dimensional reconstruction which, under favorable conditions, can extend to atomic resolution. Like X-ray crystallography, the quality of the structures are very much dependent on the order and size of the crystals. However, unlike X-ray crystallography, high-throughput methods for screening crystallization trials for electron crystallography are not in general use. In this chapter, we describe two alternative and potentially complementary methods for high-throughput screening of membrane protein crystallization within the lipid bilayer. The first method relies on the conventional use of dialysis for removing detergent and thus reconstituting the bilayer; an array of dialysis wells in the standard 96-well format allows the use of a liquid-handling robot and greatly increases throughput. The second method relies on detergent complexation by cyclodextrin; a specialized pipetting robot has been designed not only to titrate cyclodextrin, but to use light scattering to monitor the reconstitution process. In addition, the use of liquid-handling robots for making negatively stained grids and methods for automatically imaging samples in the electron microscope are described. PMID:23132066

  10. Selection and evolution in macromolecular systems.

    PubMed

    Demetrius, L

    1983-08-21

    The notion of a quasi-species represents the ensemble of macromolecular sequences derived by the mechanism of mutation and replication from a single wild type. In Eigen (1971) and Eigen & Schuster (1979), the deterministic evolution of this ensemble under constant environmental conditions is given in terms of continuous models which describe the dynamics of the distribution of polynucleotides. This paper starts from a discrete model of macromolecular evolution and introduces the notion of a genealogy in order to study the dynamics of the quasi-species in constant and variable environments. We introduce, in terms of these genealogies, the notions of entropy and adaptive value of a quasi-species and the notion of capacity of the environment. We discuss the significance of these indices as measures of selective value and we analyse the conditions under which these measures coincide with the growth rate of the quasi-species.

  11. Growth and Dissolution of Macromolecular Markov Chains

    NASA Astrophysics Data System (ADS)

    Gaspard, Pierre

    2016-07-01

    The kinetics and thermodynamics of free living copolymerization are studied for processes with rates depending on k monomeric units of the macromolecular chain behind the unit that is attached or detached. In this case, the sequence of monomeric units in the growing copolymer is a kth-order Markov chain. In the regime of steady growth, the statistical properties of the sequence are determined analytically in terms of the attachment and detachment rates. In this way, the mean growth velocity as well as the thermodynamic entropy production and the sequence disorder can be calculated systematically. These different properties are also investigated in the regime of depolymerization where the macromolecular chain is dissolved by the surrounding solution. In this regime, the entropy production is shown to satisfy Landauer's principle.

  12. MAD2γ, a novel MAD2 isoform, reduces mitotic arrest and is associated with resistance in testicular germ cell tumors

    PubMed Central

    López-Saavedra, Alejandro; Ramírez-Otero, Miguel; Díaz-Chávez, José; Cáceres-Gutiérrez, Rodrigo; Justo-Garrido, Monserrat; Andonegui, Marco A.; Mendoza, Julia; Downie-Ruíz, Ángela; Cortés-González, Carlo; Reynoso, Nancy; Castro-Hernández, Clementina; Domínguez-Gómez, Guadalupe; Santibáñez, Miguel; Fabián-Morales, Eunice; Pruefer, Franz; Luna-Maldonado, Fernando; González-Barrios, Rodrigo; Herrera, Luis A.

    2016-01-01

    ABSTRACT Background: Prolonged mitotic arrest in response to anti-cancer chemotherapeutics, such as DNA-damaging agents, induces apoptosis, mitotic catastrophe, and senescence. Disruptions in mitotic checkpoints contribute resistance to DNA-damaging agents in cancer. MAD2 has been associated with checkpoint failure and chemotherapy response. In this study, a novel splice variant of MAD2, designated MAD2γ, was identified, and its association with the DNA damage response was investigated. Methods: Endogenous expression of MAD2γ and full-length MAD2 (MAD2α) was measured using RT-PCR in cancer cell lines, normal foreskin fibroblasts, and tumor samples collected from patients with testicular germ cell tumors (TGCTs). A plasmid expressing MAD2γ was transfected into HCT116 cells, and its intracellular localization and checkpoint function were evaluated according to immunofluorescence and mitotic index. Results: MAD2γ was expressed in several cancer cell lines and non-cancerous fibroblasts. Ectopically expressed MAD2γ localized to the nucleus and reduced the mitotic index, suggesting checkpoint impairment. In patients with TGCTs, the overexpression of endogenous MAD2γ, but not MAD2α, was associated with resistance to cisplatin-based chemotherapy. Likewise, cisplatin induced the overexpression of endogenous MAD2γ, but not MAD2α, in HCT116 cells. Conclusions: Overexpression of MAD2γ may play a role in checkpoint disruption and is associated with resistance to cisplatin-based chemotherapy in TGCTs. PMID:27315568

  13. The structural dynamics of macromolecular processes

    PubMed Central

    Russel, Daniel; Lasker, Keren; Phillips, Jeremy; Schneidman-Duhovny, Dina; Velázquez-Muriel, Javier A.; Sali, Andrej

    2009-01-01

    Summary Dynamic processes involving macromolecular complexes are essential to cell function. These processes take place over a wide variety of length scales from nanometers to micrometers, and over time scales from nanoseconds to many minutes. As a result, information from a variety of different experimental and computational approaches is required. We review the relevant sources of information and introduce a framework for integrating the data to produce representations of dynamic processes. PMID:19223165

  14. Macromolecular extraction based on contour evolution

    NASA Astrophysics Data System (ADS)

    Wang, Zhaobin; Guo, Miao; Zhu, Ying; Yang, Lizhen; Ma, Yi-de

    2013-03-01

    Detecting the region of interest plays an important role in the field of image processing and analysis. For the microscopic image of plant embryo slice, region of interest usually indicates various cells or macromolecules. Combining contour evolution theory and pulse coupled neural network, we propose a new method of macromolecular detection and extraction for biological microscopic image. Some existing methods are compared with the proposed method. Experimental results show the proposed method has the better performance than existing methods.

  15. Protein conformational studies for macromolecularly imprinted polymers.

    PubMed

    Kryscio, David R; Fleming, Michael Q; Peppas, Nicholas A

    2012-08-01

    CD is used to clearly show the negative impact of common ligands on the overall conformation of BSA, a typical protein template in macromolecularly imprinted polymers. This change occurs at concentrations far lower than those generally used in the literature. These findings are important as they offer insight into a potential fundamental reason for the lack of success in protein imprinting to date despite significant interest from the scientific community.

  16. Stochastic dynamics of macromolecular-assembly networks.

    NASA Astrophysics Data System (ADS)

    Saiz, Leonor; Vilar, Jose

    2006-03-01

    The formation and regulation of macromolecular complexes provides the backbone of most cellular processes, including gene regulation and signal transduction. The inherent complexity of assembling macromolecular structures makes current computational methods strongly limited for understanding how the physical interactions between cellular components give rise to systemic properties of cells. Here we present a stochastic approach to study the dynamics of networks formed by macromolecular complexes in terms of the molecular interactions of their components [1]. Exploiting key thermodynamic concepts, this approach makes it possible to both estimate reaction rates and incorporate the resulting assembly dynamics into the stochastic kinetics of cellular networks. As prototype systems, we consider the lac operon and phage λ induction switches, which rely on the formation of DNA loops by proteins [2] and on the integration of these protein-DNA complexes into intracellular networks. This cross-scale approach offers an effective starting point to move forward from network diagrams, such as those of protein-protein and DNA-protein interaction networks, to the actual dynamics of cellular processes. [1] L. Saiz and J.M.G. Vilar, submitted (2005). [2] J.M.G. Vilar and L. Saiz, Current Opinion in Genetics & Development, 15, 136-144 (2005).

  17. Mad3 and Mad4: novel Max-interacting transcriptional repressors that suppress c-myc dependent transformation and are expressed during neural and epidermal differentiation.

    PubMed Central

    Hurlin, P J; Quéva, C; Koskinen, P J; Steingrímsson, E; Ayer, D E; Copeland, N G; Jenkins, N A; Eisenman, R N

    1995-01-01

    The basic helix-loop-helix-leucine zipper (bHLHZip) protein Max associates with members of the Myc family, as well as with the related proteins Mad (Mad1) and Mxi1. Whereas both Myc:Max and Mad:Max heterodimers bind related E-box sequences, Myc:Max activates transcription and promotes proliferation while Mad:Max represses transcription and suppresses Myc dependent transformation. Here we report the identification and characterization of two novel Mad1- and Mxi1-related proteins, Mad3 and Mad4. Mad3 and Mad4 interact with both Max and mSin3 and repress transcription from a promoter containing CACGTG binding sites. Using a rat embryo fibroblast transformation assay, we show that both Mad3 and Mad4 inhibit c-Myc dependent cell transformation. An examination of the expression patterns of all mad genes during murine embryogenesis reveals that mad1, mad3 and mad4 are expressed primarily in growth-arrested differentiating cells. mxi1 is also expressed in differentiating cells, but is co-expressed with either c-myc, N-myc, or both in proliferating cells of the developing central nervous system and the epidermis. In the developing central nervous system and epidermis, downregulation of myc genes occurs concomitant with upregulation of mad family genes. These expression patterns, together with the demonstrated ability of Mad family proteins to interfere with the proliferation promoting activities of Myc, suggest that the regulated expression of Myc and Mad family proteins function in a concerted fashion to regulate cell growth in differentiating tissues. Images PMID:8521822

  18. Sustained Mps1 activity is required in mitosis to recruit O-Mad2 to the Mad1–C-Mad2 core complex

    PubMed Central

    Hewitt, Laura; Tighe, Anthony; Santaguida, Stefano; White, Anne M.; Jones, Clifford D.; Musacchio, Andrea; Green, Stephen

    2010-01-01

    Mps1 is an essential component of the spindle assembly checkpoint. In this study, we describe a novel Mps1 inhibitor, AZ3146, and use it to probe the role of Mps1’s catalytic activity during mitosis. When Mps1 is inhibited before mitotic entry, subsequent recruitment of Mad1 and Mad2 to kinetochores is abolished. However, if Mps1 is inhibited after mitotic entry, the Mad1–C-Mad2 core complex remains kinetochore bound, but O-Mad2 is not recruited to the core. Although inhibiting Mps1 also interferes with chromosome alignment, we see no obvious effect on aurora B activity. In contrast, kinetochore recruitment of centromere protein E (CENP-E), a kinesin-related motor protein, is severely impaired. Strikingly, inhibition of Mps1 significantly increases its own abundance at kinetochores. Furthermore, we show that Mps1 can dimerize and transphosphorylate in cells. We propose a model whereby Mps1 transphosphorylation results in its release from kinetochores, thus facilitating recruitment of O-Mad2 and CENP-E and thereby simultaneously promoting checkpoint signaling and chromosome congression. PMID:20624899

  19. Post-translational regulation of rice MADS29 function: homodimerization or binary interactions with other seed-expressed MADS proteins modulate its translocation into the nucleus.

    PubMed

    Nayar, Saraswati; Kapoor, Meenu; Kapoor, Sanjay

    2014-10-01

    OsMADS29 is a seed-specific MADS-box transcription factor that affects embryo development and grain filling by maintaining hormone homeostasis and degradation of cells in the nucellus and nucellar projection. Although it has a bipartite nuclear localization signal (NLS) sequence, the transiently expressed OsMADS29 monomer does not localize specifically in the nucleus. Dimerization of the monomers alters the intracellular localization fate of the resulting OsMADS29 homodimer, which then translocates into the nucleus. By generating domain-specific deletions/mutations, we show that two conserved amino acids (lysine(23) and arginine(24)) in the NLS are important for nuclear localization of the OsMADS29 homodimer. Furthermore, the analyses involving interaction of OsMADS29 with 30 seed-expressed rice MADS proteins revealed 19 more MADS-box proteins, including five E-class proteins, which interacted with OsMADS29. Eleven of these complexes were observed to be localized in the nucleus. Deletion analysis revealed that the KC region (K-box and C-terminal domain) plays a pivotal role in homodimerization. These data suggest that the biological function of OsMADS29 may not only be regulated at the level of transcription and translation as reported earlier, but also at the post-translational level by way of the interaction between OsMADS29 monomers, and between OsMADS29 and other MADS-box proteins.

  20. Post-translational regulation of rice MADS29 function: homodimerization or binary interactions with other seed-expressed MADS proteins modulate its translocation into the nucleus

    PubMed Central

    Nayar, Saraswati; Kapoor, Meenu; Kapoor, Sanjay

    2014-01-01

    OsMADS29 is a seed-specific MADS-box transcription factor that affects embryo development and grain filling by maintaining hormone homeostasis and degradation of cells in the nucellus and nucellar projection. Although it has a bipartite nuclear localization signal (NLS) sequence, the transiently expressed OsMADS29 monomer does not localize specifically in the nucleus. Dimerization of the monomers alters the intracellular localization fate of the resulting OsMADS29 homodimer, which then translocates into the nucleus. By generating domain-specific deletions/mutations, we show that two conserved amino acids (lysine23 and arginine24) in the NLS are important for nuclear localization of the OsMADS29 homodimer. Furthermore, the analyses involving interaction of OsMADS29 with 30 seed-expressed rice MADS proteins revealed 19 more MADS-box proteins, including five E-class proteins, which interacted with OsMADS29. Eleven of these complexes were observed to be localized in the nucleus. Deletion analysis revealed that the KC region (K-box and C-terminal domain) plays a pivotal role in homodimerization. These data suggest that the biological function of OsMADS29 may not only be regulated at the level of transcription and translation as reported earlier, but also at the post-translational level by way of the interaction between OsMADS29 monomers, and between OsMADS29 and other MADS-box proteins. PMID:25096923

  1. 'The verses of madness': schizophrenia and poetry.

    PubMed

    Hankir, Ahmed Khaldoon; Holloway, David; Agius, Mark; Zaman, Rashid

    2012-12-20

    In the early 19th century, Lombroso introduced the concept of hereditary taint to describe the coexistence of 'madness' and creativity. In a recent investigation, Rust et al reported a study designed to test the traditionally assumed relationship between creativity and schizophrenia. They uncovered an association between creative originality and the positive cognitive aspects of schizotypal thinking. Poetry is not only the 'product' of psychopathology but it can also be utilised as a form of therapy: "My name is David Holloway, I am a 33 year old poet/blogger with paranoid schizophrenia. A poet called Charles Bukowski has described poetry as the 'ultimate psychiatrist', and I am a firm believer in this. The strongest part of my personality is my belief in the power of love. My recovery has relied heavily on medication, diet and exercise. However it is the power of poetry that has been my true inspiration."

  2. Automated High Throughput Drug Target Crystallography

    SciTech Connect

    Rupp, B

    2005-02-18

    The molecular structures of drug target proteins and receptors form the basis for 'rational' or structure guided drug design. The majority of target structures are experimentally determined by protein X-ray crystallography, which as evolved into a highly automated, high throughput drug discovery and screening tool. Process automation has accelerated tasks from parallel protein expression, fully automated crystallization, and rapid data collection to highly efficient structure determination methods. A thoroughly designed automation technology platform supported by a powerful informatics infrastructure forms the basis for optimal workflow implementation and the data mining and analysis tools to generate new leads from experimental protein drug target structures.

  3. The Construction of Group Theory in Crystallography

    NASA Astrophysics Data System (ADS)

    Maitte, Bernard

    This article sets out to retrace the manner in which Group Theory evolved in crystallography. To engage in this study it is necessary to select, amongst all the approaches to crystals, those which, from the point of view of modern science, mark a step towards the establishment of our current understanding. In this way it favours our current perspective. To compensate this distortion, we recall the context in which each explanation that marks history appears. It so becomes clear that notions of triperiodic assemblages and the crystallographic laws of crystal systems and symmetrical classes do not derive from "natural" observations but were compiled and belong to precise theories.

  4. Metalloprotein Crystallography: More than a Structure

    PubMed Central

    2016-01-01

    Conspectus Metal ions and metallocofactors play important roles in a broad range of biochemical reactions. Accordingly, it has been estimated that as much as 25–50% of the proteome uses transition metal ions to carry out a variety of essential functions. The metal ions incorporated within metalloproteins fulfill functional roles based on chemical properties, the diversity of which arises as transition metals can adopt different redox states and geometries, dictated by the identity of the metal and the protein environment. The coupling of a metal ion with an organic framework in metallocofactors, such as heme and cobalamin, further expands the chemical functionality of metals in biology. The three-dimensional visualization of metal ions and complex metallocofactors within a protein scaffold is often a starting point for enzymology, highlighting the importance of structural characterization of metalloproteins. Metalloprotein crystallography, however, presents a number of implicit challenges including correctly incorporating the relevant metal or metallocofactor, maintaining the proper environment for the protein to be purified and crystallized (including providing anaerobic, cold, or aphotic environments), and being mindful of the possibility of X-ray induced damage to the proteins or incorporated metal ions. Nevertheless, the incorporated metals or metallocofactors also present unique advantages in metalloprotein crystallography. The significant resonance that metals undergo with X-ray photons at wavelengths used for protein crystallography and the rich electronic properties of metals, which provide intense and spectroscopically unique signatures, allow a metalloprotein crystallographer to use anomalous dispersion to determine phases for structure solution and to use simultaneous or parallel spectroscopic techniques on single crystals. These properties, coupled with the improved brightness of beamlines, the ability to tune the wavelength of the X-ray beam, the

  5. Development of macromolecular prodrug for rheumatoid arthritis☆

    PubMed Central

    Yuan, Fang; Quan, Ling-dong; Cui, Liao; Goldring, Steven R.; Wang, Dong

    2012-01-01

    Rheumatoid arthritis (RA) is a chronic autoimmune disease that is considered to be one of the major public health problems worldwide. The development of therapies that target tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and co-stimulatory pathways that regulate the immune system have revolutionized the care of patients with RA. Despite these advances, many patients continue to experience symptomatic and functional impairment. To address this issue, more recent therapies that have been developed are designed to target intracellular signaling pathways involved in immunoregulation. Though this approach has been encouraging, there have been major challenges with respect to off-target organ side effects and systemic toxicities related to the widespread distribution of these signaling pathways in multiple cell types and tissues. These limitations have led to an increasing interest in the development of strategies for the macromolecularization of anti-rheumatic drugs, which could target them to the inflamed joints. This approach enhances the efficacy of the therapeutic agent with respect to synovial inflammation, while markedly reducing non-target organ adverse side effects. In this manuscript, we provide a comprehensive overview of the rational design and optimization of macromolecular prodrugs for treatment of RA. The superior and the sustained efficacy of the prodrug may be partially attributed to their Extravasation through Leaky Vasculature and subsequent Inflammatory cell-mediated Sequestration (ELVIS) in the arthritic joints. This biologic process provides a plausible mechanism, by which macromolecular prodrugs preferentially target arthritic joints and illustrates the potential benefits of applying this therapeutic strategy to the treatment of other inflammatory diseases. PMID:22433784

  6. Crystallization of macromolecular complexes:. stoichiometric variation screening

    NASA Astrophysics Data System (ADS)

    Stura, Enrico A.; Graille, Marc; Taussig, Michael J.; Sutton, Brian; Gore, Michael G.; Silverman, Gregg J.; Charbonnier, Jean-Baptiste

    2001-11-01

    Theoretically a crystal may contain both complexed and uncomplexed molecules simultaneously in the same lattice. Since we seldom screen for such possibilities, such occurrences are only rarely reported. Here we propose that stoichiometry should be one of the parameters to be screened in the crystallization of macromolecular complexes. By allowing for non-biologically significant stoichiometries, we may increase the chances of crystallizing a macromolecular complex and of selecting arrangements which crystallize better or yield more ordered crystals. Although biological forces tend to be stronger than lattice-building interactions, in the crystal the latter will dominate numerically. By allowing for a varied stoichiometry we permit a wider selection of lattice-building contacts and increase the probability of crystallization. From these theoretical considerations we have developed methodology compatible with classical solubility screening and other well-established crystallization principles. We discuss this technique, stoichiometric variation screening (SVS), as part of a multicomponent system for the enhancement of crystallization of macromolecular complexes. We present this technique as an extension of reverse screening and illustrate the complementarity in the methodology. We present two examples of the use of SVS: the complexes between an immunoglobulin Fab fragment and two bacterial proteins, namely the D domain of protein A from Staphylococcus aureus (SpA) and a single domain of protein L from Peptostreptococcus magnus (PpL). In the first example there are 3 Fab molecules and only 2 SpA D domains (domD) (2 complexed and 1 unliganded Fab), in the second 2 Fabs and only 1 PpL domain (1 complexed and 1 unliganded Fab). SVS has the added and unique advantage that in the same crystal we have information on both the unliganded and complexed states under precisely identical conditions: one structure, two answers. Together with a combinatorial method for complex

  7. Automated macromolecular crystal detection system and method

    DOEpatents

    Christian, Allen T.; Segelke, Brent; Rupp, Bernard; Toppani, Dominique

    2007-06-05

    An automated macromolecular method and system for detecting crystals in two-dimensional images, such as light microscopy images obtained from an array of crystallization screens. Edges are detected from the images by identifying local maxima of a phase congruency-based function associated with each image. The detected edges are segmented into discrete line segments, which are subsequently geometrically evaluated with respect to each other to identify any crystal-like qualities such as, for example, parallel lines, facing each other, similarity in length, and relative proximity. And from the evaluation a determination is made as to whether crystals are present in each image.

  8. Bovine Spongiform Encephalopathy (BSE), or Mad Cow Disease

    MedlinePlus

    ... Search The CDC Cancel Submit Search The CDC Bovine Spongiform Encephalopathy (BSE), or Mad Cow Disease Note: Javascript is ... gov . Recommend on Facebook Tweet Share Compartir BSE (bovine spongiform encephalopathy) is a progressive neurological disorder of cattle that ...

  9. An unusual presentation of mad honey poisoning: acute myocardial infarction.

    PubMed

    Akinci, Sinan; Arslan, Uğur; Karakurt, Kamber; Cengel, Atiye

    2008-09-26

    An unusual type of food poisoning is commonly seen in the Black Sea coast of Turkey due to grayanotoxin containing toxic honey so called "mad honey" ingestion. In cases of toxication bradycardia and rhythm disturbances are commonly observed. Herein, we present a case of a patient who was admitted to the hospital because of acute myocardial infarction with normal coronary arteries after "mad honey" ingestion.

  10. Mad Cow Disease and U.S. Beef Trade

    DTIC Science & Technology

    2006-12-06

    foreign markets that banned U.S. beef when a cow in Washington state tested positive for bovine spongiform encephalopathy (BSE, or mad cow disease) in...1 For additional details and background see CRS Report RS22345, BSE (“Mad Cow Disease:): A Brief Overview, and CRS Report RL32199, Bovine Spongiform ...rejection of three shipments of U.S. beef because of the presence of bone fragments. CRS-4 8 See also CRS Report RL32932, Bovine Spongiform

  11. MeshAndCollect: an automated multi-crystal data-collection workflow for synchrotron macromolecular crystallography beamlines

    SciTech Connect

    Zander, Ulrich; Bourenkov, Gleb; Popov, Alexander N.; Sanctis, Daniele de; Svensson, Olof; McCarthy, Andrew A.; Round, Ekaterina; Gordeliy, Valentin; Mueller-Dieckmann, Christoph; Leonard, Gordon A.

    2015-10-31

    The fully automated collection and merging of partial data sets from a series of cryocooled crystals of biological macromolecules contained on the same support is presented, as are the results of test experiments carried out on various systems. Here, an automated procedure is described to identify the positions of many cryocooled crystals mounted on the same sample holder, to rapidly predict and rank their relative diffraction strengths and to collect partial X-ray diffraction data sets from as many of the crystals as desired. Subsequent hierarchical cluster analysis then allows the best combination of partial data sets, optimizing the quality of the final data set obtained. The results of applying the method developed to various systems and scenarios including the compilation of a complete data set from tiny crystals of the membrane protein bacteriorhodopsin and the collection of data sets for successful structure determination using the single-wavelength anomalous dispersion technique are also presented.

  12. Graphene-based microfluidics for serial crystallography.

    PubMed

    Sui, Shuo; Wang, Yuxi; Kolewe, Kristopher W; Srajer, Vukica; Henning, Robert; Schiffman, Jessica D; Dimitrakopoulos, Christos; Perry, Sarah L

    2016-08-02

    Microfluidic strategies to enable the growth and subsequent serial crystallographic analysis of micro-crystals have the potential to facilitate both structural characterization and dynamic structural studies of protein targets that have been resistant to single-crystal strategies. However, adapting microfluidic crystallization platforms for micro-crystallography requires a dramatic decrease in the overall device thickness. We report a robust strategy for the straightforward incorporation of single-layer graphene into ultra-thin microfluidic devices. This architecture allows for a total material thickness of only ∼1 μm, facilitating on-chip X-ray diffraction analysis while creating a sample environment that is stable against significant water loss over several weeks. We demonstrate excellent signal-to-noise in our X-ray diffraction measurements using a 1.5 μs polychromatic X-ray exposure, and validate our approach via on-chip structure determination using hen egg white lysozyme (HEWL) as a model system. Although this work is focused on the use of graphene for protein crystallography, we anticipate that this technology should find utility in a wide range of both X-ray and other lab on a chip applications.

  13. Structural physiology based on electron crystallography

    PubMed Central

    Fujiyoshi, Yoshinori

    2011-01-01

    There are many questions in brain science, which are extremely interesting but very difficult to answer. For example, how do education and other experiences during human development influence the ability and personality of the adult? The molecular mechanisms underlying such phenomena are still totally unclear. However, technological and instrumental advancements of electron microscopy have facilitated comprehension of the structures of biological components, cells, and organelles. Electron crystallography is especially good for studying the structure and function of membrane proteins, which are key molecules of signal transduction in neural and other cells. Electron crystallography is now an established technique to analyze the structures of membrane proteins in lipid bilayers, which are close to their natural biological environment. By utilizing cryo-electron microscopes with helium cooled specimen stages, which were developed through a personal motivation to understand functions of neural systems from a structural point of view, structures of membrane proteins were analyzed at a resolution higher than 3 Å. This review has four objectives. First, it is intended to introduce the new research field of structural physiology. Second, it introduces some of the personal struggles, which were involved in developing the cryo-electron microscope. Third, it discusses some of the technology for the structural analysis of membrane proteins based on cryo-electron microscopy. Finally, it reviews structural and functional analyses of membrane proteins. PMID:21416541

  14. Novel complex MAD phasing and RNase H structural insights using selenium oligonucleotides

    SciTech Connect

    Abdur, Rob; Gerlits, Oksana O.; Gan, Jianhua; Jiang, Jiansheng; Salon, Jozef; Kovalevsky, Andrey Y.; Chumanevich, Alexander A.; Weber, Irene T.; Huang, Zhen

    2014-02-01

    Selenium-derivatized oligonucleotides may facilitate phase determination and high-resolution structure determination for protein–nucleic acid crystallography. The Se atom-specific mutagenesis (SAM) strategy may also enhance the study of nuclease catalysis. The crystal structures of protein–nucleic acid complexes are commonly determined using selenium-derivatized proteins via MAD or SAD phasing. Here, the first protein–nucleic acid complex structure determined using selenium-derivatized nucleic acids is reported. The RNase H–RNA/DNA complex is used as an example to demonstrate the proof of principle. The high-resolution crystal structure indicates that this selenium replacement results in a local subtle unwinding of the RNA/DNA substrate duplex, thereby shifting the RNA scissile phosphate closer to the transition state of the enzyme-catalyzed reaction. It was also observed that the scissile phosphate forms a hydrogen bond to the water nucleophile and helps to position the water molecule in the structure. Consistently, it was discovered that the substitution of a single O atom by a Se atom in a guide DNA sequence can largely accelerate RNase H catalysis. These structural and catalytic studies shed new light on the guide-dependent RNA cleavage.

  15. Lipid monolayer and sparse matrix screening for growing two-dimensional crystals for electron crystallography: methods and examples.

    PubMed

    Yeager, Mark; Dryden, Kelly A; Ganser-Pornillos, Barbie K

    2013-01-01

    Electron microscopy provides an efficient method for rapidly assessing whether a solution of macromolecules is homogeneous and monodisperse. If the macromolecules can be induced to form two-dimensional crystals that are a single layer in thickness, then electron crystallography of frozen-hydrated crystals has the potential of achieving three-dimensional density maps at sub-nanometer or even atomic resolution. Here we describe the lipid monolayer and sparse matrix screening methods for growing two-dimensional crystals and present successful applications to soluble macromolecular complexes: carboxysome shell proteins and HIV CA, respectively. Since it is common to express recombinant proteins with poly-His tags for purification by metal affinity chromatography, the monolayer technique using bulk lipids doped with Ni(2+) lipids has the potential for broad application. Likewise, the sparse matrix method uses screening conditions for three-dimensional crystallization and is therefore of broad applicability.

  16. Enabling X-ray free electron laser crystallography for challenging biological systems from a limited number of crystals

    SciTech Connect

    Uervirojnangkoorn, Monarin; Zeldin, Oliver B.; Lyubimov, Artem Y.; Hattne, Johan; Brewster, Aaron S.; Sauter, Nicholas K.; Brunger, Axel T.; Weis, William I.

    2015-03-17

    There is considerable potential for X-ray free electron lasers (XFELs) to enable determination of macromolecular crystal structures that are difficult to solve using current synchrotron sources. Prior XFEL studies often involved the collection of thousands to millions of diffraction images, in part due to limitations of data processing methods. We implemented a data processing system based on classical post-refinement techniques, adapted to specific properties of XFEL diffraction data. When applied to XFEL data from three different proteins collected using various sample delivery systems and XFEL beam parameters, our method improved the quality of the diffraction data as well as the resulting refined atomic models and electron density maps. Moreover, the number of observations for a reflection necessary to assemble an accurate data set could be reduced to a few observations. These developments will help expand the applicability of XFEL crystallography to challenging biological systems, including cases where sample is limited.

  17. Enabling X-ray free electron laser crystallography for challenging biological systems from a limited number of crystals.

    PubMed

    Uervirojnangkoorn, Monarin; Zeldin, Oliver B; Lyubimov, Artem Y; Hattne, Johan; Brewster, Aaron S; Sauter, Nicholas K; Brunger, Axel T; Weis, William I

    2015-03-17

    There is considerable potential for X-ray free electron lasers (XFELs) to enable determination of macromolecular crystal structures that are difficult to solve using current synchrotron sources. Prior XFEL studies often involved the collection of thousands to millions of diffraction images, in part due to limitations of data processing methods. We implemented a data processing system based on classical post-refinement techniques, adapted to specific properties of XFEL diffraction data. When applied to XFEL data from three different proteins collected using various sample delivery systems and XFEL beam parameters, our method improved the quality of the diffraction data as well as the resulting refined atomic models and electron density maps. Moreover, the number of observations for a reflection necessary to assemble an accurate data set could be reduced to a few observations. These developments will help expand the applicability of XFEL crystallography to challenging biological systems, including cases where sample is limited.

  18. UV-Visible Absorption Spectroscopy Enhanced X-ray Crystallography at Synchrotron and X-ray Free Electron Laser Sources.

    PubMed

    Cohen, Aina E; Doukov, Tzanko; Soltis, Michael S

    2016-01-01

    This review describes the use of single crystal UV-Visible Absorption micro-Spectrophotometry (UV-Vis AS) to enhance the design and execution of X-ray crystallography experiments for structural investigations of reaction intermediates of redox active and photosensitive proteins. Considerations for UV-Vis AS measurements at the synchrotron and associated instrumentation are described. UV-Vis AS is useful to verify the intermediate state of an enzyme and to monitor the progression of reactions within crystals. Radiation induced redox changes within protein crystals may be monitored to devise effective diffraction data collection strategies. An overview of the specific effects of radiation damage on macromolecular crystals is presented along with data collection strategies that minimize these effects by combining data from multiple crystals used at the synchrotron and with the X-ray free electron laser.

  19. Enabling X-ray free electron laser crystallography for challenging biological systems from a limited number of crystals

    DOE PAGES

    Uervirojnangkoorn, Monarin; Zeldin, Oliver B.; Lyubimov, Artem Y.; ...

    2015-03-17

    There is considerable potential for X-ray free electron lasers (XFELs) to enable determination of macromolecular crystal structures that are difficult to solve using current synchrotron sources. Prior XFEL studies often involved the collection of thousands to millions of diffraction images, in part due to limitations of data processing methods. We implemented a data processing system based on classical post-refinement techniques, adapted to specific properties of XFEL diffraction data. When applied to XFEL data from three different proteins collected using various sample delivery systems and XFEL beam parameters, our method improved the quality of the diffraction data as well as themore » resulting refined atomic models and electron density maps. Moreover, the number of observations for a reflection necessary to assemble an accurate data set could be reduced to a few observations. These developments will help expand the applicability of XFEL crystallography to challenging biological systems, including cases where sample is limited.« less

  20. Women's madness J Ussher Women's madness Harvester Wheatsheaf 342pp £13.50 0-7450-0832-1.

    PubMed

    1993-03-10

    Women's Madness: Misogyny or Mental Illness? is an extremely interesting book that examines the experiences of women with depression and the way that society deals with women's mental distress. It looks at two opposing viewpoints.

  1. Effects of macromolecular crowding on nuclear size.

    PubMed

    Rosania, G R; Swanson, J A

    1995-05-01

    The concentration of macromolecules inside cells is high, and the resultant crowding of cytoplasm can be expected to affect many interactions involving macromolecular assemblies. Here, we have examined the effect of solute size and concentration on nuclear volume in saponin-permeabilized macrophages. Nuclei swelled in the presence of small solutes and shrank reversibly in the presence of larger permeant solutes. Remarkably, the smallest solutes capable of shrinking the nucleus were not excluded by the pores in the nuclear envelope. Indeed, nuclei shrank in the presence of such solutes even after the nuclear envelope had been sheared mechanically or permeabilized with detergent. Nuclei extracted with 1% Triton X-100 shrank in the presence of very high concentrations of small solute molecules (30% w/v) as well as in lower concentrations of larger solutes. Consistent with a macromolecular crowding effect, changes in nuclear volume were dependent on solute size and not simply dependent on the colligative properties of solutes or the exclusion of solutes by the nuclear envelope. Solute size-dependent changes in nuclear volume were independent of the chemical nature of the solutes and of the activity of the ions in the buffer. Together, these observations indicate that high concentrations of macromolecules such as those found inside cells can influence the size of the nucleus by directly affecting nuclear structure.

  2. Multiscale macromolecular simulation: role of evolving ensembles.

    PubMed

    Singharoy, A; Joshi, H; Ortoleva, P J

    2012-10-22

    Multiscale analysis provides an algorithm for the efficient simulation of macromolecular assemblies. This algorithm involves the coevolution of a quasiequilibrium probability density of atomic configurations and the Langevin dynamics of spatial coarse-grained variables denoted order parameters (OPs) characterizing nanoscale system features. In practice, implementation of the probability density involves the generation of constant OP ensembles of atomic configurations. Such ensembles are used to construct thermal forces and diffusion factors that mediate the stochastic OP dynamics. Generation of all-atom ensembles at every Langevin time step is computationally expensive. Here, multiscale computation for macromolecular systems is made more efficient by a method that self-consistently folds in ensembles of all-atom configurations constructed in an earlier step, history, of the Langevin evolution. This procedure accounts for the temporal evolution of these ensembles, accurately providing thermal forces and diffusions. It is shown that efficiency and accuracy of the OP-based simulations is increased via the integration of this historical information. Accuracy improves with the square root of the number of historical timesteps included in the calculation. As a result, CPU usage can be decreased by a factor of 3-8 without loss of accuracy. The algorithm is implemented into our existing force-field based multiscale simulation platform and demonstrated via the structural dynamics of viral capsomers.

  3. Macromolecular recognition in the Protein Data Bank

    PubMed Central

    Janin, Joël; Rodier, Francis; Chakrabarti, Pinak; Bahadur, Ranjit P.

    2007-01-01

    Crystal structures deposited in the Protein Data Bank illustrate the diversity of biological macromolecular recognition: transient interactions in protein–protein and protein–DNA complexes and permanent assemblies in homodimeric proteins. The geometric and physical chemical properties of the macromolecular interfaces that may govern the stability and specificity of recognition are explored in complexes and homodimers compared with crystal-packing interactions. It is found that crystal-packing interfaces are usually much smaller; they bury fewer atoms and are less tightly packed than in specific assemblies. Standard-size interfaces burying 1200–2000 Å2 of protein surface occur in protease–inhibitor and antigen–antibody complexes that assemble with little or no conformation changes. Short-lived electron-transfer complexes have small interfaces; the larger size of the interfaces observed in complexes involved in signal transduction and homodimers correlates with the presence of conformation changes, often implicated in biological function. Results of the CAPRI (critical assessment of predicted interactions) blind prediction experiment show that docking algorithms efficiently and accurately predict the mode of assembly of proteins that do not change conformation when they associate. They perform less well in the presence of large conformation changes and the experiment stimulates the development of novel procedures that can handle such changes. PMID:17164520

  4. Magnetic macromolecular cross linked enzyme aggregates (CLEAs) of glucoamylase.

    PubMed

    Nadar, Shamraja S; Rathod, Virendra K

    2016-02-01

    This work illustrates the preparation of magnetic macromolecular glucoamylase CLEAs using dialdehydic pectin, as a cross linker instead of traditional glutaraldehyde. The effect of precipitators type and amount, cross linker concentration, cross linking time and amount of amino functionalized magnetic nanoparticles (AFMNs) on glucoamylase activity was studied. Glucoamylase magnetic macromolecular CLEAs prepared by precipitation in presence of AFMNs by ammonium sulfate were subsequently cross linked by dialdehydic pectin. After cross-linked by pectin, 95.4% activity recovery was achieved in magnetic macromolecular CLEAs, whereas in case of glutaraldehyde cross linker, 85.3% activity recovery was achieved. Magnetic macromolecular CLEAs showed 2.91 and 1.27 folds higher thermal stability as compared to free and magnetic glutaraldehyde CLEAs. In kinetics study, magnetic macromolecular CLEAs retained same Km values, whereas magnetic glutaraldehyde CLEAs showed higher Km value than free enzyme. The porous structure of magnetic macromolecular CLEAs was not only enhanced mass transfer toward macromolecular substrates, but also showed compression resistance for 5 consecutive cycles which was checked in terms of effectiveness factor. At the end, in reusability study; magnetic macromolecular CLEAs were retained 84% activity after 10(th) cycle without leaching of enzyme which is 22% higher than traditional magnetic CLEAs.

  5. X-Ray Crystallography: One Century of Nobel Prizes

    ERIC Educational Resources Information Center

    Galli, Simona

    2014-01-01

    In 2012, the United Nations General Assembly declared 2014 the International Year of Crystallography. Throughout the year 2014 and beyond, all the crystallographic associations and societies active all over the world are organizing events to attract the wider public toward crystallography and the numerous topics to which it is deeply interlinked.…

  6. In meso in situ serial X-ray crystallography of soluble and membrane proteins

    PubMed Central

    Huang, Chia-Ying; Olieric, Vincent; Ma, Pikyee; Panepucci, Ezequiel; Diederichs, Kay; Wang, Meitian; Caffrey, Martin

    2015-01-01

    The lipid cubic phase (LCP) continues to grow in popularity as a medium in which to generate crystals of membrane (and soluble) proteins for high-resolution X-ray crystallographic structure determination. To date, the PDB includes 227 records attributed to the LCP or in meso method. Among the listings are some of the highest profile membrane proteins, including the β2-adrenoreceptor–Gs protein complex that figured in the award of the 2012 Nobel Prize in Chemistry to Lefkowitz and Kobilka. The most successful in meso protocol to date uses glass sandwich crystallization plates. Despite their many advantages, glass plates are challenging to harvest crystals from. However, performing in situ X-ray diffraction measurements with these plates is not practical. Here, an alternative approach is described that provides many of the advantages of glass plates and is compatible with high-throughput in situ measurements. The novel in meso in situ serial crystallography (IMISX) method introduced here has been demonstrated with AlgE and PepT (alginate and peptide transporters, respectively) as model integral membrane proteins and with lysozyme as a test soluble protein. Structures were solved by molecular replacement and by experimental phasing using bromine SAD and native sulfur SAD methods to resolutions ranging from 1.8 to 2.8 Å using single-digit microgram quantities of protein. That sulfur SAD phasing worked is testament to the exceptional quality of the IMISX diffraction data. The IMISX method is compatible with readily available, inexpensive materials and equipment, is simple to implement and is compatible with high-throughput in situ serial data collection at macromolecular crystallography synchrotron beamlines worldwide. Because of its simplicity and effectiveness, the IMISX approach is likely to supplant existing in meso crystallization protocols. It should prove particularly attractive in the area of ligand screening for drug discovery and development. PMID

  7. In meso in situ serial X-ray crystallography of soluble and membrane proteins.

    PubMed

    Huang, Chia Ying; Olieric, Vincent; Ma, Pikyee; Panepucci, Ezequiel; Diederichs, Kay; Wang, Meitian; Caffrey, Martin

    2015-06-01

    The lipid cubic phase (LCP) continues to grow in popularity as a medium in which to generate crystals of membrane (and soluble) proteins for high-resolution X-ray crystallographic structure determination. To date, the PDB includes 227 records attributed to the LCP or in meso method. Among the listings are some of the highest profile membrane proteins, including the β2-adrenoreceptor-Gs protein complex that figured in the award of the 2012 Nobel Prize in Chemistry to Lefkowitz and Kobilka. The most successful in meso protocol to date uses glass sandwich crystallization plates. Despite their many advantages, glass plates are challenging to harvest crystals from. However, performing in situ X-ray diffraction measurements with these plates is not practical. Here, an alternative approach is described that provides many of the advantages of glass plates and is compatible with high-throughput in situ measurements. The novel in meso in situ serial crystallography (IMISX) method introduced here has been demonstrated with AlgE and PepT (alginate and peptide transporters, respectively) as model integral membrane proteins and with lysozyme as a test soluble protein. Structures were solved by molecular replacement and by experimental phasing using bromine SAD and native sulfur SAD methods to resolutions ranging from 1.8 to 2.8 Å using single-digit microgram quantities of protein. That sulfur SAD phasing worked is testament to the exceptional quality of the IMISX diffraction data. The IMISX method is compatible with readily available, inexpensive materials and equipment, is simple to implement and is compatible with high-throughput in situ serial data collection at macromolecular crystallography synchrotron beamlines worldwide. Because of its simplicity and effectiveness, the IMISX approach is likely to supplant existing in meso crystallization protocols. It should prove particularly attractive in the area of ligand screening for drug discovery and development.

  8. Developing advanced x-ray scattering methods combined with crystallography and computation

    PubMed Central

    Perry, J. Jefferson P.; Tainer, John A.

    2013-01-01

    The extensive use of small angle x-ray scattering (SAXS) over the last few years is rapidly providing new insights into protein interactions, complex formation and conformational states in solution. This SAXS methodology allows for detailed biophysical quantification of samples of interest. Initial analyses provide a judgment of sample quality, revealing the potential presence of aggregation, the overall extent of folding or disorder, the radius of gyration, maximum particle dimensions and oligomerization state. Structural characterizations include ab initio approaches from SAXS data alone, and when combined with previously determined crystal/NMR, atomistic modeling can further enhance structural solutions and assess validity. This combination can provide definitions of architectures, spatial organizations of protein domains within a complex, including those not determined by crystallography or NMR, as well as defining key conformational states of a protein interaction. SAXS is not generally constrained by macromolecule size, and the rapid collection of data in a 96-well plate format provides methods to screen sample conditions. This includes screening for co-factors, substrates, differing protein or nucleotide partners or small molecule inhibitors, to more fully characterize the variations within assembly states and key conformational changes. Such analyses may be useful for screening constructs and conditions to determine those most likely to promote crystal growth of a complex under study. Moreover, these high throughput structural determinations can be leveraged to define how polymorphisms affect assembly formations and activities. This is in addition to potentially providing architectural characterizations of complexes and interactions for systems biology-based research, and distinctions in assemblies and interactions in comparative genomics. Thus, SAXS combined with crystallography/NMR and computation provides a unique set of tools that should be considered

  9. Multi-crystal Anomalous Diffraction for Low-resolution Macromolecular Phasing

    SciTech Connect

    Q Liu; Z Zhang; W Hendrickson

    2011-12-31

    Multiwavelength anomalous diffraction (MAD) and single-wavelength anomalous diffraction (SAD) are the two most commonly used methods for de novo determination of macromolecular structures. Both methods rely on the accurate extraction of anomalous signals; however, because of factors such as poor intrinsic order, radiation damage, inadequate anomalous scatterers, poor diffraction quality and other noise-causing factors, the anomalous signal from a single crystal is not always good enough for structure solution. In this study, procedures for extracting more accurate anomalous signals by merging data from multiple crystals are devised and tested. SAD phasing tests were made with a relatively large (1456 ordered residues) poorly diffracting (d{sub min} = 3.5 {angstrom}) selenomethionyl protein (20 Se). It is quantified that the anomalous signal, success in substructure determination and accuracy of phases and electron-density maps all improve with an increase in the number of crystals used in merging. Structure solutions are possible when no single crystal can support structural analysis. It is proposed that such multi-crystal strategies may be broadly useful when only weak anomalous signals are available.

  10. The design of the MAD Design Program

    SciTech Connect

    Niederer, J.

    1992-12-31

    The study of long term stability in particle accelerators has long been served by a group of widely circulated computer programs. The progress in these programs has mirrored the growth and versatility in accelerator size, complexity, and purpose, as well as evolving technologies in computing software and hardware. A number of large accelerator projects during the last decade were designed with the aid of physics programs either written for, or tailored for the project at hand, each invariably benefiting from contributions of previous workers. This paper outlines the recent history of of expample of an accelerator lattice model tool kit, the Methodical Accelerator Design (MAD) Program, which has tried to knit together this collective wisdom of the accelerator community, The ideas behind the software design of the program itself are traced here; the accelerator physics contents and origins are thoroughly documented elsewhere. These informal notes have a Brookhaven flavor, in part because of early BNL efforts to generalize the ways that technical problems are organized and presented to computers. Some recent BNL applications not covered in the extensive CERN documentation are also included.

  11. The design of the MAD Design Program

    SciTech Connect

    Niederer, J.

    1992-01-01

    The study of long term stability in particle accelerators has long been served by a group of widely circulated computer programs. The progress in these programs has mirrored the growth and versatility in accelerator size, complexity, and purpose, as well as evolving technologies in computing software and hardware. A number of large accelerator projects during the last decade were designed with the aid of physics programs either written for, or tailored for the project at hand, each invariably benefiting from contributions of previous workers. This paper outlines the recent history of of expample of an accelerator lattice model tool kit, the Methodical Accelerator Design (MAD) Program, which has tried to knit together this collective wisdom of the accelerator community, The ideas behind the software design of the program itself are traced here; the accelerator physics contents and origins are thoroughly documented elsewhere. These informal notes have a Brookhaven flavor, in part because of early BNL efforts to generalize the ways that technical problems are organized and presented to computers. Some recent BNL applications not covered in the extensive CERN documentation are also included.

  12. Simulation of MAD Cow Disease Propagation

    NASA Astrophysics Data System (ADS)

    Magdoń-Maksymowicz, M. S.; Maksymowicz, A. Z.; Gołdasz, J.

    Computer simulation of dynamic of BSE disease is presented. Both vertical (to baby) and horizontal (to neighbor) mechanisms of the disease spread are considered. The game takes place on a two-dimensional square lattice Nx×Ny = 1000×1000 with initial population randomly distributed on the net. The disease may be introduced either with the initial population or by a spontaneous development of BSE in an item, at a small frequency. Main results show a critical probability of the BSE transmission above which the disease is present in the population. This value is vulnerable to possible spatial clustering of the population and it also depends on the mechanism responsible for the disease onset, evolution and propagation. A threshold birth rate below which the population is extinct is seen. Above this threshold the population is disease free at equilibrium until another birth rate value is reached when the disease is present in population. For typical model parameters used for the simulation, which may correspond to the mad cow disease, we are close to the BSE-free case.

  13. Mad about Physics: Braintwisters, Paradoxes, and Curiosities

    NASA Astrophysics Data System (ADS)

    Jargodzki, Christopher; Potter, Franklin

    2000-11-01

    Why is there eight times more ice in Antarctica than in the Arctic? Why can you warm your hands by blowing gently, and cool your hands by blowing hard? Why would a pitcher scuff a baseball?Which weighs more-a pound of feathers or a pound of iron? Let science experts Christopher Jargodzki and Franklin Potter guide you through the curiosities of physics and you'll find the answers to these and hundreds of other quirky conundrums. You'll discover why sounds carry well over water (especially in the summer), how a mouse can be levitated in a magnetic field, why backspin is so important when shooting a basketball, and whether women are indeed as strong as men. With nearly 400 questions and answers on everything from race cars to jumping fleas to vanishing elephants, Mad about Physics presents a comprehensive collection of braintwisters and paradoxes that will challenge and entertain even the brainiest of science lovers. Whether you're a physicist by trade or just want to give your brain a power workout, this collection of intriguing and unusual physics challenges will send you on a highly entertaining ride that reveals the relevance of physics in our everyday lives.

  14. Crystallography of Alumina-YAG-Eutectic

    NASA Technical Reports Server (NTRS)

    Farmer, Serene C.; Sayir, Ali; Dickerson, Robert M.; Matson, Lawrence E.

    2000-01-01

    Multiple descriptions of the alumina-YAG eutectic crystallography appear in the ceramic literature. The orientation between two phases in a eutectic system has direct impact on residual stress, morphology, microstructural stability, and high temperature mechanical properties. A study to demonstrate that the different crystallographic relationships can be correlated with different growth constraints was undertaken. Fibers produced by Laser-Heated Float Zone (LHFZ) and Edge-defined Film-fed Growth (EFG) were examined. A map of the orientation relationship between Al2O3 and Y3Al5O12 and their relationship to the fiber growth axis as a function of pull rate are presented. Regions in which a single orientation predominates are identified.

  15. Busting out of crystallography's Sisyphean prison

    NASA Astrophysics Data System (ADS)

    Cranswick, L. M. D.

    2008-01-01

    The history of crystallographic computing and use of crystallographic software is one which traces the escape from the drudgery of manual human calculations to a world where the user delegates most of the travail to electronic computers. This review traces the development of small-molecule single-crystal and powder diffraction hardware, starting with the use of Hollerith tabulators of the late 1930's through to today's use of high-performance personal computers. It also emphasizes that the main challenge for current and future crystallography computing is not that of hardware development, or even specific scientific challenges, but rather in maintaining a critical mass of human expertise with which these computational challenges can be undertaken.

  16. Nitric Oxide Release Part I. Macromolecular Scaffolds

    PubMed Central

    Riccio, Daniel A.; Schoenfisch, Mark H.

    2012-01-01

    Summary The roles of nitric oxide (NO) in physiology and pathophysiology merit the use of NO as a therapeutic for certain biomedical applications. Unfortunately, limited NO payloads, too rapid NO release, and the lack of targeted NO delivery have hindered the clinical utility of NO gas and low molecular weight NO donor compounds. A wide-variety of NO-releasing macromolecular scaffolds has thus been developed to improve NO’s pharmacological potential. In this tutorial review, we provide an overview of the most promising NO release scaffolds including protein, organic, inorganic, and hybrid organic-inorganic systems. The NO release vehicles selected for discussion were chosen based on their enhanced NO storage, tunable NO release characteristics, and potential as therapeutics. PMID:22362355

  17. Macromolecular crowding explains overflow metabolism in cells

    PubMed Central

    Vazquez, Alexei; Oltvai, Zoltán N.

    2016-01-01

    Overflow metabolism is a metabolic phenotype of cells characterized by mixed oxidative phosphorylation (OxPhos) and fermentative glycolysis in the presence of oxygen. Recently, it was proposed that a combination of a protein allocation constraint and a higher proteome fraction cost of energy generation by OxPhos relative to fermentation form the basis of overflow metabolism in the bacterium, Escherichia coli. However, we argue that the existence of a maximum or optimal macromolecular density is another essential requirement. Here we re-evaluate our previous theory of overflow metabolism based on molecular crowding following the proteomic fractions formulation. We show that molecular crowding is a key factor in explaining the switch from OxPhos to overflow metabolism. PMID:27484619

  18. Simulation and display of macromolecular complexes

    NASA Technical Reports Server (NTRS)

    Nir, S.; Garduno, R.; Rein, R.; Macelroy, R. D.

    1977-01-01

    In association with an investigation of the interaction of proteins with DNA and RNA, an interactive computer program for building, manipulating, and displaying macromolecular complexes has been designed. The system provides perspective, planar, and stereoscopic views on the computer terminal display, as well as views for standard and nonstandard observer locations. The molecule or its parts may be rotated and/or translated in any direction; bond connections may be added or removed by the viewer. Molecular fragments may be juxtaposed in such a way that given bonds are aligned, and given planes and points coincide. Another subroutine provides for the duplication of a given unit such as a DNA or amino-acid base.

  19. Macromolecular recognition and macroscopic interactions by cyclodextrins.

    PubMed

    Harada, Akira; Takashima, Yoshinori

    2013-10-01

    Herein macromolecular recognition by cyclodextrins (CDs) is summarized. Recognition of macromolecules by CDs is classified as main-chain recognition or side-chain recognition. We found that CDs form inclusion complexes with various polymers with high selectivity. Polyrotaxanes in which many CDs are entrapped in a polymer chain were prepared. Tubular polymers were prepared from the polyrotaxanes. CDs were found to recognize side-chains of polymers selectively. CD host polymers were found to form gels with guest polymers in water. These gels showed self-healing properties. When azobenzene was used as a guest, the gel showed sol-gel transition by photoirradiation. When ferrocene was used, redox-responsive gels were obtained. Macroscopic self-assembly through molecular recognition has been discovered. Photoswitchable gel association and dissociation have been observed.

  20. MAD1L1 Arg558His and MAD2L1 Leu84Met interaction with smoking increase the risk of colorectal cancer

    PubMed Central

    Zhong, Rong; Chen, Xiaohua; Chen, Xueqin; Zhu, Beibei; Lou, Jiao; Li, Jiaoyuan; Shen, Na; Yang, Yang; Gong, Yajie; Zhu, Ying; Yuan, Jing; Xia, Xiaoping; Miao, Xiaoping

    2015-01-01

    The spindle assembly checkpoint (SAC) has been established as an important mechanism of driving aneuploidy, which occurs at a high frequency in the colorectal tumorigenesis. Two important components of SAC are MAD1L1 and MAD2L1, which function together in an interactive manner to initiate the checkpoint signal. We hypothesize that genetic variants in the binding domains of MAD1L1 and MAD2L1 may modulate protein structures and eventually contribute to CRC susceptibility. A case-control study including 710 CRC cases and 735 controls was performed to examine MAD1L1 Arg558His and MAD2L1 Leu84Met’s conferring susceptibility to CRC. Cytokinesis-block micronucleus cytome assays were applied to assess the effect of two functional variants on chromosomal instability (CIN). Significant associations with CRC risk were observed for MAD1L1 Arg558His (OR = 1.38,95% CI: 1.09–1.75) and MAD2L1 Leu84Met in a dominant model (OR = 1.48,95% CI: 1.09–2.01). Moreover, significant multiplicative gene-smoking interactions were found in MAD1L1 Arg558His (P = 0.019) and MAD2L184 Leu/Met (P = 0.016) to enhance CRC risk. Additionally, the frequencies of lymphocytic micro-nucleated binucleated cells for MAD1L1 Arg558His polymorphism were significantly different in the exposed group (P = 0.013), but not in the control group. The study emphasized that MAD1L1 Arg558His and MAD2L1 Leu84Met can significantly interact with smoking to enhance CRC risk, and the genetic effects of MAD1L1Arg558His on CIN need to be further clarified in follow-up studies. PMID:26183163

  1. Functional Characterization of OsMADS18, a Member of the AP1/SQUA Subfamily of MADS Box Genes1[w

    PubMed Central

    Fornara, Fabio; Pařenicová, Lucie; Falasca, Giuseppina; Pelucchi, Nilla; Masiero, Simona; Ciannamea, Stefano; Lopez-Dee, Zenaida; Altamura, Maria Maddalena; Colombo, Lucia; Kater, Martin M.

    2004-01-01

    MADS box transcription factors controlling flower development have been isolated and studied in a wide variety of organisms. These studies have shown that homologous MADS box genes from different species often have similar functions. OsMADS18 from rice (Oryza sativa) belongs to the phylogenetically defined AP1/SQUA group. The MADS box genes of this group have functions in plant development, like controlling the transition from vegetative to reproductive growth, determination of floral organ identity, and regulation of fruit maturation. In this paper we report the functional analysis of OsMADS18. This rice MADS box gene is widely expressed in rice with its transcripts accumulated to higher levels in meristems. Overexpression of OsMADS18 in rice induced early flowering, and detailed histological analysis revealed that the formation of axillary shoot meristems was accelerated. Silencing of OsMADS18 using an RNA interference approach did not result in any visible phenotypic alteration, indicating that OsMADS18 is probably redundant with other MADS box transcription factors. Surprisingly, overexpression of OsMADS18 in Arabidopsis caused a phenotype closely resembling the ap1 mutant. We show that the ap1 phenotype is not caused by down-regulation of AP1 expression. Yeast two-hybrid experiments showed that some of the natural partners of AP1 interact with OsMADS18, suggesting that the OsMADS18 overexpression phenotype in Arabidopsis is likely to be due to the subtraction of AP1 partners from active transcription complexes. Thus, when compared to AP1, OsMADS18 during evolution seems to have conserved the mechanistic properties of protein-protein interactions, although it cannot complement the AP1 function. PMID:15299121

  2. In meso in situ serial X-ray crystallography of soluble and membrane proteins

    SciTech Connect

    Huang, Chia-Ying; Olieric, Vincent; Ma, Pikyee; Panepucci, Ezequiel; Diederichs, Kay; Wang, Meitian; Caffrey, Martin

    2015-05-14

    macromolecular crystallography synchrotron beamlines worldwide. Because of its simplicity and effectiveness, the IMISX approach is likely to supplant existing in meso crystallization protocols. It should prove particularly attractive in the area of ligand screening for drug discovery and development.

  3. Phylogenetic Diversity in the Macromolecular Composition of Microalgae

    PubMed Central

    Finkel, Zoe V.; Follows, Mick J.; Liefer, Justin D.; Brown, Chris M.; Benner, Ina; Irwin, Andrew J.

    2016-01-01

    The elemental stoichiometry of microalgae reflects their underlying macromolecular composition and influences competitive interactions among species and their role in the food web and biogeochemistry. Here we provide a new estimate of the macromolecular composition of microalgae using a hierarchical Bayesian analysis of data compiled from the literature. The median macromolecular composition of nutrient-sufficient exponentially growing microalgae is 32.2% protein, 17.3% lipid, 15.0% carbohydrate, 17.3% ash, 5.7% RNA, 1.1% chlorophyll-a and 1.0% DNA as percent dry weight. Our analysis identifies significant phylogenetic differences in macromolecular composition undetected by previous studies due to small sample sizes and the large inherent variability in macromolecular pools. The phylogenetic differences in macromolecular composition lead to variations in carbon-to-nitrogen ratios that are consistent with independent observations. These phylogenetic differences in macromolecular and elemental composition reflect adaptations in cellular architecture and biochemistry; specifically in the cell wall, the light harvesting apparatus, and storage pools. PMID:27228080

  4. Phylogenetic Diversity in the Macromolecular Composition of Microalgae.

    PubMed

    Finkel, Zoe V; Follows, Mick J; Liefer, Justin D; Brown, Chris M; Benner, Ina; Irwin, Andrew J

    2016-01-01

    The elemental stoichiometry of microalgae reflects their underlying macromolecular composition and influences competitive interactions among species and their role in the food web and biogeochemistry. Here we provide a new estimate of the macromolecular composition of microalgae using a hierarchical Bayesian analysis of data compiled from the literature. The median macromolecular composition of nutrient-sufficient exponentially growing microalgae is 32.2% protein, 17.3% lipid, 15.0% carbohydrate, 17.3% ash, 5.7% RNA, 1.1% chlorophyll-a and 1.0% DNA as percent dry weight. Our analysis identifies significant phylogenetic differences in macromolecular composition undetected by previous studies due to small sample sizes and the large inherent variability in macromolecular pools. The phylogenetic differences in macromolecular composition lead to variations in carbon-to-nitrogen ratios that are consistent with independent observations. These phylogenetic differences in macromolecular and elemental composition reflect adaptations in cellular architecture and biochemistry; specifically in the cell wall, the light harvesting apparatus, and storage pools.

  5. Kinetic protein crystallography: a tool to watch proteins in action

    SciTech Connect

    Bourgeois, D.; Weik, M.

    2009-04-14

    Many proteins function in the crystalline state, making crystallography a tool that, beside structure, can address mechanism. By initiating biological turnover in the crystal, transient structural species form, which may be filmed 'on the fly' by Laue diffraction or captured by trapping methods. These strategies are jointly referred to as 'kinetic crystallography'. In this article, we review the general concepts of kinetic crystallography in the context of the conformational energy landscape of a protein. Whereas Laue diffraction is best suited to the investigation of cyclic, ultra-fast and light-triggered reactions, trapping approaches, on the other hand, are applicable to a wider range of biological systems but require care to avoid artefacts. Complementary methods - mainly UV/visible single-crystal spectroscopy - have proven essential to design, interpret and validate kinetic crystallography experiments. Achievements in the field as well as remaining puzzling questions are considered through the examination of recently published work: real-time-resolved crystallography of dimeric haemoglobin based on pump-probe Laue diffraction, temperature-trapping crystallography of acetylcholinesterase based on photo- and radio-induced ligand cleavage, and lattice-trapping crystallography of superoxide reductase based on product soaking and the combined use of X-ray diffraction and Raman spectroscopy.

  6. Selenium Derivatization of Nucleic Acids for Crystallography

    SciTech Connect

    Jiang,J.; Sheng, J.; Carrasco, N.; Huang, Z.

    2007-01-01

    The high-resolution structure of the DNA (5'-GTGTACA-C-3') with the selenium derivatization at the 2'-position of T2 was determined via MAD and SAD phasing. The selenium-derivatized structure (1.28 {angstrom} resolution) with the 2'-Se modification in the minor groove is isomorphorous to the native structure (2.0 {angstrom}). To directly compare with the conventional bromine derivatization, we incorporated bromine into the 5-postion of T4, determined the bromine-derivatized DNA structure at 1.5 {angstrom} resolution, and found that the local backbone torsion angles and solvent hydration patterns were altered in the structure with the Br incorporation in the major groove. Furthermore, while the native and Br-derivatized DNAs needed over a week to form reasonable-size crystals, we observed that the Se-derivatized DNAs grew crystals overnight with high-diffraction quality, suggesting that the Se derivatization facilitated the crystal formation. In addition, the Se-derivatized DNA sequences crystallized under a broader range of buffer conditions, and generally had a faster crystal growth rate. Our experimental results indicate that the selenium derivatization of DNAs may facilitate the determination of nucleic acid X-ray crystal structures in phasing and high-quality crystal growth. In addition, our results suggest that the Se derivatization can be an alternative to the conventional Br derivatization.

  7. Possibilities for serial femtosecond crystallography sample delivery at future light sourcesa)

    PubMed Central

    Chavas, L. M. G.; Gumprecht, L.; Chapman, H. N.

    2015-01-01

    Serial femtosecond crystallography (SFX) uses X-ray pulses from free-electron laser (FEL) sources that can outrun radiation damage and thereby overcome long-standing limits in the structure determination of macromolecular crystals. Intense X-ray FEL pulses of sufficiently short duration allow the collection of damage-free data at room temperature and give the opportunity to study irreversible time-resolved events. SFX may open the way to determine the structure of biological molecules that fail to crystallize readily into large well-diffracting crystals. Taking advantage of FELs with high pulse repetition rates could lead to short measurement times of just minutes. Automated delivery of sample suspensions for SFX experiments could potentially give rise to a much higher rate of obtaining complete measurements than at today's third generation synchrotron radiation facilities, as no crystal alignment or complex robotic motions are required. This capability will also open up extensive time-resolved structural studies. New challenges arise from the resulting high rate of data collection, and in providing reliable sample delivery. Various developments for fully automated high-throughput SFX experiments are being considered for evaluation, including new implementations for a reliable yet flexible sample environment setup. Here, we review the different methods developed so far that best achieve sample delivery for X-ray FEL experiments and present some considerations towards the goal of high-throughput structure determination with X-ray FELs. PMID:26798808

  8. Getting More From Less: Correlated Single-Crystal Spectroscopy and X-ray Crystallography at the NSLS (456th Brookhaven Lecture)

    SciTech Connect

    Orville, Allen

    2010-04-21

    By integrating different techniques to collect complementary data at beam line X26C of the National Syncrotron Light Source (NSLS), Allen Orville and his colleagues of the Macromolecular Crystallography Research Resource are providing new insights into the structures of macromolecules. During the 456th Brookhaven Lecture, on Wednesday, April 21st, Orville will describe his approach and his findings in a talk entitled “Getting More From Less: Correlated Singe-Crystal Spectroscopy and X-Ray Crystallography at the NSLS,” beginning at 4 p.m. in Berkner Hall. Refreshments will be offered before and after the lecture. During his talk, Orville will discuss his field of protein crystallography, reviewing its landmark discoveries and explaining new ways of using the NSLS and, in the future, the NSLS-II, to build on those discoveries. Allen Orville took his Ph.D. in biology from the University of Minnesota in 1997. After completing a postdoc with the Howard Hughes Medical Institute at the University of Oregon, 1997-2000, he began an assistant professor at the Georgia Institute of Technology. Orville joined BNL's Biology Department in 2006 as an associate biophysicist and was promoted to biophysicist in 2008.

  9. The interaction of banana MADS-box protein MuMADS1 and ubiquitin-activating enzyme E-MuUBA in post-harvest banana fruit.

    PubMed

    Liu, Ju-Hua; Zhang, Jing; Jia, Cai-Hong; Zhang, Jian-Bin; Wang, Jia-Shui; Yang, Zi-Xian; Xu, Bi-Yu; Jin, Zhi-Qiang

    2013-01-01

    KEY MESSAGE : The interaction of MuMADS1 and MuUBA in banana was reported, which will help us to understand the mechanism of the MADS-box gene in regulating banana fruit development and ripening. The ubiquitin-activating enzyme E1 gene fragment MuUBA was obtained from banana (Musa acuminata L.AAA) fruit by the yeast two-hybrid method using the banana MADS-box gene MuMADS1 as bait and 2-day post-harvest banana fruit cDNA library as prey. MuMADS1 interacted with MuUBA. The interaction of MuMADS1 and MuUBA in vivo was further proved by bimolecular fluorescence complementation assay. Real-time quantitative PCR evaluation of MuMADS1 and MuUBA expression patterns in banana showed that they are highly expressed in the ovule 4 stage, but present in low levels in the stem, which suggests a simultaneously differential expression action exists for both MuMADS1 and MuUBA in different tissues and developmental fruits. MuMADS1 and MuUBA expression was highly stimulated by exogenous ethylene and suppressed by 1-methylcyclopropene. These results indicated that MuMADS1 and MuUBA were co-regulated by ethylene and might play an important role in post-harvest banana fruit ripening.

  10. Distinct responses to reduplicated chromosomes require distinct Mad2 responses.

    PubMed

    Stormo, Benjamin M; Fox, Donald T

    2016-05-09

    Duplicating chromosomes once each cell cycle produces sister chromatid pairs, which separate accurately at anaphase. In contrast, reduplicating chromosomes without separation frequently produces polytene chromosomes, a barrier to accurate mitosis. Chromosome reduplication occurs in many contexts, including: polytene tissue development, polytene tumors, and following treatment with mitosis-blocking chemotherapeutics. However, mechanisms responding to or resolving polyteny during mitosis are poorly understood. Here, using Drosophila, we uncover two distinct reduplicated chromosome responses. First, when reduplicated polytene chromosomes persist into metaphase, an anaphase delay prevents tissue malformation and apoptosis. Second, reduplicated polytene chromosomes can also separate prior to metaphase through a spindle-independent mechanism termed Separation-Into-Recent-Sisters (SIRS). Both reduplication responses require the spindle assembly checkpoint protein Mad2. While Mad2 delays anaphase separation of metaphase polytene chromosomes, Mad2's control of overall mitotic timing ensures efficient SIRS. Our results pinpoint mechanisms enabling continued proliferation after genome reduplication, a finding with implications for cancer progression and prevention.

  11. [Madness in the German cinema (1913-1933].

    PubMed

    Aulas, J J

    1980-01-01

    During these twenty years, from 1913 to 1933, of the history of the German cinema, the cinematographic representation of madness varies according to the fluctuations of the social and economical background. The political and ideological chaos of the immediate post-war years was symbolized in the allegorical imagery of unreason in the expressionist cinema. The same equivalence, the same symbolization can be found in the cinema of the thirties when the crash of Wall-Street foretells a crisis like the former. On the contrary in the course of the so-called "relative stabilization" (1924-1929) the meaning of the representation of madness is totally different from the representation of the previous period. At this period of economical restoration, madness which could henceforth be cured on the psychoanalyst's couch (acc. G. W. Pabst's film: "Geheimnisse einer Seele") became the symbol of the absolute power rediscovered by Germany.

  12. A new MADS-box gene (IbMADS10) from sweet potato (Ipomoea batatas (L.) Lam) is involved in the accumulation of anthocyanin.

    PubMed

    Lalusin, Antonio G; Nishita, Koichi; Kim, Sung-Hyung; Ohta, Masaru; Fujimura, Tatsuhito

    2006-01-01

    A new MADS-box gene designated as IbMADS10 was cloned and its expression was characterized from sweet potato (Ipomoea batatas (L.) Lam.) cv. Beniazuma. The deduced amino acid sequence of the gene indicated high homology with members of the MADS-box family of transcription factors. IbMADS10 shares high amino acid sequence similarity with the DEFH28 of Antirrhinum majus (64%) and with BpMADS4 of Betula pendula (61%) of the SQUA subfamily. Southern blot analysis revealed that the IbMADS10 is present in one or low copy number in the sweet potato genome. The gene is specifically expressed in the pigmented tissues such as in the flower bud, in the pink and in red roots, and hence, it was speculated that the IbMADS10 gene might be correlated with anthocyanin biosynthesis in sweet potato. RNA blot expression of the anthocyanin biosynthesis genes encoding for CHS, CHI, F3H, DFR, ANS and UFTG carried out in the tissues where the IbMADS10 gene was expressed revealed similar transcript levels in all tissues where the IbMADS10 gene is highly expressed, indicating that the IbMADS10 gene is highly correlated with the anthocyanin biosynthesis genes. Another important aspect is the pigmented phenotypes of transgenic calli that ectopically express the IbMADS10 gene, thereby supporting its involvement in the developmental regulation of pigment formation. Tissue printing result further strengthens the hypothesis that the IbMADS10 gene is indeed involved in anthocyanin pigmentation in sweet potato. As the purpose of the IbMADS10 gene is pigmentation, its function, therefore, resembles that of the transparent testa (tt) genes of Arabidopsis.

  13. Biological Macromolecular Structures Data from the RCSB Protein Data Bank (RCSB PDB)

    DOE Data Explorer

    The Research Collaboratory for Structural Bioinformatics (RCSB) is a non-profit consortium that works to improve understanding of the function of biological systems through the study of the 3-D structure of biological macromolecules. The RCSB PDB is one of three sites serving as deposition, data processing, and distribution sites of the Protein Data Bank Archive. Each site provides its own view of the primary data, thus providing a variety of tools and resources for the global community. RCSB is also the official keeper for the PDB archive, with sole access authority to the PDB archive directory structure and contents. The RCSB PDB Information Portal for Biological Macromolecular Structures offers online tools for search and retrieval, for visualizing structures, for depositing, validating, or downloading data, news and highlights, a discussion forum, and links to other areas of related research. The PDB archive is a repository of atomic coordinates and other information describing proteins and other important biological macromolecules. Structural biologists use methods such as X-ray crystallography, NMR spectroscopy, and cryo-electron microscopy to determine the location of each atom relative to each other in the molecule. They then deposit this information, which is then annotated and publicly released into the archive by the wwPDB. Results can be viewed as 3-D images or models.

  14. 33 CFR 147.839 - Mad Dog Truss Spar Platform safety zone.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 2 2010-07-01 2010-07-01 false Mad Dog Truss Spar Platform... SECURITY (CONTINUED) OUTER CONTINENTAL SHELF ACTIVITIES SAFETY ZONES § 147.839 Mad Dog Truss Spar Platform safety zone. (a) Description. Mad Dog Truss Spar Platform, Green Canyon 782 (GC 782), located at...

  15. 33 CFR 147.839 - Mad Dog Truss Spar Platform safety zone.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 2 2011-07-01 2011-07-01 false Mad Dog Truss Spar Platform... SECURITY (CONTINUED) OUTER CONTINENTAL SHELF ACTIVITIES SAFETY ZONES § 147.839 Mad Dog Truss Spar Platform safety zone. (a) Description. Mad Dog Truss Spar Platform, Green Canyon 782 (GC 782), located at...

  16. 33 CFR 147.839 - Mad Dog Truss Spar Platform safety zone.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 33 Navigation and Navigable Waters 2 2013-07-01 2013-07-01 false Mad Dog Truss Spar Platform... SECURITY (CONTINUED) OUTER CONTINENTAL SHELF ACTIVITIES SAFETY ZONES § 147.839 Mad Dog Truss Spar Platform safety zone. (a) Description. Mad Dog Truss Spar Platform, Green Canyon 782 (GC 782), located at...

  17. 33 CFR 147.839 - Mad Dog Truss Spar Platform safety zone.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 2 2014-07-01 2014-07-01 false Mad Dog Truss Spar Platform... SECURITY (CONTINUED) OUTER CONTINENTAL SHELF ACTIVITIES SAFETY ZONES § 147.839 Mad Dog Truss Spar Platform safety zone. (a) Description. Mad Dog Truss Spar Platform, Green Canyon 782 (GC 782), located at...

  18. 33 CFR 147.839 - Mad Dog Truss Spar Platform safety zone.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 2 2012-07-01 2012-07-01 false Mad Dog Truss Spar Platform... SECURITY (CONTINUED) OUTER CONTINENTAL SHELF ACTIVITIES SAFETY ZONES § 147.839 Mad Dog Truss Spar Platform safety zone. (a) Description. Mad Dog Truss Spar Platform, Green Canyon 782 (GC 782), located at...

  19. Genome-wide analysis of the MADS-box gene family in Brachypodium distachyon.

    PubMed

    Wei, Bo; Zhang, Rong-Zhi; Guo, Juan-Juan; Liu, Dan-Mei; Li, Ai-Li; Fan, Ren-Chun; Mao, Long; Zhang, Xiang-Qi

    2014-01-01

    MADS-box genes are important transcription factors for plant development, especially floral organogenesis. Brachypodium distachyon is a model for biofuel plants and temperate grasses such as wheat and barley, but a comprehensive analysis of MADS-box family proteins in Brachypodium is still missing. We report here a genome-wide analysis of the MADS-box gene family in Brachypodium distachyon. We identified 57 MADS-box genes and classified them into 32 MIKC(c)-type, 7 MIKC*-type, 9 Mα, 7 Mβ and 2 Mγ MADS-box genes according to their phylogenetic relationships to the Arabidopsis and rice MADS-box genes. Detailed gene structure and motif distribution were then studied. Investigation of their chromosomal localizations revealed that Brachypodium MADS-box genes distributed evenly across five chromosomes. In addition, five pairs of type II MADS-box genes were found on synteny blocks derived from whole genome duplication blocks. We then performed a systematic expression analysis of Brachypodium MADS-box genes in various tissues, particular floral organs. Further detection under salt, drought, and low-temperature conditions showed that some MADS-box genes may also be involved in abiotic stress responses, including type I genes. Comparative studies of MADS-box genes among Brachypodium, rice and Arabidopsis showed that Brachypodium had fewer gene duplication events. Taken together, this work provides useful data for further functional studies of MADS-box genes in Brachypodium distachyon.

  20. John Locke on madness: redressing the intellectualist bias.

    PubMed

    Charland, Louis C

    2014-06-01

    Locke is famous for defining madness as an intellectual disorder in the realm of ideas. Numerous commentators take this to be his main and only contribution to the history of psychiatry. However, a detailed exegetical review of all the relevant textual evidence suggests that this intellectualist interpretation of Locke's account of madness is both misleading and incomplete. Affective states of various sorts play an important role in that account and are in fact primordial in the determination of human conduct generally. Locke's legacy in this domain must therefore be revised and the intellectualist bias that dominates discussions of his views must be redressed.

  1. [The representation of madness in William Shakespeare's characters].

    PubMed

    Stompe, Thomas; Ritter, Kristina; Friedmann, Alexander

    2006-08-01

    Shakespeare is one of the great creators of human characters of the 16(th) century. Like for many of his contemporaries madness was a central topic of his work. The first part of this paper discusses the sociocultural environment and the semantic field of madness in the Elizabethan age, which forms the background for Shakespeare's characters. In the second part we try to analyze the clinical pictures of the fictive characters of Othello, Hamlet, Lear and Macbeth. While we find melancholy, delusions and hallucinations, other diseases such as schizophrenia are missing entirely. Schizophrenia only appears in the literature more than two hundred years later, in the beginning of modern age.

  2. MADNESS: A Multiresolution, Adaptive Numerical Environment for Scientific Simulation

    SciTech Connect

    Harrison, Robert J.; Beylkin, Gregory; Bischoff, Florian A.; Calvin, Justus A.; Fann, George I.; Fosso-Tande, Jacob; Galindo, Diego; Hammond, Jeff R.; Hartman-Baker, Rebecca; Hill, Judith C.; Jia, Jun; Kottmann, Jakob S.; Yvonne Ou, M-J.; Pei, Junchen; Ratcliff, Laura E.; Reuter, Matthew G.; Richie-Halford, Adam C.; Romero, Nichols A.; Sekino, Hideo; Shelton, William A.; Sundahl, Bryan E.; Thornton, W. Scott; Valeev, Edward F.; Vázquez-Mayagoitia, Álvaro; Vence, Nicholas; Yanai, Takeshi; Yokoi, Yukina

    2016-01-01

    MADNESS (multiresolution adaptive numerical environment for scientific simulation) is a high-level software environment for solving integral and differential equations in many dimensions that uses adaptive and fast harmonic analysis methods with guaranteed precision based on multiresolution analysis and separated representations. Underpinning the numerical capabilities is a powerful petascale parallel programming environment that aims to increase both programmer productivity and code scalability. This paper describes the features and capabilities of MADNESS and briefly discusses some current applications in chemistry and several areas of physics.

  3. MADNESS: A Multiresolution, Adaptive Numerical Environment for Scientific Simulation

    SciTech Connect

    Harrison, Robert J.; Beylkin, Gregory; Bischoff, Florian A.; Calvin, Justus A.; Fann, George I.; Fosso-Tande, Jacob; Galindo, Diego; Hammond, Jeff R.; Hartman-Baker, Rebecca; Hill, Judith C.; Jia, Jun; Kottmann, Jakob S.; Yvonne Ou, M-J.; Pei, Junchen; Ratcliff, Laura E.; Reuter, Matthew G.; Richie-Halford, Adam C.; Romero, Nichols A.; Sekino, Hideo; Shelton, William A.; Sundahl, Bryan E.; Thornton, W. Scott; Valeev, Edward F.; Vázquez-Mayagoitia, Álvaro; Vence, Nicholas; Yanai, Takeshi; Yokoi, Yukina

    2016-01-01

    We present MADNESS (multiresolution adaptive numerical environment for scientific simulation) that is a high-level software environment for solving integral and differential equations in many dimensions that uses adaptive and fast harmonic analysis methods with guaranteed precision that are based on multiresolution analysis and separated representations. Underpinning the numerical capabilities is a powerful petascale parallel programming environment that aims to increase both programmer productivity and code scalability. This paper describes the features and capabilities of MADNESS and briefly discusses some current applications in chemistry and several areas of physics.

  4. Phase Equilibria and Crystallography of Ceramic Oxides

    PubMed Central

    Wong-Ng, W.; Roth, R. S.; Vanderah, T. A.; McMurdie, H. F.

    2001-01-01

    Research in phase equilibria and crystallography has been a tradition in the Ceramics Division at National Bureau of Standards/National Institute of Standatrds and Technology (NBS/NIST) since the early thirties. In the early years, effort was concentrated in areas of Portland cement, ceramic glazes and glasses, instrument bearings, and battery materials. In the past 40 years, a large portion of the work was related to electronic materials, including ferroelectrics, piezoelectrics, ionic conductors, dielectrics, microwave dielectrics, and high-temperature superconductors. As a result of the phase equilibria studies, many new compounds have been discovered. Some of these discoveries have had a significant impact on US industry. Structure determinations of these new phases have often been carried out as a joint effort among NBS/NIST colleagues and also with outside collaborators using both single crystal and neutron and x-ray powder diffraction techniques. All phase equilibria diagrams were included in Phase Diagrams for Ceramists, which are collaborative publications between The American Ceramic Society (ACerS) and NBS/NIST. All x-ray powder diffraction patterns have been included in the Powder Diffraction File (PDF). This article gives a brief account of the history of the development of the phase equilibria and crystallographic research on ceramic oxides in the Ceramics Division. Represented systems, particularly electronic materials, are highlighted. PMID:27500068

  5. Crystallography, evolution, and the structure of viruses.

    PubMed

    Rossmann, Michael G

    2012-03-16

    My undergraduate education in mathematics and physics was a good grounding for graduate studies in crystallographic studies of small organic molecules. As a postdoctoral fellow in Minnesota, I learned how to program an early electronic computer for crystallographic calculations. I then joined Max Perutz, excited to use my skills in the determination of the first protein structures. The results were even more fascinating than the development of techniques and provided inspiration for starting my own laboratory at Purdue University. My first studies on dehydrogenases established the conservation of nucleotide-binding structures. Having thus established myself as an independent scientist, I could start on my most cherished ambition of studying the structure of viruses. About a decade later, my laboratory had produced the structure of a small RNA plant virus and then, in another six years, the first structure of a human common cold virus. Many more virus structures followed, but soon it became essential to supplement crystallography with electron microscopy to investigate viral assembly, viral infection of cells, and neutralization of viruses by antibodies. A major guide in all these studies was the discovery of evolution at the molecular level. The conservation of three-dimensional structure has been a recurring theme, from my experiences with Max Perutz in the study of hemoglobin to the recognition of the conserved nucleotide-binding fold and to the recognition of the jelly roll fold in the capsid protein of a large variety of viruses.

  6. Crystallography, Evolution, and the Structure of Viruses

    PubMed Central

    Rossmann, Michael G.

    2012-01-01

    My undergraduate education in mathematics and physics was a good grounding for graduate studies in crystallographic studies of small organic molecules. As a postdoctoral fellow in Minnesota, I learned how to program an early electronic computer for crystallographic calculations. I then joined Max Perutz, excited to use my skills in the determination of the first protein structures. The results were even more fascinating than the development of techniques and provided inspiration for starting my own laboratory at Purdue University. My first studies on dehydrogenases established the conservation of nucleotide-binding structures. Having thus established myself as an independent scientist, I could start on my most cherished ambition of studying the structure of viruses. About a decade later, my laboratory had produced the structure of a small RNA plant virus and then, in another six years, the first structure of a human common cold virus. Many more virus structures followed, but soon it became essential to supplement crystallography with electron microscopy to investigate viral assembly, viral infection of cells, and neutralization of viruses by antibodies. A major guide in all these studies was the discovery of evolution at the molecular level. The conservation of three-dimensional structure has been a recurring theme, from my experiences with Max Perutz in the study of hemoglobin to the recognition of the conserved nucleotide-binding fold and to the recognition of the jelly roll fold in the capsid protein of a large variety of viruses. PMID:22318719

  7. Fourier-Space Crystallography as Group Cohomology

    NASA Astrophysics Data System (ADS)

    Rabson, David; Fisher, Benji

    2001-03-01

    David Mermin has argued persuasively that the theoretical significance of quasicrystals lies not so much in relaxing the requirement of periodicity as in replacing exact identity of a density function (e.g., electronic or mass) under symmetry operations with indistinguishability of correlation functions, as expressed in Fourier space.(N.D. Mermin, Phys. Stat. Sol. (a) 151), 275 (1995) and references. After reviewing the formalism of Fourier-space crystallography (phase functions and gauge transformations), we present a new formulation in the language of cohomology of groups. First we reexpress the classification of space groups in terms of a first cohomology group; we then show how recent work by König and Mermin(A. König and N.D. Mermin, Am. J. Phys. 68), 525 (2000). on band sticking in nonsymmorphic crystals derives naturally from a first homology group and discuss its connection to a second cohomology group. The new language lets us prove generally several theorems previously known only in special cases. Finally, we let the listener decide whether we're just ``speaking prose.''(N.D. Mermin, Rev. Mod. Phys. 64), 3 (1992).

  8. Phase Equilibria and Crystallography of Ceramic Oxides.

    PubMed

    Wong-Ng, W; Roth, R S; Vanderah, T A; McMurdie, H F

    2001-01-01

    Research in phase equilibria and crystallography has been a tradition in the Ceramics Division at National Bureau of Standards/National Institute of Standatrds and Technology (NBS/NIST) since the early thirties. In the early years, effort was concentrated in areas of Portland cement, ceramic glazes and glasses, instrument bearings, and battery materials. In the past 40 years, a large portion of the work was related to electronic materials, including ferroelectrics, piezoelectrics, ionic conductors, dielectrics, microwave dielectrics, and high-temperature superconductors. As a result of the phase equilibria studies, many new compounds have been discovered. Some of these discoveries have had a significant impact on US industry. Structure determinations of these new phases have often been carried out as a joint effort among NBS/NIST colleagues and also with outside collaborators using both single crystal and neutron and x-ray powder diffraction techniques. All phase equilibria diagrams were included in Phase Diagrams for Ceramists, which are collaborative publications between The American Ceramic Society (ACerS) and NBS/NIST. All x-ray powder diffraction patterns have been included in the Powder Diffraction File (PDF). This article gives a brief account of the history of the development of the phase equilibria and crystallographic research on ceramic oxides in the Ceramics Division. Represented systems, particularly electronic materials, are highlighted.

  9. Molecular cloning, identification, and chromosomal localization of two MADS box genes in peach (Prunus persica).

    PubMed

    Zhang, Lin; Xu, Yong; Ma, Rongcai

    2008-06-01

    MADS box proteins play an important role in floral development. To find genes involved in the floral transition of Prunus species, cDNAs for two MADS box genes, PpMADS1 and PpMADS10, were cloned using degenerate primers and 5'- and 3'-RACE based on the sequence database of P. persica and P. dulcis. The full length of PpMADS1 cDNA is 1,071 bp containing an open reading frame (ORF) of 717 bp and coding for a polypeptide of 238 amino acid residues. The full length of PpMADS10 cDNA is 937 bp containing an ORF of 633 bp and coding for a polypeptide of 210 amino acid residues. Sequence comparison revealed that PpMADS1 and PpMADS10 were highly homologous to genes AP1 and PI in Arabidopsis, respectively. Phylogenetic analysis indicated that PpMADS1 belongs to the euAP1 clade of class A, and PpMADS10 is a member of GLO/PI clade of class B. RT-PCR analysis showed that PpMADS1 was expressed in sepal, petal, carpel, and fruit, which was slightly different from the expression pattern of AP1; PpMADS10 was expressed in petal and stamen, which shared the same expression pattern as PI. Using selective mapping strategy, PpMADS1 was assigned onto the Bin1:50 on the G1 linkage group between the markers MCO44 and TSA2, and PpMADS10 onto the Bin1:73 on the same linkage group between the markers Lap-1 and FGA8. Our results provided the basis for further dissection of the two MADS box gene function.

  10. Transcriptome-wide analysis of the MADS-box gene family in the orchid Erycina pusilla.

    PubMed

    Lin, Choun-Sea; Hsu, Chen-Tran; Liao, De-Chih; Chang, Wan-Jung; Chou, Ming-Lun; Huang, Yao-Ting; Chen, Jeremy J W; Ko, Swee-Suak; Chan, Ming-Tsair; Shih, Ming-Che

    2016-01-01

    Orchids exhibit a range of unique flower shapes and are a valuable ornamental crop. MADS-box transcription factors are key regulatory components in flower initiation and development. Changing the flower shape and flowering time can increase the value of the orchid in the ornamental horticulture industry. In this study, 28 MADS-box genes were identified from the transcriptome database of the model orchid Erycina pusilla. The full-length genomic sequences of these MADS-box genes were obtained from BAC clones. Of these, 27 were MIKC-type EpMADS (two truncated forms) and one was a type I EpMADS. Eleven EpMADS genes contained introns longer than 10 kb. Phylogenetic analysis classified the 24 MIKC(c) genes into nine subfamilies. Three specific protein motifs, AG, FUL and SVP, were identified and used to classify three subfamilies. The expression profile of each EpMADS gene correlated with its putative function. The phylogenetic analysis was highly correlated with the protein domain identification and gene expression results. Spatial expression of EpMADS6, EpMADS12 and EpMADS15 was strongly detected in the inflorescence meristem, floral bud and seed via in situ hybridization. The subcellular localization of the 28 EpMADS proteins was also investigated. Although EpMADS27 lacks a complete MADS-box domain, EpMADS27-YFP was localized in the nucleus. This characterization of the orchid MADS-box family genes provides useful information for both orchid breeding and studies of flowering and evolution.

  11. Macromolecular Crystal Growth by Means of Microfluidics

    NASA Technical Reports Server (NTRS)

    vanderWoerd, Mark; Ferree, Darren; Spearing, Scott; Monaco, Lisa; Molho, Josh; Spaid, Michael; Brasseur, Mike; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    We have performed a feasibility study in which we show that chip-based, microfluidic (LabChip(TM)) technology is suitable for protein crystal growth. This technology allows for accurate and reliable dispensing and mixing of very small volumes while minimizing bubble formation in the crystallization mixture. The amount of (protein) solution remaining after completion of an experiment is minimal, which makes this technique efficient and attractive for use with proteins, which are difficult or expensive to obtain. The nature of LabChip(TM) technology renders it highly amenable to automation. Protein crystals obtained in our initial feasibility studies were of excellent quality as determined by X-ray diffraction. Subsequent to the feasibility study, we designed and produced the first LabChip(TM) device specifically for protein crystallization in batch mode. It can reliably dispense and mix from a range of solution constituents into two independent growth wells. We are currently testing this design to prove its efficacy for protein crystallization optimization experiments. In the near future we will expand our design to incorporate up to 10 growth wells per LabChip(TM) device. Upon completion, additional crystallization techniques such as vapor diffusion and liquid-liquid diffusion will be accommodated. Macromolecular crystallization using microfluidic technology is envisioned as a fully automated system, which will use the 'tele-science' concept of remote operation and will be developed into a research facility for the International Space Station as well as on the ground.

  12. The solvent component of macromolecular crystals

    SciTech Connect

    Weichenberger, Christian X.; Kantardjieff, Katherine; Rupp, Bernhard

    2015-04-30

    On average, the mother liquor or solvent and its constituents occupy about 50% of a macromolecular crystal. Ordered as well as disordered solvent components need to be accurately accounted for in modelling and refinement, often with considerable complexity. The mother liquor from which a biomolecular crystal is grown will contain water, buffer molecules, native ligands and cofactors, crystallization precipitants and additives, various metal ions, and often small-molecule ligands or inhibitors. On average, about half the volume of a biomolecular crystal consists of this mother liquor, whose components form the disordered bulk solvent. Its scattering contributions can be exploited in initial phasing and must be included in crystal structure refinement as a bulk-solvent model. Concomitantly, distinct electron density originating from ordered solvent components must be correctly identified and represented as part of the atomic crystal structure model. Herein, are reviewed (i) probabilistic bulk-solvent content estimates, (ii) the use of bulk-solvent density modification in phase improvement, (iii) bulk-solvent models and refinement of bulk-solvent contributions and (iv) modelling and validation of ordered solvent constituents. A brief summary is provided of current tools for bulk-solvent analysis and refinement, as well as of modelling, refinement and analysis of ordered solvent components, including small-molecule ligands.

  13. Macromolecular components of tomato fruit pectin.

    PubMed

    Fishman, M L; Gross, K C; Gillespie, D T; Sondey, S M

    1989-10-01

    Chelate and alkaline-soluble pectin extracted from cell walls of pericarp tissue from mature green, turning, and red ripe (Lycopersicon esculentum Mill.) fruit (cv. Rutgers), were studied by high-performance size-exclusion chromatography. Computer-aided curve fitting of the chromatograms to a series of Gaussian-shaped components revealed that pectin from all fractions was composed of a linear combination of five macromolecular-sized species. The relative sizes of these macromolecules as obtained from their radii of gyration were 1:2:4:8:16. Dialysis against 0.05 M NaCl induced partial dissociation of the biopolymers. Apparently, the weight fraction of smaller sized species increased at the expense of larger ones. Also, the dissociation produced low-molecular-weight fragments. Behavior in the presence of 0.05 M NaCl led to the conclusion that cell wall pectin acted as if it were an aggregated mosaic, held together at least partially through noncovalent interactions.

  14. The regulation of MADS-box gene expression during ripening of banana and their regulatory interation with ethylene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    MADS-box genes (MaMADS1-6), potential components of the developmental control of ripening have been cloned from Grand Nain banana cultivar. Similarity of these genes to tomato LeRIN is very low and neither MaMADS2 nor MaMADS1 complement the tomato rin mutation. Nevertheless, the expression patterns...

  15. TrMADS3, a new MADS-box gene, from a perennial species Taihangia rupestris (Rosaceae) is upregulated by cold and experiences seasonal fluctuation in expression level.

    PubMed

    Du, Xiaoqiu; Xiao, Qiying; Zhao, Ran; Wu, Feng; Xu, Qijiang; Chong, Kang; Meng, Zheng

    2008-06-01

    In many temperate perennial plants, floral transition is initiated in the first growth season but the development of flower is arrested during the winter to ensure production of mature flowers in the next spring. The molecular mechanisms of the process remain poorly understood with few well-characterized regulatory genes. Here, a MADS-box gene, named as TrMADS3, was isolated from the overwintering inflorescences of Taihangia rupestris, a temperate perennial in the rose family. Phylogenetic analysis reveals that TrMADS3 is more closely related to the homologs of the FLOWERING LOCUS C lineage than to any of the other MIKC-type MADS-box lineages known from Arabidopsis. The TrMADS3 transcripts are extensively distributed in inflorescences, roots, and leaves during the winter. In controlled conditions, the TrMADS3 expression level is upregulated by a chilling exposure for 1 to 2 weeks and remains high for a longer period of time in warm conditions after cold treatment. In situ hybridization reveals that TrMADS3 is predominantly expressed in the vegetative and reproductive meristems. Ectopic expression of TrMADS3 in Arabidopsis promotes seed germination on the media containing relatively high NaCl or mannitol concentrations. These data indicate that TrMADS3 in a perennial species might have its role in both vegetative and reproductive meristems in response to cold.

  16. Banana Ovate Family Protein MaOFP1 and MADS-Box Protein MuMADS1 Antagonistically Regulated Banana Fruit Ripening

    PubMed Central

    Hu, Wei; Miao, Hongxia; Zhang, Jianbin; Jia, Caihong; Wang, Zhuo; Xu, Biyu; Jin, Zhiqiang

    2015-01-01

    The ovate family protein named MaOFP1 was identified in banana (Musa acuminata L.AAA) fruit by a yeast two-hybrid (Y2H) method using the banana MADS-box gene MuMADS1 as bait and a 2 day postharvest (DPH) banana fruit cDNA library as prey. The interaction between MuMADS1 and MaOFP1 was further confirmed by Y2H and Bimolecular Fluorescence Complementation (BiFC) methods, which showed that the MuMADS1 K domain interacted with MaOFP1. Real-time quantitative PCR evaluation of MuMADS1 and MaOFP1 expression patterns in banana showed that they are highly expressed in 0 DPH fruit, but present in low levels in the stem, which suggests that simultaneous but different expression patterns exist for both MuMADS1 and MaOFP1 in different tissues and developing fruits. Meanwhile, MuMADS1 and MaOFP1 expression was highly stimulated and greatly suppressed, respectively, by exogenous ethylene. In contrast, MaOFP1 expression was highly stimulated while MuMADS1 was greatly suppressed by the ethylene competitor 1-methylcyclopropene (1-MCP). These results indicate that MuMADS1 and MaOFP1 are antagonistically regulated by ethylene and might play important roles in postharvest banana fruit ripening. PMID:25886169

  17. Genome-wide identification and analysis of the MADS-box gene family in apple.

    PubMed

    Tian, Yi; Dong, Qinglong; Ji, Zhirui; Chi, Fumei; Cong, Peihua; Zhou, Zongshan

    2015-01-25

    The MADS-box gene family is one of the most widely studied families in plants and has diverse developmental roles in flower pattern formation, gametophyte cell division and fruit differentiation. Although the genome-wide analysis of this family has been performed in some species, little is known regarding MADS-box genes in apple (Malus domestica). In this study, 146 MADS-box genes were identified in the apple genome and were phylogenetically clustered into six subgroups (MIKC(c), MIKC*, Mα, Mβ, Mγ and Mδ) with the MADS-box genes from Arabidopsis and rice. The predicted apple MADS-box genes were distributed across all 17 chromosomes at different densities. Additionally, the MADS-box domain, exon length, gene structure and motif compositions of the apple MADS-box genes were analysed. Moreover, the expression of all of the apple MADS-box genes was analysed in the root, stem, leaf, flower tissues and five stages of fruit development. All of the apple MADS-box genes, with the exception of some genes in each group, were expressed in at least one of the tissues tested, which indicates that the MADS-box genes are involved in various aspects of the physiological and developmental processes of the apple. To the best of our knowledge, this report describes the first genome-wide analysis of the apple MADS-box gene family, and the results should provide valuable information for understanding the classification, cloning and putative functions of this family.

  18. Reviewing the Link between Creativity and Madness: A Postmodern Perspective

    ERIC Educational Resources Information Center

    Koh, Caroline

    2006-01-01

    Researchers on creativity and psychology have long been fascinated with the high incidence of psychotic behavior amongst geniuses and individuals of exceptional creativity. The aims of this paper are first, to review the existing findings for a better insight into the socio-contextual factors underpinning the mad genius conundrum, and secondly, to…

  19. March Money Madness: The Coaches vs. the Professors

    ERIC Educational Resources Information Center

    Cottle, Thomas

    2009-01-01

    College basketball's March Madness has come at a time when one prominent coach's salary has been held up for inspection. Apparently, the fact that the $1.6-million annual income of the University of Connecticut's Jim Calhoun makes him the highest-paid public employee in his state has rankled some people. Or are they more upset that he was caught…

  20. Cassandra in the Classroom: Teaching and Moral Madness

    ERIC Educational Resources Information Center

    Santoro, Doris A.

    2017-01-01

    Moral madness is a symptom of the moral violence experienced by teachers who are expected to exercise responsibility for their students and their work, but whose moral voice is misrecognized as self-interest and whose moral agency is suppressed. I conduct a feminist ethical analysis of the figure of Cassandra to examine the ways in which teachers…

  1. Globalizing Education, Educating the Local: How Method Made Us Mad

    ERIC Educational Resources Information Center

    Edwards, Richard; Carney, Stephen; Ambrosius, Ulla; Lauder, Hugh

    2012-01-01

    This article presents the authors' review of "Globalizing education, educating the local: how method made us mad," by Ian Stronach. In the opening chapter of their highly influential 1997 book "Education Research Undone: The Postmodern Embrace," Ian Stronach and Maggie MacLure draw upon the work of Derrida to argue for…

  2. E-Science and Protein Crystallography

    SciTech Connect

    Miller, Laniece E.; Powell, James E. Jr.

    2012-08-09

    Dr. Zoe Fisher is the instrument scientist for the Protein Crystallography Station (PCS) at the Los Alamos Neutron Science Center's (LANSC) Lujan Neutron Scattering Center. She helps schedule researchers who intend to use the instrument to collect data, and provides in depth support for their activities. Users submit proposals for beam/instrument time via LANSCE proposal review system. In 2012, there were about 20 proposals submitted for this instrument. The instrument scientists review the proposals online. Accepted proposals are scheduled via an aggregate calendar which takes into account staff and resource availability, and the scientist is notified via email when their proposal is accepted and their requested time is scheduled. The entire PCS data acquisition and processing workflow is streamlined through various locally developed and commercial software packages. One 24 hour period produces one 200 Mb file, giving a total of maybe 2-5 Gb of data for the entire run. This data is then transferred to a hard disk in Dr. Fisher's office where she views the data with the customer and compresses the data to a text format which she sends them. This compression translates the data from an electron density to structural coordinates, which are the products submitted to a protein structure database. As noted above, the raw experimental data is stored onsite at LANSCE on workstations maintained by the instrument scientist. It is extraordinarily rare for anyone to request this data, although the remote possibility of an audit by a funding organization motivates its limited preservation. The raw data is not rigorously backed up, but only stored on a single hard drive. Interestingly, only about 50% of the experimental data actually ends up deposited and described in peer reviewed publications; the data that is not published tends to either not be viable structures or is calibration data. Dr. Fisher does protein crystallography research using both neutron and x-ray scattering

  3. Genome-wide identification and analysis of the MADS-box gene family in sesame.

    PubMed

    Wei, Xin; Wang, Linhai; Yu, Jingyin; Zhang, Yanxin; Li, Donghua; Zhang, Xiurong

    2015-09-10

    MADS-box genes encode transcription factors that play crucial roles in plant growth and development. Sesame (Sesamum indicum L.) is an oil crop that contributes to the daily oil and protein requirements of almost half of the world's population; therefore, a genome-wide analysis of the MADS-box gene family is needed. Fifty-seven MADS-box genes were identified from 14 linkage groups of the sesame genome. Analysis of phylogenetic relationships with Arabidopsis thaliana, Utricularia gibba and Solanum lycopersicum MADS-box genes was performed. Sesame MADS-box genes were clustered into four groups: 28 MIKC(c)-type, 5 MIKC(⁎)-type, 14 Mα-type and 10 Mγ-type. Gene structure analysis revealed from 1 to 22 exons of sesame MADS-box genes. The number of exons in type II MADS-box genes greatly exceeded the number in type I genes. Motif distribution analysis of sesame MADS-box genes also indicated that type II MADS-box genes contained more motifs than type I genes. These results suggested that type II sesame MADS-box genes had more complex structures. By analyzing expression profiles of MADS-box genes in seven sesame transcriptomes, we determined that MIKC(C)-type MADS-box genes played significant roles in sesame flower and seed development. Although most MADS-box genes in the same clade showed similar expression features, some gene functions were diversified from the orthologous Arabidopsis genes. This research will contribute to uncovering the role of MADS-box genes in sesame development.

  4. Interactions of OsMADS1 with Floral Homeotic Genes in Rice Flower Development.

    PubMed

    Hu, Yun; Liang, Wanqi; Yin, Changsong; Yang, Xuelian; Ping, Baozhe; Li, Anxue; Jia, Ru; Chen, Mingjiao; Luo, Zhijing; Cai, Qiang; Zhao, Xiangxiang; Zhang, Dabing; Yuan, Zheng

    2015-09-01

    During reproductive development, rice plants develop unique flower organs which determine the final grain yield. OsMADS1, one of SEPALLATA-like MADS-box genes, has been unraveled to play critical roles in rice floral organ identity specification and floral meristem determinacy. However, the molecular mechanisms underlying interactions of OsMADS1 with other floral homeotic genes in regulating flower development remains largely elusive. In this work, we studied the genetic interactions of OsMADS1 with B-, C-, and D-class genes along with physical interactions among their proteins. We show that the physical and genetic interactions between OsMADS1 and OsMADS3 are essential for floral meristem activity maintenance and organ identity specification; while OsMADS1 physically and genetically interacts with OsMADS58 in regulating floral meristem determinacy and suppressing spikelet meristem reversion. We provided important genetic evidence to support the neofunctionalization of two rice C-class genes (OsMADS3 and OsMADS58) during flower development. Gene expression profiling and quantitative RT-PCR analyses further revealed that OsMADS1 affects the expression of many genes involved in floral identity and hormone signaling, and chromatin immunoprecipitation (ChIP)-PCR assay further demonstrated that OsMADS17 is a direct target gene of OsMADS1. Taken together, these results reveal that OsMADS1 has diversified regulatory functions in specifying rice floral organ and meristem identity, probably through its genetic and physical interactions with different floral homeotic regulators.

  5. Macromolecular networks and intelligence in microorganisms

    PubMed Central

    Westerhoff, Hans V.; Brooks, Aaron N.; Simeonidis, Evangelos; García-Contreras, Rodolfo; He, Fei; Boogerd, Fred C.; Jackson, Victoria J.; Goncharuk, Valeri; Kolodkin, Alexey

    2014-01-01

    Living organisms persist by virtue of complex interactions among many components organized into dynamic, environment-responsive networks that span multiple scales and dimensions. Biological networks constitute a type of information and communication technology (ICT): they receive information from the outside and inside of cells, integrate and interpret this information, and then activate a response. Biological networks enable molecules within cells, and even cells themselves, to communicate with each other and their environment. We have become accustomed to associating brain activity – particularly activity of the human brain – with a phenomenon we call “intelligence.” Yet, four billion years of evolution could have selected networks with topologies and dynamics that confer traits analogous to this intelligence, even though they were outside the intercellular networks of the brain. Here, we explore how macromolecular networks in microbes confer intelligent characteristics, such as memory, anticipation, adaptation and reflection and we review current understanding of how network organization reflects the type of intelligence required for the environments in which they were selected. We propose that, if we were to leave terms such as “human” and “brain” out of the defining features of “intelligence,” all forms of life – from microbes to humans – exhibit some or all characteristics consistent with “intelligence.” We then review advances in genome-wide data production and analysis, especially in microbes, that provide a lens into microbial intelligence and propose how the insights derived from quantitatively characterizing biomolecular networks may enable synthetic biologists to create intelligent molecular networks for biotechnology, possibly generating new forms of intelligence, first in silico and then in vivo. PMID:25101076

  6. Macromolecular Topography Leaps into the Digital Age

    NASA Technical Reports Server (NTRS)

    Lovelace, J.; Bellamy, H.; Snell, E. H.; Borgstahl, G.

    2003-01-01

    A low-cost, real-time digital topography system is under development which will replace x-ray film and nuclear emulsion plates. The imaging system is based on an inexpensive surveillance camera that offers a 1000x1000 array of 8 im square pixels, anti-blooming circuitry, and very quick read out. Currently, the system directly converts x-rays to an image with no phosphor. The system is small and light and can be easily adapted to work with other crystallographic equipment. Preliminary images have been acquired of cubic insulin at the NSLS x26c beam line. NSLS x26c was configured for unfocused monochromatic radiation. Six reflections were collected with stills spaced from 0.002 to 0.001 degrees apart across the entire oscillation range that the reflections were in diffracting condition. All of the reflections were rotated to the vertical to reduce Lorentz and beam related effects. This particular CCD is designed for short exposure applications (much less than 1 sec) and so has a relatively high dark current leading to noisy raw images. The images are processed to remove background and other system noise with a multi-step approach including the use of wavelets, histogram, and mean window filtering. After processing, animations were constructed with the corresponding reflection profile to show the diffraction of the crystal volume vs. the oscillation angle as well as composite images showing the parts of the crystal with the strongest diffraction for each reflection. The final goal is to correlate features seen in reflection profiles captured with fine phi slicing to those seen in the topography images. With this development macromolecular topography finally comes into the digital age.

  7. Macromolecular Antioxidants and Dietary Fiber in Edible Seaweeds.

    PubMed

    Sanz-Pintos, Nerea; Pérez-Jiménez, Jara; Buschmann, Alejandro H; Vergara-Salinas, José Rodrigo; Pérez-Correa, José Ricardo; Saura-Calixto, Fulgencio

    2017-02-01

    Seaweeds are rich in different bioactive compounds with potential uses in drugs, cosmetics and the food industry. The objective of this study was to analyze macromolecular antioxidants or nonextractable polyphenols, in several edible seaweed species collected in Chile (Gracilaria chilensis, Callophyllis concepcionensis, Macrocystis pyrifera, Scytosyphon lomentaria, Ulva sp. and Enteromorpha compressa), including their 1st HPLC characterization. Macromolecular antioxidants are commonly ignored in studies of bioactive compounds. They are associated with insoluble dietary fiber and exhibit significant biological activity, with specific features that are different from those of both dietary fiber and extractable polyphenols. We also evaluated extractable polyphenols and dietary fiber, given their relationship with macromolecular antioxidants. Our results show that macromolecular antioxidants are a major polyphenol fraction (averaging 42% to total polyphenol content), with hydroxycinnamic acids, hydroxybenzoic acids and flavonols being the main constituents. This fraction also showed remarkable antioxidant capacity, as determined by 2 complementary assays. The dietary fiber content was over 50% of dry weight, with some samples exhibiting the target proportionality between soluble and insoluble dietary fiber for adequate nutrition. Overall, our data show that seaweed could be an important source of commonly ignored macromolecular antioxidants.

  8. Solvent isotope effect on macromolecular dynamics in E. coli.

    PubMed

    Jasnin, Marion; Tehei, Moeava; Moulin, Martine; Haertlein, Michael; Zaccai, Giuseppe

    2008-06-01

    Elastic incoherent neutron scattering was used to explore solvent isotope effects on average macromolecular dynamics in vivo. Measurements were performed on living E. coli bacteria containing H2O and D2O, respectively, close to physiological conditions of temperature. Global macromolecular flexibility, expressed as mean square fluctuation (MSF) values, and structural resilience in a free energy potential, expressed as a mean effective force constant, [Symbol: see text]k'[Symbol: see text], were extracted in the two solvent conditions. They referred to the average contribution of all macromolecules inside the cell, mostly dominated by the internal motions of the protein fraction. Flexibility and resilience were both found to be smaller in D2O than in H2O. A difference was expected because the driving forces behind macromolecular stabilization and dynamics are different in H2O and D2O. In D2O, the hydrophobic effect is known to be stronger than in H2O: it favours the burial of non-polar surfaces as well as their van der Waals' packing in the macromolecule cores. This may lead to the observed smaller MSF values. In contrast, in H2O, macromolecules would present more water-exposed surfaces, which would give rise to larger MSF values, in particular at the macromolecular surface. The smaller [Symbol: see text]k'[Symbol: see text] value suggested a larger entropy content in the D2O case due to increased sampling of macromolecular conformational substates.

  9. ZmMADS47 Regulates Zein Gene Transcription through Interaction with Opaque2

    PubMed Central

    Qiao, Zhenyi; Qi, Weiwei; Wang, Qian; Feng, Ya’nan; Yang, Qing; Zhang, Nan; Wang, Shanshan; Tang, Yuanping; Song, Rentao

    2016-01-01

    Zeins, the predominent storage proteins in maize endosperm, are encoded by multiple genes and gene families. However, only a few transcriptional factors for zein gene regulation have been functionally characterized. In this study, a MADS-box protein, namely ZmMADS47, was identified as an Opaque2 (O2) interacting protein via yeast two-hybrid screening. The N-terminal portion of ZmMADS47 contains a nuclear localization signal (NLS), and its C-terminal portion contains a transcriptional activation domain (AD). Interestingly, the transcriptional activation activity is blocked in its full length form, suggesting conformational regulation of the AD. Molecular and RNA-seq analyses of ZmMADS47 RNAi lines revealed down regulation of α-zein and 50-kD γ-zein genes. ZmMADS47 binds the CATGT motif in promoters of these zein genes, but ZmMADS47 alone is not able to transactivate the promoters. However, when both O2 and ZmMADS47 are present, the transactivation of these promoters was greatly enhanced. This enhancement was dependent on the AD function of ZmMADS47 and the interaction between ZmMADS47 and O2, but it was independent from the AD function of O2. Therefore, it appears interaction with O2 activates ZmMADS47 on zein gene promoters. PMID:27077660

  10. Control of the spindle checkpoint by lateral kinetochore attachment and limited Mad1 recruitment

    PubMed Central

    Krefman, Nathaniel I.; Drubin, David G.; Barnes, Georjana

    2015-01-01

    We observed the dynamic recruitment of spindle checkpoint proteins Mad1 and Bub1 to detached kinetochores in budding yeast using real-time live-cell imaging and quantified recruitment in fixed cells. After induced de novo kinetochore assembly at one pair of sister centromeres, Mad1 appeared after the kinetochore protein Mtw1. Detached kinetochores were not associated with the nuclear envelope, so Mad1 does not anchor them to nuclear pore complexes (NPCs). Disrupting Mad1's NPC localization increased Mad1 recruitment to detached sister kinetochores. Conversely, increasing the number of detached kinetochores reduced the amount of Mad1 per detached kinetochore. Bub1 also relocalized completely from the spindle to detached sister centromeres after kinetochore assembly. After their capture by microtubules, Mad1 and Bub1 progressively disappeared from kinetochores. Sister chromatids that arrested with a lateral attachment to one microtubule exhibited half the Mad1 of fully detached sisters. We propose that detached kinetochores compete with alternate binding sites in the nucleus to recruit Mad1 and Bub1 from available pools that are small enough to be fully depleted by just one pair of detached kinetochores and that lateral attachment licenses Mad1 removal from kinetochores after a kinetic delay. PMID:26023090

  11. Involvement of a banana MADS-box transcription factor gene in ethylene-induced fruit ripening.

    PubMed

    Liu, Juhua; Xu, Biyu; Hu, Lifang; Li, Meiying; Su, Wei; Wu, Jing; Yang, Jinghao; Jin, Zhiqiang

    2009-01-01

    To investigate the regulation of MADS-box genes in banana (Musa acuminata L. AAA group cv. Brazilian) fruit development and postharvest ripening, we isolated from banana fruit a MADS-box gene designated MuMADS1. Amino acid alignment indicated MuMADS1 belongs to the AGAMOUS subfamily, and phylogenetic analysis indicates that this gene is most similar to class D MADS-box genes. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that MuMADS1 is expressed in the stamen and pistil of male and female flowers and in the rhizome, the vegetative reproductive organ of the banana plant. In preharvest banana fruit, MuMADS1 is likely expressed throughout banana fruit development. In postharvest banana ripening, MuMADS1 is associated with ethylene biosynthesis. Expression patterns of MuMADS1 during postharvest ripening as determined by real-time RT-PCR suggest that differential expression of MuMADS1 may not only be induced by ethylene biosynthesis associated with postharvest banana ripening, but also may be induced by exogenous ethylene.

  12. Visualization of Myc/Max/Mad family dimers and the competition for dimerization in living cells.

    PubMed

    Grinberg, Asya V; Hu, Chang-Deng; Kerppola, Tom K

    2004-05-01

    Myc and Mad family proteins play opposing roles in the control of cell growth and proliferation. We have visualized the subcellular locations of complexes formed by Myc/Max/Mad family proteins using bimolecular fluorescence complementation (BiFC) analysis. Max was recruited to different subnuclear locations by interactions with Myc versus Mad family members. Complexes formed by Max with Mxi1, Mad3, or Mad4 were enriched in nuclear foci, whereas complexes formed with Myc were more uniformly distributed in the nucleoplasm. Mad4 was localized to the cytoplasm when it was expressed separately, and Mad4 was recruited to the nucleus through dimerization with Max. The cytoplasmic localization of Mad4 was determined by a CRM1-dependent nuclear export signal located near the amino terminus. We compared the relative efficiencies of complex formation among Myc, Max, and Mad family proteins in living cells using multicolor BiFC analysis. Max formed heterodimers with the basic helix-loop-helix leucine zipper (bHLHZIP) domain of Myc (bMyc) more efficiently than it formed homodimers. Replacement of two amino acid residues in the leucine zipper of Max reversed the relative efficiencies of homo- and heterodimerization in cells. Surprisingly, Mad3 formed complexes with Max less efficiently than bMyc, whereas Mad4 formed complexes with Max more efficiently than bMyc. The distinct subcellular locations and the differences between the efficiencies of dimerization with Max indicate that Mad3 and Mad4 are likely to modulate transcription activation by Myc at least in part through distinct mechanisms.

  13. Visualization of Myc/Max/Mad Family Dimers and the Competition for Dimerization in Living Cells†

    PubMed Central

    Grinberg, Asya V.; Hu, Chang-Deng; Kerppola, Tom K.

    2004-01-01

    Myc and Mad family proteins play opposing roles in the control of cell growth and proliferation. We have visualized the subcellular locations of complexes formed by Myc/Max/Mad family proteins using bimolecular fluorescence complementation (BiFC) analysis. Max was recruited to different subnuclear locations by interactions with Myc versus Mad family members. Complexes formed by Max with Mxi1, Mad3, or Mad4 were enriched in nuclear foci, whereas complexes formed with Myc were more uniformly distributed in the nucleoplasm. Mad4 was localized to the cytoplasm when it was expressed separately, and Mad4 was recruited to the nucleus through dimerization with Max. The cytoplasmic localization of Mad4 was determined by a CRM1-dependent nuclear export signal located near the amino terminus. We compared the relative efficiencies of complex formation among Myc, Max, and Mad family proteins in living cells using multicolor BiFC analysis. Max formed heterodimers with the basic helix-loop-helix leucine zipper (bHLHZIP) domain of Myc (bMyc) more efficiently than it formed homodimers. Replacement of two amino acid residues in the leucine zipper of Max reversed the relative efficiencies of homo- and heterodimerization in cells. Surprisingly, Mad3 formed complexes with Max less efficiently than bMyc, whereas Mad4 formed complexes with Max more efficiently than bMyc. The distinct subcellular locations and the differences between the efficiencies of dimerization with Max indicate that Mad3 and Mad4 are likely to modulate transcription activation by Myc at least in part through distinct mechanisms. PMID:15121849

  14. Applied Crystallography - Proceedings of the XVth Conference

    NASA Astrophysics Data System (ADS)

    Morawiec, H.; Ströż, D.

    1993-06-01

    The Table of Contents for the full book PDF is as follows: * Foreword * The International Centre for Diffraction Data and Its Future Developments * The Rietveld Method - A Historical Perspective * Real Structure in Quantitative Powder Diffraction Phase Analysis * Neutron Focusing Optics in Applied Crystallography * The Crystal Structures of Oxygen Deficient Rare Earth Oxides * Short-Range Order in Layer-Structured Ba1-xSrxBi2Nb2O9 Ferroelectrics * Radial Distribution Function as a Tool of Structural Studies on Noncrystalline Materials * Determination of Radial Distribution Function (RDF) of Electrodeposited Cu-Cd Alloys After Annealing * Spheres Packing as a Factor Describing the Local Environment and Structure Stability * X-Ray Stress Measurement of Samples Combined with Diffraction Line Analysis * Phase Stability and Martensitic Transformation in Cu-Zn and Cu-Zn-Al Single Crystals * Order, Defects, Precipitates and the Martensitic Transformation in β Cu-Zn-Al * Effect of γ Precipitates on the Martensitic Transformation in Cu-Zn-Al Alloys * Phase Transitions and Shape Memory Effect in a Thermomechanically Treated NiTi Alloy * Structure of Martensite and Bainite in CuAlMn Alloys * Glass-Ceramics * Mechanism of Texture Formation at the Rolling of Low Stacking Fault Energy Metals and Alloys * Shear Texture of Zinc and the Conditions of Its Occuring * The Development of Texture of ZnAlMg Sheets Depending on Deformation Geometry * Texture Stability of the D.S. NiAlMoCrTi Alloy After Heat Treatment * X-Ray Diffraction Method for Controlling of Texture Evolution in Layers * Texture and Lattice Imperfections Study of Some Low Alloyed Copper Alloys * Selected Examples of the Calculation of the Orientation Distribution Function for Low Crystal and Sample Symmetries * Automatical X-Ray Quantitative Phase Analysis * Application of a PC Computer for Crystallographic Calculations * Electron Diffraction Analysis using a Personal Computer * CA.R.INE Crystallography Version 2

  15. RECENT ADVANCES IN MACROMOLECULAR HYDRODYNAMIC MODELING

    PubMed Central

    Aragon, Sergio R.

    2010-01-01

    The modern implementation of the boundary element method (S.R. Aragon, J. Comput. Chem. 25(2004)1191–12055) has ushered unprecedented accuracy and precision for the solution of the Stokes equations of hydrodynamics with stick boundary conditions. This article begins by reviewing computations with the program BEST of smooth surface objects such as ellipsoids, the dumbbell, and cylinders that demonstrate that the numerical solution of the integral equation formulation of hydrodynamics yields very high precision and accuracy. When BEST is used for macromolecular computations, the limiting factor becomes the definition of the molecular hydrodynamic surface and the implied effective solvation of the molecular surface. Studies on 49 different proteins, ranging in molecular weight from 9 to over 400 kDa, have shown that a model using a 1.1 A thick hydration layer describes all protein transport properties very well for the overwhelming majority of them. In addition, this data implies that the crystal structure is an excellent representation of the average solution structure for most of them. In order to investigate the origin of a handful of significant discrepancies in some multimeric proteins (over −20% observed in the intrinsic viscosity), the technique of Molecular Dynamics simulation (MD) has been incorporated into the research program. A preliminary study of dimeric α-chymotrypsin using approximate implicit water MD is presented. In addition I describe the successful validation of modern protein force fields, ff03 and ff99SB, for the accurate computation of solution structure in explicit water simulation by comparison of trajectory ensemble average computed transport properties with experimental measurements. This work includes small proteins such as lysozyme, ribonuclease and ubiquitin using trajectories around 10 ns duration. We have also studied a 150 kDa flexible monoclonal IgG antibody, trastuzumab, with multiple independent trajectories encompassing over

  16. Polysaccharide-based micro/nanohydrogels for delivering macromolecular therapeutics.

    PubMed

    Ganguly, Kuntal; Chaturvedi, Kiran; More, Uttam A; Nadagouda, Mallikarjuna N; Aminabhavi, Tejraj M

    2014-11-10

    Increased interest in developing novel micro/nanohydrogel based formulations for delivering macromolecular therapeutics has led to multiple choices of biodegradable and biocompatible natural polymers. This interest is largely due to the availability of large number of highly pure recombinant proteins and peptides with tunable properties as well as RNA interference technology that are used in treating some of the deadly diseases that were difficult to be treated by the conventional approaches. The majority of marketed drugs that are now available are in the form of injectables that pose limited patient compliance and convenience. On the other hand, micro/nanotechnology based macromolecular delivery formulations offer many alternative routes of administration and advantages with improved patient compliance and efficient or targeted delivery of intracellular therapeutics to the site of action. This review outlines and critically evaluates the research findings on micro and nano-carrier polymeric hydrogels for the delivery of macromolecular therapeutics.

  17. Crystallography Open Databases and Preservation: a World-wide Initiative

    NASA Astrophysics Data System (ADS)

    Chateigner, Daniel

    In 2003, an international team of crystallographers proposed the Crystallography Open Database (COD), a fully-free collection of crystal structure data, in the aim of ensuring their preservation. With nearly 250000 entries, this database represents a large open set of data for crystallographers, academics and industrials, located at five different places world-wide, and included in Thomson-Reuters’ ISI. As a large step towards data preservation, raw data can now be uploaded along with «digested» structure files, and COD can be questioned by most of the crystallography-linked industrial software. The COD initiative work deserves several other open developments.

  18. Cryo-crystallography: diffraction at low temperature and more.

    PubMed

    Macchi, Piero

    2012-01-01

    This chapter comments on the motivations and the methods of crystallographic studies at low temperature. Cry-crystallography is a brunch of Crystallography, a science that is too often confused with a technique. On the other hand, the scientific background to study crystal phases at low temperature is here provided, together with a survey of many possible techniques that provide complementary or supplementary information. Several applications are discussed, in particular in relation with highly accurate studies like electron density determination or phase transition mechanisms.

  19. Native sulfur/chlorine SAD phasing for serial femtosecond crystallography

    SciTech Connect

    Nakane, Takanori; Song, Changyong; Suzuki, Mamoru; Nango, Eriko; Kobayashi, Jun; Masuda, Tetsuya; Inoue, Shigeyuki; Mizohata, Eiichi; Nakatsu, Toru; Tanaka, Tomoyuki; Tanaka, Rie; Shimamura, Tatsuro; Tono, Kensuke; Joti, Yasumasa; Kameshima, Takashi; Hatsui, Takaki; Yabashi, Makina; Nureki, Osamu; Iwata, So; Sugahara, Michihiro

    2015-11-27

    Sulfur SAD phasing facilitates the structure determination of diverse native proteins using femtosecond X-rays from free-electron lasers via serial femtosecond crystallography. Serial femtosecond crystallography (SFX) allows structures to be determined with minimal radiation damage. However, phasing native crystals in SFX is not very common. Here, the structure determination of native lysozyme from single-wavelength anomalous diffraction (SAD) by utilizing the anomalous signal of sulfur and chlorine at a wavelength of 1.77 Å is successfully demonstrated. This sulfur SAD method can be applied to a wide range of proteins, which will improve the determination of native crystal structures.

  20. Electrostatics in the self-assembly of macromolecular surfactants

    NASA Astrophysics Data System (ADS)

    Mendes, E.; Schädler, V.; Marques, C. M.; Lindner, P.; Wiesner, U.

    1997-12-01

    We report on a small-angle neutron scattering (SANS) study of dilute solutions of neutral and charged polystyrene-polyisoprene (PS-b-PI) diblock copolymers in dimethyl acetamide (DMAc), a polar selective solvent for PS. This is a model macromolecular surfactant system: the low glass temperature of the PI block ensures that thermodynamic equilibrium can be attained; the ionic character of the copolymers is provided by a single sulfonate group at the free chain end of the PI block. The crossover from ionic to non-ionic behaviour is investigated by addition of salt. The results are compared to theoretical predictions for micellization of these model macromolecular systems.

  1. Histone modification and signalling cascade of the dormancy-associated MADS-box gene, PpMADS13-1, in Japanese pear (Pyrus pyrifolia) during endodormancy.

    PubMed

    Saito, Takanori; Bai, Songling; Imai, Tsuyoshi; Ito, Akiko; Nakajima, Ikuko; Moriguchi, Takaya

    2015-06-01

    Dormancy-associated MADS-box (DAM) genes play an important role in endodormancy phase transition. We investigated histone modification in the DAM homolog (PpMADS13-1) from Japanese pear, via chromatin immunoprecipitation-quantitative PCR, to understand the mechanism behind the reduced expression of the PpMADS13-1 gene towards endodormancy release. Our results indicated that the reduction in the active histone mark by trimethylation of the histone H3 tail at lysine 4 contributed to the reduction of PpMADS13-1 expression towards endodormancy release. In contrast, the inactive histone mark by trimethylation of the histone H3 tail at lysine 27 in PpMADS13-1 locus was quite low, and these levels were more similar to a negative control [normal mouse immunoglobulin G (IgG)] than to a positive control (AGAMOUS) in endodormancy phase transition. The loss of histone variant H2A.Z also coincided with the down-regulation of PpMADS13-1. Subsequently, we investigated the PpMADS13-1 signalling cascade and found that PpCBF2, a pear C-repeated binding factor, regulated PpMADS13-1 expression via interaction of PpCBF2 with the 5'-upstream region of PpMADS13-1 by transient reporter assay. Furthermore, transient reporter assay confirmed no interaction between the PpMADS13-1 protein and the pear FLOWERING LOCUS T genes. Taken together, our results enhance understanding of the molecular mechanisms underlying endodormancy phase transition in Japanese pear.

  2. Cryo-electron microscopy and X-ray crystallography: complementary approaches to structural biology and drug discovery.

    PubMed

    Vénien-Bryan, Catherine; Li, Zhuolun; Vuillard, Laurent; Boutin, Jean Albert

    2017-04-01

    The invention of the electron microscope has greatly enhanced the view scientists have of small structural details. Since its implementation, this technology has undergone considerable evolution and the resolution that can be obtained for biological objects has been extended. In addition, the latest generation of cryo-electron microscopes equipped with direct electron detectors and software for the automated collection of images, in combination with the use of advanced image-analysis methods, has dramatically improved the performance of this technique in terms of resolution. While calculating a sub-10 Å resolution structure was an accomplishment less than a decade ago, it is now common to generate structures at sub-5 Å resolution and even better. It is becoming possible to relatively quickly obtain high-resolution structures of biological molecules, in particular large ones (>500 kDa) which, in some cases, have resisted more conventional methods such as X-ray crystallography or nuclear magnetic resonance (NMR). Such newly resolved structures may, for the first time, shed light on the precise mechanisms that are essential for cellular physiological processes. The ability to attain atomic resolution may support the development of new drugs that target these proteins, allowing medicinal chemists to understand the intimacy of the relationship between their molecules and targets. In addition, recent developments in cryo-electron microscopy combined with image analysis can provide unique information on the conformational variability of macromolecular complexes. Conformational flexibility of macromolecular complexes can be investigated using cryo-electron microscopy and multiconformation reconstruction methods. However, the biochemical quality of the sample remains the major bottleneck to routine cryo-electron microscopy-based determination of structures at very high resolution.

  3. In meso in situ serial X-ray crystallography of soluble and membrane proteins at cryogenic temperatures

    PubMed Central

    Huang, Chia-Ying; Olieric, Vincent; Ma, Pikyee; Howe, Nicole; Vogeley, Lutz; Liu, Xiangyu; Warshamanage, Rangana; Weinert, Tobias; Panepucci, Ezequiel; Kobilka, Brian; Diederichs, Kay; Wang, Meitian; Caffrey, Martin

    2016-01-01

    Here, a method for presenting crystals of soluble and membrane proteins growing in the lipid cubic or sponge phase for in situ diffraction data collection at cryogenic temperatures is introduced. The method dispenses with the need for the technically demanding and inefficient crystal-harvesting step that is an integral part of the lipid cubic phase or in meso method of growing crystals. Crystals are dispersed in a bolus of mesophase sandwiched between thin plastic windows. The bolus contains tens to hundreds of crystals, visible with an in-line microscope at macromolecular crystallography synchrotron beamlines and suitably disposed for conventional or serial crystallographic data collection. Wells containing the crystal-laden boluses are removed individually from hermetically sealed glass plates in which crystallization occurs, affixed to pins on goniometer bases and excess precipitant is removed from around the mesophase. The wells are snap-cooled in liquid nitrogen, stored and shipped in Dewars, and manually or robotically mounted on a goniometer in a cryostream for diffraction data collection at 100 K, as is performed routinely with standard, loop-harvested crystals. The method is a variant on the recently introduced in meso in situ serial crystallography (IMISX) method that enables crystallo­graphic measurements at cryogenic temperatures where crystal lifetimes are enormously enhanced whilst reducing protein consumption dramatically. The new approach has been used to generate high-resolution crystal structures of a G-protein-coupled receptor, α-helical and β-barrel transporters and an enzyme as model integral membrane proteins. Insulin and lysozyme were used as test soluble proteins. The quality of the data that can be generated by this method was attested to by performing sulfur and bromine SAD phasing with two of the test proteins. PMID:26894538

  4. In meso in situ serial X-ray crystallography of soluble and membrane proteins at cryogenic temperatures.

    PubMed

    Huang, Chia Ying; Olieric, Vincent; Ma, Pikyee; Howe, Nicole; Vogeley, Lutz; Liu, Xiangyu; Warshamanage, Rangana; Weinert, Tobias; Panepucci, Ezequiel; Kobilka, Brian; Diederichs, Kay; Wang, Meitian; Caffrey, Martin

    2016-01-01

    Here, a method for presenting crystals of soluble and membrane proteins growing in the lipid cubic or sponge phase for in situ diffraction data collection at cryogenic temperatures is introduced. The method dispenses with the need for the technically demanding and inefficient crystal-harvesting step that is an integral part of the lipid cubic phase or in meso method of growing crystals. Crystals are dispersed in a bolus of mesophase sandwiched between thin plastic windows. The bolus contains tens to hundreds of crystals, visible with an in-line microscope at macromolecular crystallography synchrotron beamlines and suitably disposed for conventional or serial crystallographic data collection. Wells containing the crystal-laden boluses are removed individually from hermetically sealed glass plates in which crystallization occurs, affixed to pins on goniometer bases and excess precipitant is removed from around the mesophase. The wells are snap-cooled in liquid nitrogen, stored and shipped in Dewars, and manually or robotically mounted on a goniometer in a cryostream for diffraction data collection at 100 K, as is performed routinely with standard, loop-harvested crystals. The method is a variant on the recently introduced in meso in situ serial crystallography (IMISX) method that enables crystallographic measurements at cryogenic temperatures where crystal lifetimes are enormously enhanced whilst reducing protein consumption dramatically. The new approach has been used to generate high-resolution crystal structures of a G-protein-coupled receptor, α-helical and β-barrel transporters and an enzyme as model integral membrane proteins. Insulin and lysozyme were used as test soluble proteins. The quality of the data that can be generated by this method was attested to by performing sulfur and bromine SAD phasing with two of the test proteins.

  5. Bent Diamond Crystals and Multilayer Based Optics at the new 5-Station Protein Crystallography Beamline 'Cassiopeia' at MAX-lab

    SciTech Connect

    Mammen, Christian B.; Als-Nielsen, Jens; Ursby, Thomas; Thunnissen, Marjolein

    2004-05-12

    A new 5-station beamline for protein crystallography is being commissioned at the Swedish synchrotron light source MAX-II at Lund University. Of the 2K/{gamma} = 14 mrad horizontal wiggler fan, the central 2 mrad are used and split in three parts. The central 1 mrad will be used for a station optimized for MAD experiments and on each side of the central fan, from 0.5 mrad to 1 mrad, there are two fixed energy stations using different energies of the same part of the beam. These, in total five stations, can be used simultaneously and independently for diffraction data collection. The two upstream monochromators for the side stations are meridionally bent asymmetric diamond(111) crystals in Laue transmission geometry. The monochromators for the downstream side stations are bent Ge(111) crystals in asymmetric Bragg reflection geometry. Curved multilayer mirrors inserted in the monochromatic beams provide focusing in the vertical plane. The first side station is under commissioning, and a preliminary test protein data set has been collected.

  6. Distinct responses to reduplicated chromosomes require distinct Mad2 responses

    PubMed Central

    Stormo, Benjamin M; Fox, Donald T

    2016-01-01

    Duplicating chromosomes once each cell cycle produces sister chromatid pairs, which separate accurately at anaphase. In contrast, reduplicating chromosomes without separation frequently produces polytene chromosomes, a barrier to accurate mitosis. Chromosome reduplication occurs in many contexts, including: polytene tissue development, polytene tumors, and following treatment with mitosis-blocking chemotherapeutics. However, mechanisms responding to or resolving polyteny during mitosis are poorly understood. Here, using Drosophila, we uncover two distinct reduplicated chromosome responses. First, when reduplicated polytene chromosomes persist into metaphase, an anaphase delay prevents tissue malformation and apoptosis. Second, reduplicated polytene chromosomes can also separate prior to metaphase through a spindle-independent mechanism termed Separation-Into-Recent-Sisters (SIRS). Both reduplication responses require the spindle assembly checkpoint protein Mad2. While Mad2 delays anaphase separation of metaphase polytene chromosomes, Mad2’s control of overall mitotic timing ensures efficient SIRS. Our results pinpoint mechanisms enabling continued proliferation after genome reduplication, a finding with implications for cancer progression and prevention. DOI: http://dx.doi.org/10.7554/eLife.15204.001 PMID:27159240

  7. The petunia MADS box gene FBP11 determines ovule identity.

    PubMed Central

    Colombo, L; Franken, J; Koetje, E; van Went, J; Dons, H J; Angenent, G C; van Tunen, A J

    1995-01-01

    In contrast to the wealth of information relating to genes regulating floral meristem and floral organ identity, only limited data are available concerning genes that are involved in determining and regulating the identity and development of an ovule. We have recently isolated the floral binding protein 11 (FBP11) MADS box gene from petunia and found that it is expressed exclusively in ovule primordia and subsequently in the ovules, suggesting a role for this gene in ovule formation. To test this hypothesis, we constructed a recombinant gene in which the full-size FBP11 cDNA was placed under the control of a strong cauliflower mosaic virus 35S promoter. Transgenic petunia plants expressing this chimeric gene have ovulelike structures on the adaxial side of the sepals and the abaxial side of the petals. Detailed morphological studies showed that these ovulelike structures are true ovules. RNA gel blot analysis was performed to investigate ectopic FBP11 expression in relation to the expression of the closely related FBP7 gene and the putative petunia class C-type homeotic genes FBP6 and pMADS3. Our results indicate that FBP11 represents an ovule identity gene. A new model describing the mode of action of FBP11 as an additional class D MADS box gene is presented. PMID:8535139

  8. Effects of Mad Honey on Some Biochemical Parameters in Rats.

    PubMed

    Sahin, Huseyin; Yildiz, Oktay; Kolayli, Sevgi

    2016-10-01

    The aims of this study were to determine grayanotoxin (GTX-III) toxin level in mad honey using liquid chromatography-tandem mass spectrometry and examine the dynamic changes of certain biochemical parameters in blood serum of rats that consumed mad honey. For the experimental animal study, 20 Sprague-Dawley female rats were divided into 5 groups of 4 rats each, with one group being the control group (Group 1) and the others being the experimental groups (Groups 2-5). Groups 2, 3, 4, and 5 were, respectively, given mad honey extract at doses of 0.3, 0.6, 1.2, and 2.4 mg/g body weight/day via oral gavage for 8 days. According to results, the quantity of GTX-III found in the honey sample as 39.949 ± 0.020 μg GTX-III/g honey, and the biochemical analysis of the tested parameters (aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, creatine kinase, and creatine kinase muscle and brain) showed a significant elevation with increasing concentration of honey. In conclusion, the use of increasing concentrations of Rhododendron honey was seen as a source of enzymatic symptoms.

  9. Crystallization of macromolecular complexes: combinatorial complex crystallization

    NASA Astrophysics Data System (ADS)

    Stura, Enrico A.; Graille, Marc; Charbonnier, Jean-Baptiste

    2001-11-01

    The usefulness of antibody complexation, as a way of increasing the chances of crystallization needs to be re-evaluated after many antibody complexes have been crystallized and their structure determined. It is somewhat striking that among these, only a small number is a complex with a large protein antigen. The problem is that the effort of raising, cleaving and purifying an Fab is rewarded only by an extra chance of getting crystals; depending on the relative likelihood of crystallization of the complexed and uncomplexed protein. The example of the complex between HIV gp120, CD4 and an Fab fragment from a neutralizing antibody suggests that further complexation of an antigen-antibody complex with a third protein could, by increasing the number of possible combinations, improve the likelihood of crystallization. We propose the use of Ig-binding proteins as a way of extending the method from HIV gp120 to all proteins for which there are monoclonal antibodies. We discuss this technique, combinatorial complex crystallization (CCC), as part of a multi-component system for the enhancement of crystallization of macromolecular complexes. The method makes use of single Ig-binding domains from Staphylococcus aureus protein A (SpA), Peptostreptococcus magnus protein L (PpL) and the streptococcal protein G (SpG). The generality of the method depends on the ability of these domains to interact with a large repertoire of antibodies without affecting antigen binding. There is strong evidence to suggest that these Ig-binding domains bind outside the antigen-combining site of the antibody without perturbing antigen binding. It is clear from the crystal structure of the single SpG domain complexed with an Fab that the interaction involves mainly the immunoglobulin CH1 domain, a region not involved in antigen recognition. We have recently determined the structure of the complex between a human Fab and the domain D from SpA and found that steric hindrance is unlikely even for large

  10. Single-step Antibody-based Affinity Cryo-Electron Microscopy for Imaging and Structural Analysis of Macromolecular Assemblies

    PubMed Central

    Yu, Guimei; Vago, Frank; Zhang, Dongsheng; Snyder, Jonathan E.; Yan, Rui; Zhang, Ci; Benjamin, Christopher; Jiang, Xi; Kuhn, Richard J.; Serwer, Philip; Thompson, David H.; Jiang, Wen

    2014-01-01

    Single particle cryo-electron microscopy (cryo-EM) is an emerging powerful tool for structural studies of macromolecular assemblies (i.e., protein complexes and viruses). Although single particle cryo-EM requires less concentrated and smaller amounts of samples than X-ray crystallography, it remains challenging to study specimens that are low-abundance, low-yield, or short-lived. The recent development of affinity grid techniques can potentially further extend single particle cryo-EM to these challenging samples by combining sample purification and cryo-EM grid preparation into a single step. Here we report a new design of affinity cryo-EM approach, cryo-SPIEM, that applies a traditional pathogen diagnosis tool Solid Phase Immune Electron Microscopy (SPIEM) to the single particle cryo-EM method. This approach provides an alternative, largely simplified and easier to use affinity grid that directly works with most native macromolecular complexes with established antibodies, and enables cryo-EM studies of native samples directly from cell cultures. In the present work, we extensively tested the feasibility of cryo-SPIEM with multiple samples including those of high or low molecular weight, macromolecules with low or high symmetry, His-tagged or native particles, and high- or low-yield macromolecules. Results for all these samples (nonpurified His-tagged bacteriophage T7, His-tagged E. coli ribosomes, native Sindbis virus, and purified but low-concentration native Tulane virus) demonstrated the capability of cryo-SPIEM approach in specifically trapping and concentrating target particles on TEM grids with minimal view constraints for cryo-EM imaging and determination of 3D structures. PMID:24780590

  11. Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies.

    PubMed

    Yu, Guimei; Vago, Frank; Zhang, Dongsheng; Snyder, Jonathan E; Yan, Rui; Zhang, Ci; Benjamin, Christopher; Jiang, Xi; Kuhn, Richard J; Serwer, Philip; Thompson, David H; Jiang, Wen

    2014-07-01

    Single particle cryo-electron microscopy (cryo-EM) is an emerging powerful tool for structural studies of macromolecular assemblies (i.e., protein complexes and viruses). Although single particle cryo-EM requires less concentrated and smaller amounts of samples than X-ray crystallography, it remains challenging to study specimens that are low-abundance, low-yield, or short-lived. The recent development of affinity grid techniques can potentially further extend single particle cryo-EM to these challenging samples by combining sample purification and cryo-EM grid preparation into a single step. Here we report a new design of affinity cryo-EM approach, cryo-SPIEM, that applies a traditional pathogen diagnosis tool Solid Phase Immune Electron Microscopy (SPIEM) to the single particle cryo-EM method. This approach provides an alternative, largely simplified and easier to use affinity grid that directly works with most native macromolecular complexes with established antibodies, and enables cryo-EM studies of native samples directly from cell cultures. In the present work, we extensively tested the feasibility of cryo-SPIEM with multiple samples including those of high or low molecular weight, macromolecules with low or high symmetry, His-tagged or native particles, and high- or low-yield macromolecules. Results for all these samples (non-purified His-tagged bacteriophage T7, His-tagged Escherichiacoli ribosomes, native Sindbis virus, and purified but low-concentration native Tulane virus) demonstrated the capability of cryo-SPIEM approach in specifically trapping and concentrating target particles on TEM grids with minimal view constraints for cryo-EM imaging and determination of 3D structures.

  12. Regulation of AURORA B function by mitotic checkpoint protein MAD2.

    PubMed

    Shandilya, Jayasha; Medler, Kathryn F; Roberts, Stefan G E

    2016-08-17

    Cell cycle checkpoint signaling stringently regulates chromosome segregation during cell division. MAD2 is one of the key components of the spindle and mitotic checkpoint complex that regulates the fidelity of cell division along with MAD1, CDC20, BUBR1, BUB3 and MAD3. MAD2 ablation leads to erroneous attachment of kinetochore-spindle fibers and defective chromosome separation. A potential role for MAD2 in the regulation of events beyond the spindle and mitotic checkpoints is not clear. Together with active spindle assembly checkpoint signaling, AURORA B kinase activity is essential for chromosome condensation as cells enter mitosis. AURORA B phosphorylates histone H3 at serine 10 and serine 28 to facilitate the formation of condensed metaphase chromosomes. In the absence of functional AURORA B cells escape mitosis despite the presence of misaligned chromosomes. In this study we report that silencing of MAD2 results in a drastic reduction of metaphase-specific histone H3 phosphorylation at serine 10 and serine 28. We demonstrate that this is due to mislocalization of AURORA B in the absence of MAD2. Conversely, overexpression of MAD2 concentrated the localization of AURORA B at the metaphase plate and caused hyper-phosphorylation of histone H3. We find that MAD1 plays a minor role in influencing the MAD2-dependent regulation of AURORA B suggesting that the effects of MAD2 on AURORA B are independent of the spindle checkpoint complex. Our findings reveal that, in addition to its role in checkpoint signaling, MAD2 ensures chromosome stability through the regulation of AURORA B.

  13. The regulation of MADS-box gene expression during ripening of banana and their regulatory interaction with ethylene

    PubMed Central

    Elitzur, Tomer; Vrebalov, Julia; Giovannoni, James J.; Goldschmidt, Eliezer E.; Friedman, Haya

    2010-01-01

    Six MaMADS-box genes have been cloned from the banana fruit cultivar Grand Nain. The similarity of these genes to tomato LeRIN is low and neither MaMADS2 nor MaMADS1 complement the tomato rin mutation. Nevertheless, the expression patterns, specifically in fruit and the induction during ripening and in response to ethylene and 1-MCP, suggest that some of these genes may participate in ripening. MaMADS1, 2, and 3, are highly expressed in fruit only, while the others are expressed in fruit as well as in other organs. Moreover, the suites of MaMADS-box genes and their temporal expression differ in peel and pulp during ripening. In the pulp, the increase in MaMADS2, 3, 4, and 5 expression preceded an increase in ethylene production, but coincides with the CO2 peak. However, MaMADS1 expression in pulp coincided with ethylene production, but a massive increase in its expression occurred late during ripening, together with a second wave in the expression of MaMADS2, 3, and 4. In the peel, on the other hand, an increase in expression of MaMADS1, 3, and to a lesser degree also of MaMADS4 and 2 coincided with an increase in ethylene production. Except MaMADS3, which was induced by ethylene in pulp and peel, only MaMADS4, and 5 in pulp and MaMADS1 in peel were induced by ethylene. 1-MCP applied at the onset of the increase in ethylene production, increased the levels of MaMADS4 and MaMADS1 in pulp, while it decreased MaMADS1, 3, 4, and 5 in peel, suggesting that MaMADS4 and MaMADS1 are negatively controlled by ethylene at the onset of ethylene production only in pulp. Only MaMADS2 is neither induced by ethylene nor by 1-MCP, and it is expressed mainly in pulp. Our results suggest that two independent ripening programs are employed in pulp and peel which involve the activation of mainly MaMADS2, 4, and 5 and later on also MaMADS1 in pulp, and mainly MaMADS1, and 3 in peel. Hence, our results are consistent with MaMADS2, a SEP3 homologue, acting in the pulp upstream of the

  14. The nucleoporin Nup153 affects spindle checkpoint activity due to an association with Mad1

    PubMed Central

    Shimi, Takeshi

    2010-01-01

    The nucleoporin Nup153 is known to play pivotal roles in nuclear import and export in interphase cells and as the cell transitions into mitosis, Nup153 is involved in nuclear envelope breakdown. In this study, we demonstrate that the interaction of Nup153 with the spindle assembly checkpoint protein Mad1 is important in the regulation of the spindle checkpoint. Overexpression of human Nup153 in HeLa cells leads to the appearance of multinucleated cells and induces the formation of multipolar spindles. Importantly, it causes inactivation of the spindle checkpoint due to hypophosphorylation of Mad1. Depletion of Nup153 using RNA interference results in the decline of Mad1 at nuclear pores during interphase and more significantly causes a delayed dissociation of Mad1 from kinetochores in metaphase and an increase in the number of unresolved midbodies. In the absence of Nup153 the spindle checkpoint remains active. In vitro studies indicate direct binding of Mad1 to the N-terminal domain of Nup153. Importantly, Nup153 binding to Mad1 affects Mad1's phosphorylation status, but not its ability to interact with Mad2. Our data suggest that Nup153 levels regulate the localization of Mad1 during the metaphase/anaphase transition thereby affecting its phoshorylation status and in turn spindle checkpoint activity and mitotic exit. PMID:21327106

  15. The vocabulary of madness from Homer to Hippocrates. Part 1: the verbal group of mualphaiotanuomicronmualphaiota.

    PubMed

    Perdicoyianni-Paléologou, Hélène

    2009-09-01

    In Part 1 of this two-part paper, I examine the evolution of the concept of madness expressed by the various forms--verbal and nominal, simple and compound--of the verbal group of mualphaiotanuomicronmualphaiota in the archaic and classical periods. I point out how the divine madness is contrasted to pathological madness considered as a psychic and mental disease and foreseeable by doctors as well as curable by medications. This new procedure highlights rational knowledge of the Greeks about the cause and the medical care of madness.

  16. Using Two-Dimensional Colloidal Crystals to Understand Crystallography

    ERIC Educational Resources Information Center

    Bosse, Stephanie A.; Loening, Nikolaus M.

    2008-01-01

    X-ray crystallography is an essential technique for modern chemistry and biochemistry, but it is infrequently encountered by undergraduate students owing to lack of access to equipment, the time-scale for generating diffraction-quality molecular crystals, and the level of mathematics involved in analyzing the resulting diffraction patterns.…

  17. Tinker Toys, Crystallography, and the Introductory Mineralogy Course

    ERIC Educational Resources Information Center

    Buseck, Peter R.

    1970-01-01

    Describes the use of Tinker Toys to construct three dimensional models of crystals useful in illustrating many concepts of crystallography. Space lattices representing all of the Bravais types can be constructed. Also discusses the use of appropriate models to demonstrate the various symmetry operations. Bibliography. (LC)

  18. Models as an Aid to Courses in Crystallography and Mineralogy.

    ERIC Educational Resources Information Center

    Brady, K. T.

    1983-01-01

    Three models used in teaching crystallography/mineralogy at the University of Technology (Papua, New Guinea) are described. These include stereographic projection model, optical indicatrix models for Istropic/Anisotropic minerals, and model showing effect of anisotropic minerals under crossed polars. Photographs of the models are also included.…

  19. Two-Dimensional Crystallography Introduced by the Sprinkler Watering Problem

    ERIC Educational Resources Information Center

    De Toro, Jose A.; Calvo, Gabriel F.; Muniz, Pablo

    2012-01-01

    The problem of optimizing the number of circular sprinklers watering large fields is used to introduce, from a purely elementary geometrical perspective, some basic concepts in crystallography and comment on a few size effects in condensed matter physics. We examine square and hexagonal lattices to build a function describing the, so-called, dry…

  20. Fab Chaperone-Assisted RNA Crystallography (Fab CARC).

    PubMed

    Sherman, Eileen; Archer, Jennifer; Ye, Jing-Dong

    2016-01-01

    Recent discovery of structured RNAs such as ribozymes and riboswitches shows that there is still much to learn about the structure and function of RNAs. Knowledge learned can be employed in both biochemical research and clinical applications. X-ray crystallography gives unparalleled atomic-level structural detail from which functional inferences can be deduced. However, the difficulty in obtaining high-quality crystals and their phasing information make it a very challenging task. RNA crystallography is particularly arduous due to several factors such as RNA's paucity of surface chemical diversity, lability, repetitive anionic backbone, and flexibility, all of which are counterproductive to crystal packing. Here we describe Fab chaperone assisted RNA crystallography (CARC), a systematic technique to increase RNA crystallography success by facilitating crystal packing as well as expediting phase determination through molecular replacement of conserved Fab domains. Major steps described in this chapter include selection of a synthetic Fab library displayed on M13 phage against a structured RNA crystallization target, ELISA for initial choice of binding Fabs, Fab expression followed by protein A affinity then cation exchange chromatography purification, final choice of Fab by binding specificity and affinity as determined by a dot blot assay, and lastly gel filtration purification of a large quantity of chosen Fabs for crystallization.

  1. Expression and genomic structure of the dormancy-associated MADS box genes MADS13 in Japanese pears (Pyrus pyrifolia Nakai) that differ in their chilling requirement for endodormancy release.

    PubMed

    Saito, Takanori; Bai, Songling; Ito, Akiko; Sakamoto, Daisuke; Saito, Toshihiro; Ubi, Banjamin Ewa; Imai, Tsuyoshi; Moriguchi, Takaya

    2013-06-01

    We isolated three dormancy-associated MADS-box (DAM) genes (MADS13-1, MADS13-2 and MADS13-3) and showed regulated expression concomitant with endodormancy establishment and release in the leaf buds of Japanese pear 'Kosui'. Comparative analysis between 'Kosui' and Taiwanese pear TP-85-119 ('Hengshanli'), a less dormant pear cultivar, showed reduction of MADS13-1 expression level in 'Hengshanli' earlier than in 'Kosui' towards endodormancy release, suggesting the possible relationship between chilling requirement and MADS13-1 expression. Application of hydrogen cyanamide accelerated endodormancy release with a reduction in MADS13 expression, whereas heat treatment in autumn inhibited endodormancy establishment without induction of MADS13 expression, indicating a close relationship between the MADS13 expression pattern and endodormancy phase transitions. Moreover, both the cis-acting regulatory elements and the methylation status in the 5' upstream region of the MADS13-1 gene were not largely different between 'Kosui' and 'Hengshanli'. Genomic structures of MADS13-1 from 'Kosui' and 'Hengshanli' revealed a 3218 bp insertion in the first intron of 'Hengshanli' that might be ascribed to the lower expression of MADS13-1tw; however, this insertion was also found in pear genotypes with a high chilling requirement. These results indicated that the low expression of MADS13-1 in 'Hengshanli' towards endodormancy release could not be explained by the identified cis-acting regulatory elements, the methylation status of the putative promoter or by intron insertion.

  2. Macromolecular Crowding Regulates the Gene Expression Profile by Limiting Diffusion

    PubMed Central

    Golkaram, Mahdi; Hellander, Stefan; Drawert, Brian; Petzold, Linda R.

    2016-01-01

    We seek to elucidate the role of macromolecular crowding in transcription and translation. It is well known that stochasticity in gene expression can lead to differential gene expression and heterogeneity in a cell population. Recent experimental observations by Tan et al. have improved our understanding of the functional role of macromolecular crowding. It can be inferred from their observations that macromolecular crowding can lead to robustness in gene expression, resulting in a more homogeneous cell population. We introduce a spatial stochastic model to provide insight into this process. Our results show that macromolecular crowding reduces noise (as measured by the kurtosis of the mRNA distribution) in a cell population by limiting the diffusion of transcription factors (i.e. removing the unstable intermediate states), and that crowding by large molecules reduces noise more efficiently than crowding by small molecules. Finally, our simulation results provide evidence that the local variation in chromatin density as well as the total volume exclusion of the chromatin in the nucleus can induce a homogenous cell population. PMID:27893768

  3. Macromolecular Pt(IV) Prodrugs from Poly(organo)phosphazenes

    PubMed Central

    Banfić, Jelena; Theiner, Sarah; Körner, Wilfried; Brüggemann, Oliver; Berger, Walter; Keppler, Bernhard K.; Heffeter, Petra; Teasdale, Ian

    2016-01-01

    The preparation of novel macromolecular prodrugs via the conjugation of two platinum(IV) complexes to suitably functionalized poly(organo)phosphazenes is presented. The inorganic/organic polymers provide carriers with controlled dimensions due to the use of living cationic polymerization and allow the preparation of conjugates with excellent aqueous solubility but long-term hydrolytic degradability. The macromolecular Pt(IV) prodrugs are designed to undergo intracellular reduction and simultaneous release from the macromolecular carrier to present the active Pt(II) drug derivatives. In vitro investigations show a significantly enhanced intracellular uptake of Pt for the macromolecular prodrugs when compared to small molecule Pt complexes, which is also reflected in an increase in cytotoxicity. Interestingly, drug-resistant sublines also show a significantly smaller resistance against the conjugates compared to clinically established platinum drugs, indicating that an alternative uptake route of the Pt(IV) conjugates might also be able to overcome acquired resistance against Pt(II) drugs. In vivo studies of a selected conjugate show improved tumor shrinkage compared to the respective Pt(IV) complex. PMID:27169668

  4. Effects of macromolecular crowding and osmolyte on human Tau fibrillation.

    PubMed

    Wu, Yingying; Teng, Ningning; Li, Sen

    2016-09-01

    Tau fibrillation is reported to be involved in neurodegenerative disorders, such as Alzheimer's disease, in which the natural environment is very crowded in the cells. Understanding the role of crowding environments in regulating Tau fibrillation is of great importance for elucidating the etiology of these diseases. In this experiment, the effects of macromolecular crowding and osmolyte reagents in the crowding environment on Tau fibrillation were studied by thioflavin T binding, SDS-PAGE and TEM assays. Ficoll 70 and Dextran 70 of different concentrations were used as macromolecular crowding reagents inside the cells and showed a strong enhancing effect on the fibrillation of normal and hyperphosphorylated Tau. The enhancing effect of Dextran is stronger than that of Ficoll 70 at the same concentration. In addition, the cellular osmolyte sucrose was found to protect Tau against fibrillation, and inhibit the enhancing effect of macromolecular crowding on Tau fibrillation. A possible model for the fibrillation process of Tau and the effect of macromolecular crowding and osmolyte on this process was proposed based on these experimental results. The information obtained from our study can enhance the understanding of how proteins aggregate and avoid aggregation in crowded physiological environments and might lead to a better understanding of the molecular mechanisms of Alzheimer's disease in vivo.

  5. Combined Effects of Agitation, Macromolecular Crowding, and Interfaces on Amyloidogenesis*

    PubMed Central

    Lee, Chiu Fan; Bird, Sarah; Shaw, Michael; Jean, Létitia; Vaux, David J.

    2012-01-01

    Amyloid formation and accumulation is a hallmark of protein misfolding diseases and is associated with diverse pathologies including type II diabetes and Alzheimer's disease (AD). In vitro, amyloidogenesis is widely studied in conditions that do not simulate the crowded and viscous in vivo environment. A high volume fraction of most biological fluids is occupied by various macromolecules, a phenomenon known as macromolecular crowding. For some amyloid systems (e.g. α-synuclein) and under shaking condition, the excluded volume effect of macromolecular crowding favors aggregation, whereas increased viscosity reduces the kinetics of these reactions. Amyloidogenesis can also be catalyzed by hydrophobic-hydrophilic interfaces, represented by the air-water interface in vitro and diverse heterogeneous interfaces in vivo (e.g. membranes). In this study, we investigated the effects of two different crowding polymers (dextran and Ficoll) and two different experimental conditions (with and without shaking) on the fibrilization of amyloid-β peptide, a major player in AD pathogenesis. Specifically, we demonstrate that, during macromolecular crowding, viscosity dominates over the excluded volume effect only when the system is spatially non homogeneous (i.e. an air-water interface is present). We also show that the surfactant activity of the crowding agents can critically influence the outcome of macromolecular crowding and that the structure of the amyloid species formed may depend on the polymer used. This suggests that, in vivo, the outcome of amyloidogenesis may be affected by both macromolecular crowding and spatial heterogeneity (e.g. membrane turn-over). More generally, our work suggests that any factors causing changes in crowding may be susceptibility factors in AD. PMID:22988239

  6. Kinetics of myc-max-mad gene expression during hepatocyte proliferation in vivo: Differential regulation of mad family and stress-mediated induction of c-myc.

    PubMed

    Mauleon, Itsaso; Lombard, Marie-Noëlle; Muñoz-Alonso, Maria J; Cañelles, Matilde; Leon, Javier

    2004-02-01

    Mad proteins (Mad1, Mxi1, Mad3, Mad4, Mnt/Rox) are biochemical and biological antagonists of c-Myc oncoprotein. Mad-Max dimers repress the transcription of the same target genes activated by Myc-Max dimers. Despite the critical role of Max and Mad proteins as modulators of c-Myc functions, there are no comparative data on their regulation in vivo. We carried out a systematic analysis of c-myc, max, and mad family expression in a model of synchronized cell proliferation in vivo in adult tissues, that is, rat hepatocytes after partial hepatectomy. We confirmed the previously reported early peak of c-myc expression after hepatectomy but we show that it did not correlate with hepatocyte proliferation as it also occurred in sham-operated animals as a result of surgical stresses. A second peak of c-myc expression was observed later, at the time of the wave of DNA synthesis. No such expression was detected in sham-operated rat quiescent hepatocytes. max expression increased around 4-16 h after hepatectomy, before the peaks of c-myc and DNA synthesis. mxi1 and mad4 were slightly downregulated during liver regeneration. mnt/rox expression did not change. These expression patterns suggest a role of Myc-Max for efficient mitogenic response of hepatocytes. We also analyzed the effects of Myc and Max ectopic expression on the clonogenic growth of the rat hepatoma cells. Expression of c-Myc and Max increased clonogenic growth, whereas the reduction of c-Myc levels by an antisense vector decreased growth. The results suggest nonredundant roles for mad genes in hepatocyte proliferation and point to c-Myc as a putative target for anticancer therapy of liver cancer.

  7. Three MADS-box genes similar to APETALA1 and FRUITFULL from silver birch (Betula pendula).

    PubMed

    Elo, Annakaisa; Lemmetyinen, Juha; Turunen, Marja-Leena; Tikka, Liisa; Sopanen, Tuomas

    2001-05-01

    Despite intensive research on genetic regulation of flower development there are still only a few studies on the early phases of this process in perennial plants like trees. The aim of this study has been to identify genes that regulate early stages of inflorescence development in silver birch (Betula pendula Roth) and to follow the expression of these genes during development of the unisexual birch inflorescences. Here we describe the cloning and characterization of 3 cDNAs representing MADS-box genes designated BpMADS3, BpMADS4 and BpMADS5, all belonging to the AP1/SQUA group of plant MADS-box genes. According to RNA blot analysis, all 3 genes are active during the development of both male and female inflorescences. However, differences in patterns of expression suggest that they play different roles. BpMADS3 is most similar in sequence to AP1 and SQUA, but it seems to have the highest expression at late developmental stages. BpMADS4 is most similar in sequence to the Arabidopsis gene FRUITFULL, but is expressed, in addition to developing inflorescences, in shoots and roots. BpMADS5 is also similar to FRUITFULL; its expression seems to be inflorescence-specific and continues during fruit development. Ectopic expression of either BpMADS3, BpMADS4 or BpMADS5 with the CaMV 35S promoter in tobacco results in extremely early flowering. All of these birch genes seem to act early during the transition to reproductive phase and might be involved in the determination of the identity of the inflorescence or flower meristem. They could apparently be used to accelerate flowering in various plant species.

  8. Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

    PubMed Central

    Wan, Qun; Parks, Jerry M.; Hanson, B. Leif; Fisher, Suzanne Zoe; Ostermann, Andreas; Schrader, Tobias E.; Graham, David E.; Coates, Leighton; Langan, Paul; Kovalevsky, Andrey

    2015-01-01

    Glycoside hydrolase (GH) enzymes apply acid/base chemistry to catalyze the decomposition of complex carbohydrates. These ubiquitous enzymes accept protons from solvent and donate them to substrates at close to neutral pH by modulating the pKa values of key side chains during catalysis. However, it is not known how the catalytic acid residue acquires a proton and transfers it efficiently to the substrate. To better understand GH chemistry, we used macromolecular neutron crystallography to directly determine protonation and ionization states of the active site residues of a family 11 GH at multiple pD (pD = pH + 0.4) values. The general acid glutamate (Glu) cycles between two conformations, upward and downward, but is protonated only in the downward orientation. We performed continuum electrostatics calculations to estimate the pKa values of the catalytic Glu residues in both the apo- and substrate-bound states of the enzyme. The calculated pKa of the Glu increases substantially when the side chain moves down. The energy barrier required to rotate the catalytic Glu residue back to the upward conformation, where it can protonate the glycosidic oxygen of the substrate, is 4.3 kcal/mol according to free energy simulations. These findings shed light on the initial stage of the glycoside hydrolysis reaction in which molecular motion enables the general acid catalyst to obtain a proton from the bulk solvent and deliver it to the glycosidic oxygen. PMID:26392527

  9. Enabling X-ray free electron laser crystallography for challenging biological systems from a limited number of crystals

    PubMed Central

    Uervirojnangkoorn, Monarin; Zeldin, Oliver B; Lyubimov, Artem Y; Hattne, Johan; Brewster, Aaron S; Sauter, Nicholas K; Brunger, Axel T; Weis, William I

    2015-01-01

    There is considerable potential for X-ray free electron lasers (XFELs) to enable determination of macromolecular crystal structures that are difficult to solve using current synchrotron sources. Prior XFEL studies often involved the collection of thousands to millions of diffraction images, in part due to limitations of data processing methods. We implemented a data processing system based on classical post-refinement techniques, adapted to specific properties of XFEL diffraction data. When applied to XFEL data from three different proteins collected using various sample delivery systems and XFEL beam parameters, our method improved the quality of the diffraction data as well as the resulting refined atomic models and electron density maps. Moreover, the number of observations for a reflection necessary to assemble an accurate data set could be reduced to a few observations. These developments will help expand the applicability of XFEL crystallography to challenging biological systems, including cases where sample is limited. DOI: http://dx.doi.org/10.7554/eLife.05421.001 PMID:25781634

  10. Computational identification and analysis of MADS box genes in Camellia sinensis.

    PubMed

    Gogoi, Madhurjya; Borchetia, Sangeeta; Bandyopadhyay, Tanoy

    2015-01-01

    MADS (Minichromosome Maintenance1 Agamous Deficiens Serum response factor) box genes encode transcription factors and they play a key role in growth and development of flowering plants. There are two types of MADS box genes- Type I (serum response factor (SRF)-like) and Type II (myocyte enhancer factor 2 (MEF2)-like). Type II MADS box genes have a conserved MIKC domain (MADS DNA-binding domain, intervening domain, keratin-like domain, and c-terminal domain) and these were extensively studied in plants. Compared to other plants very little is known about MADS box genes in Camellia sinensis. The present study aims at identifying and analyzing the MADS-box genes present in Camellia sinensis. A comparative bioinformatics and phylogenetic analysis of the Camellia sinensis sequences along with Arabidopsis thaliana MADS box sequences available in the public domain databases led to the identification of 16 genes which were orthologous to Type II MADS box gene family members. The protein sequences were classified into distinct clades which are associated with the conserved function of flower and seed development. The identified genes may be used for gene expression and gene manipulation studies to elucidate their role in the development and flowering of tea which may pave the way to improve the crop productivity.

  11. Computational identification and analysis of MADS box genes in Camellia sinensis

    PubMed Central

    Gogoi, Madhurjya; Borchetia, Sangeeta; Bandyopadhyay, Tanoy

    2015-01-01

    MADS (Minichromosome Maintenance1 Agamous Deficiens Serum response factor) box genes encode transcription factors and they play a key role in growth and development of flowering plants. There are two types of MADS box genes- Type I (serum response factor (SRF)-like) and Type II (myocyte enhancer factor 2 (MEF2)-like). Type II MADS box genes have a conserved MIKC domain (MADS DNA-binding domain, intervening domain, keratin-like domain, and c-terminal domain) and these were extensively studied in plants. Compared to other plants very little is known about MADS box genes in Camellia sinensis. The present study aims at identifying and analyzing the MADS-box genes present in Camellia sinensis. A comparative bioinformatics and phylogenetic analysis of the Camellia sinensis sequences along with Arabidopsis thaliana MADS box sequences available in the public domain databases led to the identification of 16 genes which were orthologous to Type II MADS box gene family members. The protein sequences were classified into distinct clades which are associated with the conserved function of flower and seed development. The identified genes may be used for gene expression and gene manipulation studies to elucidate their role in the development and flowering of tea which may pave the way to improve the crop productivity. PMID:25914445

  12. MADS-box genes in maize: Frequent targets of selection during domestication

    Technology Transfer Automated Retrieval System (TEKTRAN)

    MADS-box genes encode transcription factors that are key regulators of plant inflorescence and flower development. We examined DNA sequence variation in 32 maize MADS-box genes and 32 random loci from the maize genome and investigated their involvement in maize domestication and improvement. Using n...

  13. A pavement Moisture Accelerated Distress (MAD) identification system, volume 2

    NASA Astrophysics Data System (ADS)

    Carpenter, S. H.; Darter, M. I.; Dempsey, B. J.

    1981-09-01

    A users manual is designed which provides the engineer with a rational method of examining a pavement and determining rehabilitation needs that are related to the causes of the existing distress, particularly moisture related distress. The key elements in this procedure are the MAD Index developed in Volume 1, the Pavement Condition Index (PCI) and the Moisture Distress Index (MDI). Step by step procedures are presented for calculating each parameter. Complete distress identification manuals are included for asphalt surfaced highways and jointed reinforced concrete highways with pictures and descriptions of all major distress types. Descriptions of the role moisture plays in the development of each distress type are included.

  14. Does UV CETI Suffer from the Mad Syndrome?

    NASA Technical Reports Server (NTRS)

    Drake, Jeremy

    1999-01-01

    Photometric data have been analyzed and searched for events of flaring and other variability. Some flaring has been detected, though probably not at a level that will hinder our continuing spectral analysis. X-ray diagnostics for the very hot coronal emission measure are under investigation in order to determine whether or not the very hot coronal plasma contributes significantly to the observed X-ray flux in the (EUV) Extreme Ultraviolet Radiation. The key test of the MAD syndrome lies in whether or not the coronal lines indicate a depletion in metals in the corona relative to the underlying photosphere.

  15. Stable folding intermediates prevent fast interconversion between the closed and open states of Mad2 through its denatured state.

    PubMed

    Zhao, Yuanyuan; Li, Lianghui; Wu, Chunfei; Jiang, Xiaoyong; Ge, Baosheng; Ren, Hao; Huang, Fang

    2016-01-01

    Different states of metamorphic proteins can interconvert under physiological conditions to realize corresponding functions. The mechanism behind the conversion is critical for understanding how these proteins work. We report a combined thermodynamic and kinetic study on the folding/unfolding process of the open and closed conformers of mitotic arrest deficient protein 2 (Mad2), a metamorphic protein. It has been observed that open Mad2 (O-Mad2) can convert to closed Mad2 (C-Mad2). Our results show that O-Mad2 and C-Mad2 have similar thermodynamic stability, which explains the presence of metamorphosis. The folding/unfolding kinetics suggest that the conversion between O-Mad2 and C-Mad2 would be much faster than that reported previously if this conversion goes through the denatured state (U) directly, i.e. through an O-Mad2-denatured state (U)-C-Mad2 (O-U-C) pathway. This inconsistency implies that there exist stable intermediates in between the native and denatured states of Mad2, which would either slow down the O-U-C interconversion or prevent it going through the denatured state.

  16. Expression of the mad gene during cell differentiation in vivo and its inhibition of cell growth in vitro

    PubMed Central

    1995-01-01

    Mad is a basic region helix-loop-helix leucine zipper transcription factor which can dimerize with the Max protein and antagonize transcriptional activation by the Myc-Max transcription factor heterodimer. While the expression of Myc is necessary for cell proliferation, the expression of Mad is induced upon differentiation of at least some leukemia cell lines. Here, the expression of the mad gene has been explored in developing mouse tissues. During organogenesis in mouse embryos mad mRNA was predominantly expressed in the liver and in the mantle layer of the developing brain. At later stages mad expression was detected in neuroretina, epidermis, and whisker follicles, and in adult mice mad was expressed at variable levels in most organs analyzed. Interestingly, in the skin mad was highly expressed in the differentiating epidermal keratinocytes, but not in the underlying proliferating basal keratinocyte layer. Also, in the gut mad mRNA was abundant in the intestinal villi, where cells cease proliferation and differentiate, but not in the crypts, where the intestinal epithelial cells proliferate. In the testis, mad expression was associated with the completion of meiosis and early development of haploid cells. In cell culture, Mad inhibited colony formation of a mouse keratinocyte cell line and rat embryo fibroblast transformation by Myc and Ras. The pattern of mad expression in tissues and its ability to inhibit cell growth in vitro suggests that Mad can cause the cessation of cell proliferation associated with cell differentiation in vivo. PMID:7896882

  17. The emerging importance of type I MADS box transcription factors for plant reproduction.

    PubMed

    Masiero, Simona; Colombo, Lucia; Grini, Paul E; Schnittger, Arp; Kater, Martin M

    2011-03-01

    Based on their evolutionary origin, MADS box transcription factor genes have been divided into two classes, namely, type I and II. The plant-specific type II MIKC MADS box genes have been most intensively studied and shown to be key regulators of developmental processes, such as meristem identity, flowering time, and fruit and seed development. By contrast, very little is known about type I MADS domain transcription factors, and they have not attracted interest for a long time. A number of recent studies have now indicated a key regulatory role for type I MADS box factors in plant reproduction, in particular in specifying female gametophyte, embryo, and endosperm development. These analyses have also suggested that type I MADS box factors are decisive for setting reproductive boundaries between species.

  18. Rapid visualization of hydrogen positions in neutron protein crystallography structures

    SciTech Connect

    Blakeley, Matthew P.; Meilleur, Flora; Myles, Dean A A; Weiss, Kevin L; Munshi, Parthapratim; Shang-Lin, Chung

    2012-01-01

    Neutron crystallography is a powerful technique to visualize experimentally the position of light atoms, including hydrogen and its isotope deuterium. Over the last several years, structural biologists have shown an increasing interest for the technique as it uniquely complements X-ray crystallographic data by revealing the position of hydrogen/deuterium atoms in macromolecules. With this regained interest, access to macromolecule neutron crystallography beam lines is becoming a limiting step. In this report we show that rapid data collection could be a valuable alternative to long data collection time when appropriate. Comparison of perdeuterated Rubredoxin structures refined against neutron data sets collected over hours and up to five days shows that rapid neutron data collection in just 14 hours is sufficient to provide the position of 262 hydrogen positions atoms without ambiguity.

  19. Quantification of detergent using colorimetric methods in membrane protein crystallography.

    PubMed

    Prince, Chelsy; Jia, Zongchao

    2015-01-01

    Membrane protein crystallography has the potential to greatly aid our understanding of membrane protein biology. Yet, membrane protein crystals remain challenging to produce. Although robust methods for the expression and purification of membrane proteins continue to be developed, the detergent component of membrane protein samples is equally important to crystallization efforts. This chapter describes the development of three colorimetric assays for the quantitation of detergent in membrane protein samples and provides detailed protocols. All of these techniques use small sample volumes and have potential applications in crystallography. The application of these techniques in crystallization prescreening, detergent concentration modification, and detergent exchange experiments is demonstrated. It has been observed that the concentration of detergent in a membrane protein sample can be just as important as the protein concentration when attempting to reproduce crystallization lead conditions.

  20. Lipidic cubic phase serial millisecond crystallography using synchrotron radiation

    PubMed Central

    Nogly, Przemyslaw; James, Daniel; Wang, Dingjie; White, Thomas A.; Zatsepin, Nadia; Shilova, Anastasya; Nelson, Garrett; Liu, Haiguang; Johansson, Linda; Heymann, Michael; Jaeger, Kathrin; Metz, Markus; Wickstrand, Cecilia; Wu, Wenting; Båth, Petra; Berntsen, Peter; Oberthuer, Dominik; Panneels, Valerie; Cherezov, Vadim; Chapman, Henry; Schertler, Gebhard; Neutze, Richard; Spence, John; Moraes, Isabel; Burghammer, Manfred; Standfuss, Joerg; Weierstall, Uwe

    2015-01-01

    Lipidic cubic phases (LCPs) have emerged as successful matrixes for the crystallization of membrane proteins. Moreover, the viscous LCP also provides a highly effective delivery medium for serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs). Here, the adaptation of this technology to perform serial millisecond crystallography (SMX) at more widely available synchrotron microfocus beamlines is described. Compared with conventional microcrystallography, LCP-SMX eliminates the need for difficult handling of individual crystals and allows for data collection at room temperature. The technology is demonstrated by solving a structure of the light-driven proton-pump bacteriorhodopsin (bR) at a resolution of 2.4 Å. The room-temperature structure of bR is very similar to previous cryogenic structures but shows small yet distinct differences in the retinal ligand and proton-transfer pathway. PMID:25866654

  1. Maintaining network security: how macromolecular structures cross the peptidoglycan layer.

    PubMed

    Scheurwater, Edie M; Burrows, Lori L

    2011-05-01

    Peptidoglycan plays a vital role in bacterial physiology, maintaining cell shape and resisting cellular lysis from high internal turgor pressures. Its integrity is carefully maintained by controlled remodeling during growth and division by the coordinated activities of penicillin-binding proteins, lytic transglycosylases, and N-acetylmuramyl-l-alanine amidases. However, its small pore size (∼2 nm) and covalently closed structure make it a formidable barrier to the assembly of large macromolecular cell-envelope-spanning complexes involved in motility and secretion. Here, we review the strategies used by Gram-negative bacteria to assemble such macromolecular complexes across the peptidoglycan layer, while preserving its essential structural role. In addition, we discuss evidence that suggests that peptidoglycan can be integrated into cell-envelope-spanning complexes as a structural and functional extension of their architecture.

  2. The growth of filaments under macromolecular confinement using scaling theory.

    PubMed

    Zhu, Lin; Pan, Wei; Lu, Xi; Li, Desheng; Zhao, Jiang; Liang, Dehai

    2015-11-14

    Quantitatively describing macromolecular confinement is still a challenge. Using the assembly of DNA tiles in a polyacrylamide network as a model, we studied the effect of macromolecular confinement on the growth of the filament by scaling theory. The results show that the confinement regulates the morphology, the initial growth rate v, and the eventual length of the filament Nm. The initial growth rate is dependent on the medium viscosity η as ν∝η(-0.94), and the filament adjusts its length in the given confined space as Nm∝ (ξ/Rg)(1.8), with ξ being the mesh size of the polyacrylamide solution and Rg being the radius of gyration of polyacrylamide.

  3. Controlled architecture for improved macromolecular memory within polymer networks.

    PubMed

    DiPasquale, Stephen A; Byrne, Mark E

    2016-08-01

    This brief review analyzes recent developments in the field of living/controlled polymerization and the potential of this technique for creating imprinted polymers with highly structured architecture with macromolecular memory. As a result, it is possible to engineer polymers at the molecular level with increased homogeneity relating to enhanced template binding and transport. Only recently has living/controlled polymerization been exploited to decrease heterogeneity and substantially improve the efficiency of the imprinting process for both highly and weakly crosslinked imprinted polymers. Living polymerization can be utilized to create imprinted networks that are vastly more efficient than similar polymers produced using conventional free radical polymerization, and these improvements increase the role that macromolecular memory can play in the design and engineering of new drug delivery and sensing platforms.

  4. Isotope labeling for NMR studies of macromolecular structure and interactions

    SciTech Connect

    Wright, P.E.

    1994-12-01

    Implementation of biosynthetic methods for uniform or specific isotope labeling of proteins, coupled with the recent development of powerful heteronuclear multidimensional NMR methods, has led to a dramatic increase in the size and complexity of macromolecular systems that are now amenable to NMR structural analysis. In recent years, a new technology has emerged that combines uniform {sup 13}C, {sup 15}N labeling with heteronuclear multidimensional NMR methods to allow NMR structural studies of systems approaching 25 to 30 kDa in molecular weight. In addition, with the introduction of specific {sup 13}C and {sup 15}N labels into ligands, meaningful NMR studies of complexes of even higher molecular weight have become feasible. These advances usher in a new era in which the earlier, rather stringent molecular weight limitations have been greatly surpassed and NMR can begin to address many central biological problems that involve macromolecular structure, dynamics, and interactions.

  5. Branched Macromolecular Architectures for Degradable, Multifunctional Phosphorus-Based Polymers.

    PubMed

    Henke, Helena; Brüggemann, Oliver; Teasdale, Ian

    2017-02-01

    This feature article briefly highlights some of the recent advances in polymers in which phosphorus is an integral part of the backbone, with a focus on the preparation of functional, highly branched, soluble polymers. A comparison is made between the related families of materials polyphosphazenes, phosphazene/phosphorus-based dendrimers and polyphosphoesters. The work described herein shows this to be a rich and burgeoning field, rapidly catching up with organic chemistry in terms of the macromolecular synthetic control and variety of available macromolecular architectures, whilst offering unique property combinations not available with carbon backbones, such as tunable degradation rates, high multi-valency and facile post-polymerization functionalization. As an example of their use in advanced applications, we highlight some investigations into their use as water-soluble drug carriers, whereby in particular the degradability in combination with multivalent nature has made them useful materials, as underlined by some of the recent studies in this area.

  6. Stochastic reaction-diffusion algorithms for macromolecular crowding

    NASA Astrophysics Data System (ADS)

    Sturrock, Marc

    2016-06-01

    Compartment-based (lattice-based) reaction-diffusion algorithms are often used for studying complex stochastic spatio-temporal processes inside cells. In this paper the influence of macromolecular crowding on stochastic reaction-diffusion simulations is investigated. Reaction-diffusion processes are considered on two different kinds of compartmental lattice, a cubic lattice and a hexagonal close packed lattice, and solved using two different algorithms, the stochastic simulation algorithm and the spatiocyte algorithm (Arjunan and Tomita 2010 Syst. Synth. Biol. 4, 35-53). Obstacles (modelling macromolecular crowding) are shown to have substantial effects on the mean squared displacement and average number of molecules in the domain but the nature of these effects is dependent on the choice of lattice, with the cubic lattice being more susceptible to the effects of the obstacles. Finally, improvements for both algorithms are presented.

  7. Oil-free hyaluronic acid matrix for serial femtosecond crystallography

    PubMed Central

    Sugahara, Michihiro; Song, Changyong; Suzuki, Mamoru; Masuda, Tetsuya; Inoue, Shigeyuki; Nakane, Takanori; Yumoto, Fumiaki; Nango, Eriko; Tanaka, Rie; Tono, Kensuke; Joti, Yasumasa; Kameshima, Takashi; Hatsui, Takaki; Yabashi, Makina; Nureki, Osamu; Numata, Keiji; Iwata, So

    2016-01-01

    The grease matrix was originally introduced as a microcrystal-carrier for serial femtosecond crystallography and has been expanded to applications for various types of proteins, including membrane proteins. However, the grease-based matrix has limited application for oil-sensitive proteins. Here we introduce a grease-free, water-based hyaluronic acid matrix. Applications for proteinase K and lysozyme proteins were able to produce electron density maps at 2.3-Å resolution. PMID:27087008

  8. Oil-free hyaluronic acid matrix for serial femtosecond crystallography

    NASA Astrophysics Data System (ADS)

    Sugahara, Michihiro; Song, Changyong; Suzuki, Mamoru; Masuda, Tetsuya; Inoue, Shigeyuki; Nakane, Takanori; Yumoto, Fumiaki; Nango, Eriko; Tanaka, Rie; Tono, Kensuke; Joti, Yasumasa; Kameshima, Takashi; Hatsui, Takaki; Yabashi, Makina; Nureki, Osamu; Numata, Keiji; Iwata, So

    2016-04-01

    The grease matrix was originally introduced as a microcrystal-carrier for serial femtosecond crystallography and has been expanded to applications for various types of proteins, including membrane proteins. However, the grease-based matrix has limited application for oil-sensitive proteins. Here we introduce a grease-free, water-based hyaluronic acid matrix. Applications for proteinase K and lysozyme proteins were able to produce electron density maps at 2.3-Å resolution.

  9. The inhibition of mating in Phycomyces blakesleeanus by light is dependent on the MadA-MadB complex that acts in a sex-specific manner.

    PubMed

    Shakya, Viplendra P S; Idnurm, Alexander

    2017-04-01

    Light is an environmental signal that influences reproduction in the Mucoromycotina fungi, as it does in many other species of fungi. Mating in Phycomyces blakesleeanus is inhibited by light, but the molecular mechanisms for this inhibition are uncharacterized. In this analysis, the role of the light-sensing MadA-MadB complex in mating was tested. The MadA-MadB complex is homologous to the Neurospora crassa White Collar complex. Three genes required for cell type determination in the sex locus or pheromone biosynthesis are transcriptionally-regulated by light and are controlled by MadA and MadB. This regulation acts through the plus partner, indicating that the inhibitory effect of light on mating is executed through only one of the two sexes. These results are an example whereby the mating types of fungi have acquired sex-specific properties beyond their role in conferring cell-type identity, and provide insight into how sex-determining chromosomal regions can expand the traits they control.

  10. Genome-wide analysis of the MADS-box gene family in Brassica rapa (Chinese cabbage).

    PubMed

    Duan, Weike; Song, Xiaoming; Liu, Tongkun; Huang, Zhinan; Ren, Jun; Hou, Xilin; Li, Ying

    2015-02-01

    The MADS-box gene family is an ancient and well-studied transcription factor family that functions in almost every developmental process in plants. There are a number of reports about the MADS-box family in different plant species, but systematic analysis of the MADS-box transcription factor family in Brassica rapa (Chinese cabbage) is still lacking. In this study, 160 MADS-box transcription factors were identified from the entire Chinese cabbage genome and compared with the MADS-box factors from 21 other representative plant species. A detailed list of MADS proteins from these 22 species was sorted. Phylogenetic analysis of the BrMADS genes, together with their Arabidopsis and rice counterparts, showed that the BrMADS genes were categorised into type I (Mα, Mβ, Mγ) and type II (MIKC(C), MIKC*) groups, and the MIKC(C) proteins were further divided into 13 subfamilies. The Chinese cabbage type II group has 95 members, which is twice as much as the Arabidopsis type II group, indicating that the Chinese cabbage type II genes have been retained more frequently than the type I genes. Finally, RNA-seq transcriptome data and quantitative real-time PCR analysis revealed that BrMADS genes are expressed in a tissue-specific manner similar to Arabidopsis. Interestingly, a number of BrMIKC genes showed responses to different abiotic stress treatments, suggesting a function for some of the genes in these processes as well. Taken together, the characterization of the B. rapa MADS-box family presented here, will certainly help in the selection of appropriate candidate genes and further facilitate functional studies in Chinese cabbage.

  11. A 3D cellular context for the macromolecular world

    PubMed Central

    Patwardhan, Ardan; Ashton, Alun; Brandt, Robert; Butcher, Sarah; Carzaniga, Raffaella; Chiu, Wah; Collinson, Lucy; Doux, Pascal; Duke, Elizabeth; Ellisman, Mark H; Franken, Erik; Grünewald, Kay; Heriche, Jean-Karim; Koster, Abraham; Kühlbrandt, Werner; Lagerstedt, Ingvar; Larabell, Carolyn; Lawson, Catherine L; Saibil, Helen R; Sanz-García, Eduardo; Subramaniam, Sriram; Verkade, Paul; Swedlow, Jason R; Kleywegt, Gerard J

    2015-01-01

    We report the outcomes of the discussion initiated at the workshop entitled A 3D Cellular Context for the Macromolecular World and propose how data from emerging three-dimensional (3D) cellular imaging techniques—such as electron tomography, 3D scanning electron microscopy and soft X-ray tomography—should be archived, curated, validated and disseminated, to enable their interpretation and reuse by the biomedical community. PMID:25289590

  12. Ultrashort Laser Pulse Induced Electromagnetic Stress on Biological Macromolecular Systems.

    DTIC Science & Technology

    1979-11-01

    ULTRASHORT LASER PULSE INDUCED ~~~~~ ELECTROMAGNET IC STRESS ON BIOLOGICAL MACROMOLECULAR SYSTEMS Adam P. Bruckner , Ph.D. ( i~iiCJ. Michael ...AFSC, Brooks Air Force Base, Texas. Dr. John Taboada (RZL) was the Laboratory Project Scientjst..in...Charge When U.S. Goverrijie~t drawings...available to the general public , including foreignnations. Thi s technical report has been reviewed and is approved for publ i-cation. OHN TABOADA , Ph.D

  13. A strategy for dissecting the architectures of native macromolecular assemblies

    PubMed Central

    Shi, Yi; Pellarin, Riccardo; Fridy, Peter C.; Fernandez-Martinez, Javier; Thompson, Mary K.; Li, Yinyin; Wang, Qing Jun; Sali, Andrej; Rout, Michael P.; Chait, Brian T.

    2015-01-01

    Despite the central role of large multi-protein complexes in many biological processes, it remains challenging to elucidate their structures and particularly problematic to define the structures of native macromolecular assemblies, which are often of low abundance. Here, we present a strategy for isolating such complexes and for extracting distance restraints that allow the determination of their molecular architectures. The method was optimized to allow facile use of the extensive global resources of GFP-tagged transgenic cells and animals. PMID:26436480

  14. Neutron Crystallography for the Study of Hydrogen Bonds in Macromolecules.

    PubMed

    Oksanen, Esko; Chen, Julian C-H; Fisher, Suzanne Zoë

    2017-04-07

    Abstract: The hydrogen bond (H bond) is one of the most important interactions that form the foundation of secondary and tertiary protein structure. Beyond holding protein structures together, H bonds are also intimately involved in solvent coordination, ligand binding, and enzyme catalysis. The H bond by definition involves the light atom, H, and it is very difficult to study directly, especially with X-ray crystallographic techniques, due to the poor scattering power of H atoms. Neutron protein crystallography provides a powerful, complementary tool that can give unambiguous information to structural biologists on solvent organization and coordination, the electrostatics of ligand binding, the protonation states of amino acid side chains and catalytic water species. The method is complementary to X-ray crystallography and the dynamic data obtainable with NMR spectroscopy. Also, as it gives explicit H atom positions, it can be very valuable to computational chemistry where exact knowledge of protonation and solvent orientation can make a large difference in modeling. This article gives general information about neutron crystallography and shows specific examples of how the method has contributed to structural biology, structure-based drug design; and the understanding of fundamental questions of reaction mechanisms.

  15. VQLM: A visual query language for macromolecular structural databases

    SciTech Connect

    Cohen, D.; Dickinson, B.; Salem, H.

    1994-12-31

    Databases of macromolecular structures allow researchers to identify general principles of molecular behavior. They do this by providing a variety of data obtained under a number of different experimental conditions. Many new tools have been developed recently to aid in exploratory analysis of structural data. However, some queries of interest still require considerable manual filtering of data. In particular, studies attempting to make generalizations about complex arrangements of atoms or building blocks in macro-molecular structures cannot be approached directly with existing tools. Such studies are frequently carried out on only a few structures or else require a labor-intensive process. To address this problem, we have developed a visual language, VQLM (Visual Query Language for Macromolecules). A query is formulated in this language by drawing an abstract picture of sub-structures to be searched for in the database and specifying constraints on the objects in them. To illustrate the usefulness of our language, we show how to encode a number of queries that were found scientifically interesting in the published literature in molecular biology. VQLM relies on VQL, a new database language, as its underlying engine for database retrieval and computation. We believe that VQLM will make macromolecular structural data more accessible to scientists, enabling faster and deeper data analysis.

  16. Macromolecular Assemblage in the Design of a Synthetic AIDS Vaccine

    NASA Astrophysics Data System (ADS)

    Defoort, Jean-Philippe; Nardelli, Bernardetta; Huang, Wolin; Ho, David D.; Tam, James P.

    1992-05-01

    We describe a peptide vaccine model based on the mimicry of surface coat protein of a pathogen. This model used a macromolecular assemblage approach to amplify peptide antigens in liposomes or micelles. The key components of the model consisted of an oligomeric lysine scaffolding to amplify peptide antigens covalently 4-fold and a lipophilic membrane-anchoring group to further amplify noncovalently the antigens many-fold in liposomal or micellar form. A peptide antigen derived from the third variable domain of glycoprotein gp120 of human immunodeficiency virus type 1 (HIV-1), consisting of neutralizing, T-helper, and T-cytotoxic epitopes, was used in a macromolecular assemblage model (HIV-1 linear peptide amino acid sequence 308-331 in a tetravalent multiple antigen peptide system linked to tripalmitoyl-S-glycerylcysteine). The latter complex, in liposome or micelle, was used to immunize mice and guinea pigs without any adjuvant and found to induce gp120-specific antibodies that neutralize virus infectivity in vitro, elicit cytokine production, and prime CD8^+ cytotoxic T lymphocytes in vivo. Our results show that the macromolecular assemblage approach bears immunological mimicry of the gp120 of HIV virus and may lead to useful vaccines against HIV infection.

  17. Cryo-electron tomography for structural characterization of macromolecular complexes.

    PubMed

    Cope, Julia; Heumann, John; Hoenger, Andreas

    2011-08-01

    Cryo-electron tomography (cryo-ET) is an emerging 3-D reconstruction technology that combines the principles of tomographic 3-D reconstruction with the unmatched structural preservation of biological matter embedded in vitreous ice. Cryo-ET is particularly suited to investigating cell-biological samples and large macromolecular structures that are too polymorphic to be reconstructed by classical averaging-based 3-D reconstruction procedures. This unit aims to make cryo-ET accessible to newcomers and discusses the specialized equipment required, as well as relevant advantages and hurdles associated with sample preparation by vitrification and cryo-ET. Protocols describe specimen preparation, data recording and 3-D data reconstruction for cryo-ET, with a special focus on macromolecular complexes. A step-by-step procedure for specimen vitrification by plunge freezing is provided, followed by the general practicalities of tilt-series acquisition for cryo-ET, including advice on how to select an area appropriate for acquiring a tilt series. A brief introduction to the underlying computational reconstruction principles applied in tomography is described, along with instructions for reconstructing a tomogram from cryo-tilt series data. Finally, a method is detailed for extracting small subvolumes containing identical macromolecular structures from tomograms for alignment and averaging as a means to increase the signal-to-noise ratio and eliminate missing wedge effects inherent in tomographic reconstructions.

  18. Macromolecular amplification of binding response in superaptamer hydrogels.

    PubMed

    Bai, Wei; Gariano, Nicholas A; Spivak, David A

    2013-05-08

    It is becoming more important to detect ultralow concentrations of analytes for biomedical, environmental, and national security applications. Equally important is that new methods should be easy to use, inexpensive, portable, and if possible allow detection by the naked eye. By and large, detection of low concentrations of analytes cannot be achieved directly but requires signal amplification by catalysts, macromolecules, metal surfaces, or supramolecular aggregates. The rapidly progressing field of macromolecular signal amplification has been advanced using conjugated polymers, chirality in polymers, solvating polymers, and polymerization/depolymerization strategies. A new type of aptamer-based hydrogel with specific response to target proteins presented in this report demonstrates an additional category of macromolecular signal amplification. This superaptamer assembly provides the first example of using protein-specific aptamers to create volume-changing hydrogels with amplified response to the target protein. A remarkable aspect of these superaptamer hydrogels is that volume shrinking is visible to the naked eye down to femtomolar concentrations of protein. This extraordinary macromolecular amplification is attributed to a complex interplay between protein-aptamer supramolecular cross-links and the consequential reduction of excluded volume in the hydrogel. Specific recognition is even maintained in biological matrices such as urine and tears. Furthermore, the gels can be dried for long-term storage and regenerated for use without loss of activity. In practice, the ease of this biomarker detection method offers an alternative to traditional analytical techniques that require sophisticated instrumentation and highly trained personnel.

  19. Polybivalency and disordered proteins in ordering macromolecular assemblies.

    PubMed

    Barbar, Elisar; Nyarko, Afua

    2015-01-01

    Intrinsically disordered proteins (IDPs) are prevalent in macromolecular assemblies and are thought to mediate protein recognition in complex regulatory processes and signaling pathways. The formation of a polybivalent scaffold is a key process by which IDPs drive early steps in macromolecular assemblies. Three intrinsically disordered proteins, IC, Swallow and Nup159, are core components, respectively, of cytoplasmic dynein, bicoid mRNA localization apparatus, and nuclear pore complexes. In all three systems, the hub protein LC8 recognizes on the IDP, short linear motifs that are fully disordered in the apo form, but adopt a β-strand when bound to LC8. The IDP/LC8 complex forms a bivalent scaffold primed to bind additional bivalent ligands. Scaffold formation also promotes self-association and/or higher order organization of the IDP components at a site distant from LC8 binding. Rigorous thermodynamic analyses imply that association of additional bivalent ligands is driven by entropic effects where the first binding event is weak but subsequent binding of additional ligands occurs with higher affinity. Here, we review specific examples of macromolecular assemblies in which polybivalency of aligned IDP duplexes not only enhances binding affinity and results in formation of a stable complex but also compensates unfavorable steric and enthalpic interactions. We propose that polybivalent scaffold assembly involving IDPs and LC8-like proteins is a general process in the cell biology of a class of multi-protein structures that are stable yet fine-tuned for diverse cellular requirements.

  20. Identifying, studying and making good use of macromolecular crystals

    PubMed Central

    Calero, Guillermo; Cohen, Aina E.; Luft, Joseph R.; Newman, Janet; Snell, Edward H.

    2014-01-01

    Structural biology has contributed tremendous knowledge to the understanding of life on the molecular scale. The Protein Data Bank, a depository of this structural knowledge, currently contains over 100 000 protein structures, with the majority stemming from X-ray crystallography. As the name might suggest, crystallography requires crystals. As detectors become more sensitive and X-ray sources more intense, the notion of a crystal is gradually changing from one large enough to embellish expensive jewellery to objects that have external dimensions of the order of the wavelength of visible light. Identifying these crystals is a prerequisite to their study. This paper discusses developments in identifying these crystals during crystallization screening and distinguishing them from other potential outcomes. The practical aspects of ensuring that once a crystal is identified it can then be positioned in the X-ray beam for data collection are also addressed. PMID:25084371

  1. A MAD Model for Gamma-Ray Burst Variability

    DOE PAGES

    Lloyd-Ronning, Nicole Marie; Dolence, Joshua C.; Fryer, Christopher Lee

    2016-06-09

    Here we present a model for the temporal variability of long gamma-ray bursts during the prompt phase (the highly variable first 100 seconds or so), in the context of a magnet- ically arrested disk (MAD) around a black hole. In this state, sufficient magnetic flux is held on to the black hole such that it stalls the accretion near the inner region of the disk. The system transitions in and out of the MAD state, which we relate to the vari- able luminosity of the GRB during the prompt phase, with a characteristic timescale defined by the free fall timemore » in the region over which the accretion is arrested. We present simple analytic estimates of the relevant energetics and timescales, and com- pare them to gamma-ray burst observations. In particular, we show how this model can reproduce the characteristic one second time scale that emerges from various analyses of the prompt emission light curve. Finally, we also discuss how our model can accommodate the potentially physically important correlation between a burst quiescent time and the duration of its subsequent pulse (Ramirez-Ruiz & Merloni 2001).« less

  2. A MAD Model for Gamma-Ray Burst Variability

    SciTech Connect

    Lloyd-Ronning, Nicole Marie; Dolence, Joshua C.; Fryer, Christopher Lee

    2016-06-09

    Here we present a model for the temporal variability of long gamma-ray bursts during the prompt phase (the highly variable first 100 seconds or so), in the context of a magnet- ically arrested disk (MAD) around a black hole. In this state, sufficient magnetic flux is held on to the black hole such that it stalls the accretion near the inner region of the disk. The system transitions in and out of the MAD state, which we relate to the vari- able luminosity of the GRB during the prompt phase, with a characteristic timescale defined by the free fall time in the region over which the accretion is arrested. We present simple analytic estimates of the relevant energetics and timescales, and com- pare them to gamma-ray burst observations. In particular, we show how this model can reproduce the characteristic one second time scale that emerges from various analyses of the prompt emission light curve. Finally, we also discuss how our model can accommodate the potentially physically important correlation between a burst quiescent time and the duration of its subsequent pulse (Ramirez-Ruiz & Merloni 2001).

  3. The MAD View on the Outskirts of Disks

    NASA Astrophysics Data System (ADS)

    Carollo, C. M.; Erroz-Ferrer, S.; den Brok, M.; Fagioli, M.; Onodera, M.; Tacchella, S. MAD Collaboration

    2017-03-01

    We present the MUSE Atlas of Disks (MAD), a GTO program with the MUSE spectrograph on the ESO/VLT that is dedicated to the study of the optical spectroscopic properties of z = 0 disk galaxies on the star-forming `Main Sequence' at < 100 pc physical resolution. MUSE pointings on the MAD galaxies extend out to ~ 2 disk scale lengths, enabling to investigate the bulge and inner disk properties of galaxies with different outer disks. Here we specifically compare, at constant stellar-mass, the stellar population properties of the inner components in disks with down-bending (Type II) and up-bending (Type III) outer profiles. We highlight similarities in the inner stellar properties of such different hosts, which point at a universal inside-out growth of disks, as well as differences which suggest an additional role of stellar migration and/or gas accretion in the growth of disk galaxies with an outer up-bending profile, as expected from theoretical models.

  4. BTA Magnet Field Map Archive and MAD Model

    SciTech Connect

    Glenn,J.W.

    2008-04-01

    This note publishes some and information that has resided in private files. The attached tables were provided by Joseph Skelly from his archives. They show magnetic field measurements versus excitation current for the Booster to AGS transfer line quadrupoles and dipoles based on field measurements [we believe] were done by the Magnet Division. Also given are Ed Blesser's fifth order fits of field versus current. The results are given in 'Tesla' or T-M/M. These tables are attached to provide an archive of this data. The MAD model of the BTA line does have the same values as shown in the attached fits so the transfer was correct. MAD uses as its 'gradient' for quads Tesla per meter normalized to rigidity [B-rho]. The model of the BTA line in use uses the T-M/M given in the tables divided by the length to give T M which is then normalized by Brho. Thus, the input to the model appears to be correct. The original model is also attached as part of a memo by Skelly describing it.

  5. AGL15, a MADS domain protein expressed in developing embryos.

    PubMed Central

    Heck, G R; Perry, S E; Nichols, K W; Fernandez, D E

    1995-01-01

    To extend our knowledge of genes expressed during early embryogenesis, the differential display technique was used to identify and isolate mRNA sequences that accumulate preferentially in young Brassica napus embryos. One of these genes encodes a new member of the MADS domain family of regulatory proteins; it has been designated AGL15 (for AGAMOUS-like). AGL15 shows a novel pattern of expression that is distinct from those of previously characterized family members. RNA gel blot analyses and in situ hybridization techniques were used to demonstrate that AGL15 mRNA accumulated primarily in the embryo and was present in all embryonic tissues, beginning at least as early as late globular stage in B. napus. Genomic and cDNA clones corresponding to two AGL15 genes from B. napus and the homologous single-copy gene from Arabidopsis, which is located on chromosome 5, were isolated and analyzed. Antibodies prepared against overexpressed Brassica AGL15 lacking the conserved MADS domain were used to probe immunoblots, and AGL15-related proteins were found in embryos of a variety of angiosperms, including plants as distantly related as maize. Based on these data, we suggest that AGL15 is likely to be an important component of the regulatory circuitry directing seed-specific processes in the developing embryo. PMID:7549483

  6. Overexpression of a MADS-Box Gene from Birch (Betula platyphylla) Promotes Flowering and Enhances Chloroplast Development in Transgenic Tobacco

    PubMed Central

    Qu, Guan-Zheng; Zheng, Tangchun; Liu, Guifeng; Wang, Wenjie; Zang, Lina; Liu, Huanzhen; Yang, Chuanping

    2013-01-01

    In this study, a MADS-box gene (BpMADS), which is an ortholog of AP1 from Arabidopsis, was isolated from birch (Betula platyphylla). Transgenic Arabidopsis containing a BpMADS promoter::GUS construct was produced, which exhibited strong GUS staining in sepal tissues. Ectopic expression of BpMADS significantly enhanced the flowering of tobacco (35S::BpMADS). In addition, the chloroplasts of transgenic tobacco exhibited much higher growth and division rates, as well rates of photosynthesis, than wild-type. A grafting experiment demonstrated that the flowering time of the scion was not affected by stock that overexpressed BpMADS. In addition, the overexpression of BpMADS resulted in the upregulation of some flowering-related genes in tobacco. PMID:23691043

  7. Drosophila Dullard functions as a Mad phosphatase to terminate BMP signaling

    PubMed Central

    Urrutia, Hugo; Aleman, Abigail; Eivers, Edward

    2016-01-01

    Bone morphogenetic proteins (BMPs) are growth factors that provide essential signals for normal embryonic development and adult tissue homeostasis. A key step in initiating BMP signaling is ligand induced phosphorylation of receptor Smads (R-Smads) by type I receptor kinases, while linker phosphorylation of R-Smads has been shown to cause BMP signal termination. Here we present data demonstrating that the phosphatase Dullard is involved in dephosphorylating the Drosophila R-Smad, Mad, and is integral in controlling BMP signal duration. We show that a hypomorphic Dullard allele or Dullard knockdown leads to increased Mad phosphorylation levels, while Dullard overexpression resulted in reduced Mad phosphorylations. Co-immunoprecipitation binding assays demonstrate phosphorylated Mad and Dullard physically interact, while mutation of Dullard’s phosphatase domain still allowed Mad-Dullard interactions but abolished its ability to regulate Mad phosphorylations. Finally, we demonstrate that linker and C-terminally phosphorylated Mad can be regulated by one of two terminating mechanisms, degradation by proteasomes or dephosphorylation by the phosphatase Dullard. PMID:27578171

  8. Involvement of CNOT3 in mitotic progression through inhibition of MAD1 expression

    SciTech Connect

    Takahashi, Akinori; Kikuguchi, Chisato; Morita, Masahiro; Shimodaira, Tetsuhiro; Tokai-Nishizumi, Noriko; Yokoyama, Kazumasa; Ohsugi, Miho; Suzuki, Toru; Yamamoto, Tadashi

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer CNOT3 depletion increases the mitotic index. Black-Right-Pointing-Pointer CNOT3 inhibits the expression of MAD1. Black-Right-Pointing-Pointer CNOT3 destabilizes the MAD1 mRNA. Black-Right-Pointing-Pointer MAD1 knockdown attenuates the CNOT3 depletion-induced mitotic arrest. -- Abstract: The stability of mRNA influences the dynamics of gene expression. The CCR4-NOT complex, the major deadenylase in mammalian cells, shortens the mRNA poly(A) tail and contributes to the destabilization of mRNAs. The CCR4-NOT complex plays pivotal roles in various physiological functions, including cell proliferation, apoptosis, and metabolism. Here, we show that CNOT3, a subunit of the CCR4-NOT complex, is involved in the regulation of the spindle assembly checkpoint, suggesting that the CCR4-NOT complex also plays a part in the regulation of mitosis. CNOT3 depletion increases the population of mitotic-arrested cells and specifically increases the expression of MAD1 mRNA and its protein product that plays a part in the spindle assembly checkpoint. We showed that CNOT3 depletion stabilizes the MAD1 mRNA, and that MAD1 knockdown attenuates the CNOT3 depletion-induced increase of the mitotic index. Basing on these observations, we propose that CNOT3 is involved in the regulation of the spindle assembly checkpoint through its ability to regulate the stability of MAD1 mRNA.

  9. Tetramer formation in Arabidopsis MADS domain proteins: analysis of a protein-protein interaction network

    PubMed Central

    2014-01-01

    Background MADS domain proteins are transcription factors that coordinate several important developmental processes in plants. These proteins interact with other MADS domain proteins to form dimers, and it has been proposed that they are able to associate as tetrameric complexes that regulate transcription of target genes. Whether the formation of functional tetramers is a widespread property of plant MADS domain proteins, or it is specific to few of these transcriptional regulators remains unclear. Results We analyzed the structure of the network of physical interactions among MADS domain proteins in Arabidopsis thaliana. We determined the abundance of subgraphs that represent the connection pattern expected for a MADS domain protein heterotetramer. These subgraphs were significantly more abundant in the MADS domain protein interaction network than in randomized analogous networks. Importantly, these subgraphs are not significantly frequent in a protein interaction network of TCP plant transcription factors, when compared to expectation by chance. In addition, we found that MADS domain proteins in tetramer-like subgraphs are more likely to be expressed jointly than proteins in other subgraphs. This effect is mainly due to proteins in the monophyletic MIKC clade, as there is no association between tetramer-like subgraphs and co-expression for proteins outside this clade. Conclusions Our results support that the tendency to form functional tetramers is widespread in the MADS domain protein-protein interaction network. Our observations also suggest that this trend is prevalent, or perhaps exclusive, for proteins in the MIKC clade. Because it is possible to retrodict several experimental results from our analyses, our work can be an important aid to make new predictions and facilitates experimental research on plant MADS domain proteins. PMID:24468197

  10. BpMADS4 has a central role in inflorescence initiation in silver birch (Betula pendula).

    PubMed

    Elo, Annakaisa; Lemmetyinen, Juha; Novak, Anu; Keinonen, Kaija; Porali, Ilkka; Hassinen, Minna; Sopanen, Tuomas

    2007-09-01

    Acceleration of flowering would be beneficial for breeding trees with a long juvenile phase; conversely, inhibition of flowering would prevent the spread of transgenes from the genetically modified trees. We have previously isolated and characterized several MADS genes from silver birch (Betula pendula Roth). In this study, we investigated the more detailed function of one of them, BpMADS4, a member of the APETALA1/FRUITFULL group of MADS genes. The expression of BpMADS4 starts at very early stage of the male and female inflorescence development and the activity is high in the apex of the developing inflorescence. Later, some expression is detected in the bracts and in the flower initials. Ectopic expression of BpMADS4 accelerates flowering dramatically in normally flowering clones and also in the early-flowering birch clone, in which the earliest line flowered about 11 days after rooting, when the saplings were only 3 cm high. The birches transformed with the BpMADS4 antisense construct showed remarkable delay in flowering and the number of flowering individuals was reduced. Two of the transformed lines did not show any signs of flower development during our 2-year study, whereas all the control plants formed inflorescences within 107 days. Our results show that BpMADS4 has a critical role in the initiation of birch inflorescence development and that BpMADS4 seems to be involved in the transition from vegetative to reproductive development. Therefore, BpMADS4 provides a promising tool for the genetic enhancement of forest trees.

  11. Workshop on algorithms for macromolecular modeling. Final project report, June 1, 1994--May 31, 1995

    SciTech Connect

    Leimkuhler, B.; Hermans, J.; Skeel, R.D.

    1995-07-01

    A workshop was held on algorithms and parallel implementations for macromolecular dynamics, protein folding, and structural refinement. This document contains abstracts and brief reports from that workshop.

  12. Whatever happened to the 'mad, bad' scientist? Overturning the stereotype.

    PubMed

    Haynes, Roslynn D

    2016-01-01

    The cluster of myths relating to the pursuit of knowledge has perpetuated the archetype of the alchemist/scientist as sinister, dangerous, possibly mad and threatening to society's values. Shelley's Frankenstein provided imagery and a vocabulary universally invoked in relation to scientific discoveries and technological innovation. The reasons for the longevity of this seemingly antiquated, semiotic imagery are discussed. In the twenty-first century, this stereotype has been radically revised, even overturned. Scientists are now rarely objects of fear or mockery. Mathematicians, both real-life and fictional, are discussed here as being representative of scientists now depicted empathically. This article examines possible sociological reasons for this reversal; what the revisionist image suggests about society's changed attitudes to science; and what might be the substitute fears and sources of horror.

  13. Opera and madness: Britten's Peter Grimes--a case study.

    PubMed

    Durà-Vilà, G; Bentley, D

    2009-12-01

    In this paper, Britten's opera Peter Grimes (1945) is used as an illustrative case study through which to examine the depiction of psychiatric disorders in opera. It is argued that Peter Grimes is a powerful example of how opera, in the hands of a great composer, can become an invaluable tool for examining subjective human experience. After a brief discussion of opera as a vehicle to express emotions, various operas are drawn upon to provide a historical perspective and to demonstrate the long interconnection existing between opera and madness. An in-depth analysis of Peter Grimes, its background and central character, is then provided, in order to demonstrate how opera can elicit empathy for individuals affected by mental health problems.

  14. Low-dose fixed-target serial synchrotron crystallography.

    PubMed

    Owen, Robin L; Axford, Danny; Sherrell, Darren A; Kuo, Anling; Ernst, Oliver P; Schulz, Eike C; Miller, R J Dwayne; Mueller-Werkmeister, Henrike M

    2017-04-01

    The development of serial crystallography has been driven by the sample requirements imposed by X-ray free-electron lasers. Serial techniques are now being exploited at synchrotrons. Using a fixed-target approach to high-throughput serial sampling, it is demonstrated that high-quality data can be collected from myoglobin crystals, allowing room-temperature, low-dose structure determination. The combination of fixed-target arrays and a fast, accurate translation system allows high-throughput serial data collection at high hit rates and with low sample consumption.

  15. From crystal morphology to molecular and scale crystallography

    NASA Astrophysics Data System (ADS)

    Janner, A.; Janssen, T.

    2015-08-01

    A number of topics, ranging from morphology of aperiodic crystals to indexed enclosing forms of axial-symmetric proteins, nucleic acids and viruses, have been selected among those investigated by the authors in 50 years of research. The basic symmetries involved in fields like superspace, molecular and scale crystallography, are considered from a personal point of view in their time evolution. A number of specific subjects follow, chosen among a few highlights and presented according to the experience of the authors: snow crystals, calaverite {{AuTe}}2, the incommensurately modulated crystals {{Rb}}2{{ZnBr}}4, {[{N}{({{CH}}3)}4]}2{{ZnCl}}4 and the mitochondrial ferritin.

  16. Crystallography of lath martensite and stabilization of retained austenite

    SciTech Connect

    Sarikaya. M.

    1982-10-01

    TEM was used to study the morphology and crystallography of lath martensite in low and medium carbon steels in the as-quenched and 200/sup 0/C tempered conditions. The steels have microduplex structures of dislocated lath martensite and continuous thin films of retained austenite at the lath interfaces. Stacks of laths form the packets which are derived from different (111) variants of the same austenite grain. The residual parent austenite enables microdiffraction experiments with small electron beam spot sizes for the orientation relationships (OR) between austenite and martensite. All three most commonly observed ORs, namely Kurdjumov-Sachs, Nishiyama-Wassermann, and Greninger-Troiano, operate within the same sample.

  17. High-Resolution Protein Structure Determination by Serial Femtosecond Crystallography

    PubMed Central

    Boutet, Sébastien; Lomb, Lukas; Williams, Garth J.; Barends, Thomas R. M.; Aquila, Andrew; Doak, R. Bruce; Weierstall, Uwe; DePonte, Daniel P.; Steinbrener, Jan; Shoeman, Robert L.; Messerschmidt, Marc; Barty, Anton; White, Thomas A.; Kassemeyer, Stephan; Kirian, Richard A.; Seibert, M. Marvin; Montanez, Paul A.; Kenney, Chris; Herbst, Ryan; Hart, Philip; Pines, Jack; Haller, Gunther; Gruner, Sol M.; Philipp, Hugh T.; Tate, Mark W.; Hromalik, Marianne; Koerner, Lucas J.; van Bakel, Niels; Morse, John; Ghonsalves, Wilfred; Arnlund, David; Bogan, Michael J.; Caleman, Carl; Fromme, Raimund; Hampton, Christina Y.; Hunter, Mark S.; Johansson, Linda C.; Katona, Gergely; Kupitz, Christopher; Liang, Mengning; Martin, Andrew V.; Nass, Karol; Redecke, Lars; Stellato, Francesco; Timneanu, Nicusor; Wang, Dingjie; Zatsepin, Nadia A.; Schafer, Donald; Defever, James; Neutze, Richard; Fromme, Petra; Spence, John C. H.; Chapman, Henry N.; Schlichting, Ilme

    2013-01-01

    Structure determination of proteins and other macromolecules has historically required the growth of high-quality crystals sufficiently large to diffract x-rays efficiently while withstanding radiation damage. We applied serial femtosecond crystallography (SFX) using an x-ray free-electron laser (XFEL) to obtain high-resolution structural information from microcrystals (less than 1 micrometer by 1 micrometer by 3 micrometers) of the well-characterized model protein lysozyme. The agreement with synchrotron data demonstrates the immediate relevance of SFX for analyzing the structure of the large group of difficult-to-crystallize molecules. PMID:22653729

  18. High-resolution protein structure determination by serial femtosecond crystallography.

    PubMed

    Boutet, Sébastien; Lomb, Lukas; Williams, Garth J; Barends, Thomas R M; Aquila, Andrew; Doak, R Bruce; Weierstall, Uwe; DePonte, Daniel P; Steinbrener, Jan; Shoeman, Robert L; Messerschmidt, Marc; Barty, Anton; White, Thomas A; Kassemeyer, Stephan; Kirian, Richard A; Seibert, M Marvin; Montanez, Paul A; Kenney, Chris; Herbst, Ryan; Hart, Philip; Pines, Jack; Haller, Gunther; Gruner, Sol M; Philipp, Hugh T; Tate, Mark W; Hromalik, Marianne; Koerner, Lucas J; van Bakel, Niels; Morse, John; Ghonsalves, Wilfred; Arnlund, David; Bogan, Michael J; Caleman, Carl; Fromme, Raimund; Hampton, Christina Y; Hunter, Mark S; Johansson, Linda C; Katona, Gergely; Kupitz, Christopher; Liang, Mengning; Martin, Andrew V; Nass, Karol; Redecke, Lars; Stellato, Francesco; Timneanu, Nicusor; Wang, Dingjie; Zatsepin, Nadia A; Schafer, Donald; Defever, James; Neutze, Richard; Fromme, Petra; Spence, John C H; Chapman, Henry N; Schlichting, Ilme

    2012-07-20

    Structure determination of proteins and other macromolecules has historically required the growth of high-quality crystals sufficiently large to diffract x-rays efficiently while withstanding radiation damage. We applied serial femtosecond crystallography (SFX) using an x-ray free-electron laser (XFEL) to obtain high-resolution structural information from microcrystals (less than 1 micrometer by 1 micrometer by 3 micrometers) of the well-characterized model protein lysozyme. The agreement with synchrotron data demonstrates the immediate relevance of SFX for analyzing the structure of the large group of difficult-to-crystallize molecules.

  19. A prototype direct-detection CCD for protein crystallography.

    PubMed

    Green, Katherine S; Szebenyi, Doletha M E; Boggs, Kasey; Bredthauer, Richard; Tate, Mark W; Gruner, Sol M

    2013-08-01

    The fabrication and testing of a prototype deep-depletion direct-conversion X-ray CCD detector are described. The device is fabricated on 600 µm-thick high-resistivity silicon, with 24 × 24 µm pixels in a 4k × 4k pixel format. Calibration measurements and the results of initial protein crystallography experiments at the Cornell High Energy Synchrotron Source (CHESS) F1 beamline are described, as well as suggested improvements for future versions of the detector.

  20. Membrane-Protein Crystallography and Potentiality for Drug Design

    NASA Astrophysics Data System (ADS)

    Yamashita, Atsuko

    Structure-based drug design for membrane proteins is far behind that for soluble proteins due to difficulty in crystallographic structure determination, despite the fact that about 60% of FDA-approved drugs target membrane proteins located at the cell surface. Stable homologs for a membrane protein of interest, such as prokaryotic neurotransmitter transporter homolog LeuT, might enable cooperative analyses by crystallography and functional assays, provide useful information for functional mechanisms, and thus serve as important probes for drug design based on mechanisms as well as structures.

  1. A General Method for Modeling Macromolecular Shape in Solution

    PubMed Central

    Harding, Stephen E.

    1987-01-01

    A general method for modeling macromolecular shape in solution is described involving measurements of viscosity, radius of gyration, and the second thermodynamic virial coefficient. The method, which should be relatively straightforward to apply, does not suffer from uniqueness problems, involves shape functions that are independent of hydration, and models the gross conformation of the macromolecule in solution as a general triaxial ellipsoid. The method is illustrated by application to myosin, and the relevance and applicability of ellipsoid modeling to biological structures is discussed. PMID:19431695

  2. Bringing macromolecular machinery to life using 3D animation.

    PubMed

    Iwasa, Janet H

    2015-04-01

    Over the past decade, there has been a rapid rise in the use of three-dimensional (3D) animation to depict molecular and cellular processes. Much of the growth in molecular animation has been in the educational arena, but increasingly, 3D animation software is finding its way into research laboratories. In this review, I will discuss a number of ways in which 3d animation software can play a valuable role in visualizing and communicating macromolecular structures and dynamics. I will also consider the challenges of using animation tools within the research sphere.

  3. Macromolecular therapeutics in cancer treatment: the EPR effect and beyond.

    PubMed

    Maeda, Hiroshi

    2012-12-10

    In this review, I have discussed various issues of the cancer drug targeting primarily related to the EPR (enhanced permeability and retention) effect, which utilized nanomedicine or macromolecular drugs. The content goes back to the development of the first polymer-protein conjugate anticancer agent SMANCS and development of the arterial infusion in Lipiodol formulation into the tumor feeding artery (hepatic artery for hepatoma). The brief account on the EPR effect and its definition, factors involved, heterogeneity, and various methods of augmentation of the EPR effect, which showed remarkably improved clinical outcomes are also discussed. Various obstacles involved in drug developments and commercialization are also discussed through my personal experience and recollections.

  4. Thermodynamic signatures in macromolecular interactions involving conformational flexibility.

    PubMed

    Menzel, Anja; Neumann, Piotr; Schwieger, Christian; Stubbs, Milton T

    2014-07-01

    The energetics of macromolecular interactions are complex, particularly where protein flexibility is involved. Exploiting serendipitous differences in the plasticity of a series of closely related trypsin variants, we analyzed the enthalpic and entropic contributions accompanying interaction with L45K-eglin C. Binding of the four variants show significant differences in released heat, although the affinities vary little, in accordance with the principle of enthalpy-entropy compensation. Binding of the most disordered variant is almost entirely enthalpically driven, with practically no entropy change. As structures of the complexes reveal negligible differences in protein-inhibitor contacts, we conclude that solvent effects contribute significantly to binding affinities.

  5. C- and D-class MADS-box genes from Phalaenopsis equestris (Orchidaceae) display functions in gynostemium and ovule development.

    PubMed

    Chen, You-Yi; Lee, Pei-Fang; Hsiao, Yu-Yun; Wu, Wan-Lin; Pan, Zhao-Jun; Lee, Yung-I; Liu, Ke-Wei; Chen, Li-Jun; Liu, Zhong-Jian; Tsai, Wen-Chieh

    2012-06-01

    Gynostemium and ovule development in orchid are unique developmental processes in the plant kingdom. Characterization of C- and D-class MADS-box genes could help reveal the molecular mechanisms underlying gynostemium and ovule development in orchids. In this study, we isolated and characterized a C- and a D-class gene, PeMADS1 and PeMADS7, respectively, from Phalaenopsis equestris. These two genes showed parallel spatial and temporal expression profiles, which suggests their cooperation in gynostemium and ovule development. Furthermore, only PeMADS1 was ectopically expressed in the petals of the gylp (gynostemium-like petal) mutant, whose petals were transformed into gynostemium-like structures. Protein-protein interaction analyses revealed that neither PeMADS1 and PeMADS7 could form a homodimer or a heterodimer. An E-class protein was needed to bridge the interaction between these two proteins. A complementation test revealed that PeMADS1 could rescue the phenotype of the AG mutant. Overexpression of PeMADS7 in Arabidopsis caused typical phenotypes of the D-class gene family. Together, these results indicated that both C-class PeMADS1 and D-class PeMADS7 play important roles in orchid gynostemium and ovule development.

  6. Functional characterization of GhSOC1 and GhMADS42 homologs from upland cotton (Gossypium hirsutum L.).

    PubMed

    Zhang, Xiaohong; Wei, Jianghui; Fan, Shuli; Song, Meizhen; Pang, Chaoyou; Wei, Hengling; Wang, Chengshe; Yu, Shuxun

    2016-01-01

    In Arabidopsis flowering pathway, MADS-box genes encode transcription factors, with their structures and functions highly conserved in many species. In our study, two MADS-box genes GhSOC1 and GhMADS42 (Gossypium hirsutum L.) were cloned from upland cotton CCRI36 and transformed into Arabidopsis. GhSOC1 was additionally transformed into upland cotton. Comparative analysis demonstrated sequence conservation between GhSOC1 and GhMADS42 and genes of other plant species. Tissue-specific expression analysis of GhSOC1 and GhMADS42 revealed spatiotemporal expression patterns involving high transcript levels in leaves, shoot apical buds, and flowers. In addition, overexpression of both GhSOC1 and GhMADS42 in Arabidopsis accelerated flowering, with GhMADS42 transgenic plants showing abnormal floral organ phenotypes. Overexpression of GhSOC1 in upland cotton also produced variations in floral organs. Furthermore, chromatin immunoprecipitation assay demonstrated that GhSOC1 could regulate GhMADS41 and GhMADS42, but not FLOWERING LOCUS T, by directly binding to the genes promoter. Finally, yeast two-hybrid and bimolecular fluorescence complementation approaches were undertaken to better understand the interaction of GhSOC1 and other MADS-box factors. These experiments showed that GhSOC1 can interact with APETALA1/FRUITFULL-like proteins in cotton.

  7. Emotional and deliberative reactions to a public crisis: Mad Cow disease in France.

    PubMed

    Sinaceur, Marwan; Heath, Chip; Cole, Steve

    2005-03-01

    Although most theories of choice are cognitive, recent research has emphasized the role of emotions. We used a novel context--the Mad Cow crisis in France--to investigate how emotions alter choice even when consequences are held constant. A field study showed that individuals reduced beef consumption in months after many newspaper articles featured the emotional label "Mad Cow," but beef consumption was unaffected after articles featured scientific labels for the same disease. The reverse pattern held for the disease-related actions of a government bureaucracy. A lab study showed that the Mad Cow label induces people to make choices based solely on emotional reactions, whereas scientific labels induce people to consider their own probability judgments. Although the Mad Cow label produces less rational behavior than scientific labels, it is two to four times more common in the environment.

  8. The role of masseter muscle EMG during DISE to predict the effectiveness of MAD: preliminary results.

    PubMed

    Marchese, M R; Scarano, E; Rizzotto, G; Grippaudo, C; Paludetti, G

    2016-12-01

    The use of a mandibular advancement device (MAD) increases the activity of the temporo-mandibular (TM) complex and masseter (MM) muscles with the risk of reducing treatment compliance. Predictors of treatment outcome are of importance in selecting patients who might benefit from MAD without side effects. The role of mandibular advancement (MA) during drug-induced sleep endoscopy (DISE) is controversial. In three cases (BMI < 30) affected by non-severe OSAS (AHI < 30 e/h), we recorded the surface EMG signal of MM activity during DISE. At follow-up all cases improved the AHI, two cases that showed transient increase of MM activity did not suffer from changes of overjet and did not complain of discomfort with the use of MAD. The case that showed a continuing increase of MM activity reported TM discomfort without changes of dental occlusion. EMG of MM during DISE may contribute to ameliorate the selection of cases amenable to treatment with MAD.

  9. Characterisation and fate of grayanatoxins in mad honey produced from Rhododendron ponticum nectar.

    PubMed

    Kurtoglu, Arzum Bahar; Yavuz, Recep; Evrendilek, Gulsun Akdemir

    2014-10-15

    Mad honey from Rhododendron ponticum nectar is produced in a large quantity in the western Black Sea region of Turkey and causes poisoning due to consumption of grayanatoxins (GTX I and III). There are a few studies about characterisation of GTXs in mad honey produced from R.ponticum. This study quantified basic properties including concentrations of GTX I and GTX III in mad honey samples collected in three consecutive years. Although the chemical composition of mad honey varied annually depending on the production year, mean GTX I and GTX III contents were estimated at 20.4±1.69 and 8.20±1.93mg/kg, respectively. The concentrations of GTXs did not change significantly during storage of 6months.

  10. Role for the banana AGAMOUS-like gene MaMADS7 in regulation of fruit ripening and quality.

    PubMed

    Liu, Juhua; Liu, Lin; Li, Yujia; Jia, Caihong; Zhang, Jianbin; Miao, Hongxia; Hu, Wei; Wang, Zhuo; Xu, Biyu; Jin, Zhiqiang

    2015-11-01

    MADS-box transcription factors play important roles in organ development. In plants, most studies on MADS-box genes have mainly focused on flower development and only a few concerned fruit development and ripening. A new MADS-box gene named MaMADS7 was isolated from banana fruit by rapid amplification of cDNA ends (RACE) based on a MADS-box fragment obtained from a banana suppression subtractive hybridization (SSH) cDNA library. MaMADS7 is an AGAMOUS-like MADS-box gene that is preferentially expressed in the ovaries and fruits and in tobacco its protein product localizes to the nucleus. This study found that MaMADS7 expression can be induced by exogenous ethylene. Ectopic expression of MaMADS7 in tomato resulted in broad ripening phenotypes. The expression levels of seven ripening and quality-related genes, ACO1, ACS2, E4, E8, PG, CNR and PSY1 in MaMADS7 transgenic tomato fruits were greatly increased while the expression of the AG-like MADS-box gene TAGL1 was suppressed. Compared with the control, the contents of β-carotene, lycopene, ascorbic acid and organic acid in transformed tomato fruits were increased, while the contents of glucose and fructose were slightly decreased. MaMADS7 interacted with banana 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene 1 (MaACO1) and tomato phytoene synthase gene (LePSY1) promoters. Our results indicated that MaMADS7 plays an important role in initiating endogenous ethylene biosynthesis and fruit ripening.

  11. C-terminal region of Mad2 plays an important role during mitotic spindle checkpoint in fission yeast Schizosaccharomyces pombe.

    PubMed

    Singh, Gaurav Kumar; Karade, Sharanbasappa Shrimant; Ranjan, Rajeev; Ahamad, Nafees; Ahmed, Shakil

    2017-02-01

    The mitotic arrest deficiency 2 (Mad2) protein is an essential component of the spindle assembly checkpoint that interacts with Cdc20/Slp1 and inhibit its ability to activate anaphase promoting complex/cyclosome (APC/C). In bladder cancer cell line the C-terminal residue of the mad2 gene has been found to be deleted. In this study we tried to understand the role of the C-terminal region of mad2 on the spindle checkpoint function. To envisage the role of C-terminal region of Mad2, we truncated 25 residues of Mad2 C-terminal region in fission yeast S.pombe and characterized its effect on spindle assembly checkpoint function. The cells containing C-terminal truncation of Mad2 exhibit sensitivity towards microtubule destabilizing agent suggesting perturbation of spindle assembly checkpoint. Further, the C-terminal truncation of Mad2 exhibit reduced viability in the nda3-KM311 mutant background at non-permissive temperature. Truncation in mad2 gene also affects its foci forming ability at unattached kinetochore suggesting that the mad2-∆CT mutant is unable to maintain spindle checkpoint activation. However, in response to the defective microtubule, only brief delay of mitotic progression was observed in Mad2 C-terminal truncation mutant. In addition we have shown that the deletion of two β strands of Mad2 protein abolishes its ability to interact with APC activator protein Slp1/Cdc20. We purpose that the truncation of two β strands (β7 and β8) of Mad2 destabilize the safety belt and affect the Cdc20-Mad2 interaction leading to defects in the spindle checkpoint activation.

  12. Control system for the 2nd generation Berkeley AutoMounters (BAM2) at GM/CA CAT macromolecular crystallography beamlines.

    PubMed

    Makarov, O; Hilgart, M; Ogata, C; Pothineni, S; Cork, C

    2011-09-01

    GM/CA CAT at Sector 23 of the Advanced Photon Source (APS) is an NIH funded facility for crystallographic structure determination of biological macromolecules by X-ray diffraction.A second generation Berkeley automounter is being integrated into the beamline control system at the 23-BM experimental station. This new device replaces the previous all-pneumatic gripper motions with a combination of pneumatics and XYZ motorized linear stages. The latter adds a higher degree of flexibility to the robot including auto-alignment capability, accommodation of a larger capacity sample Dewar of arbitrary shape, and support for advanced operations such as crystal washing, while preserving the overall simplicity and efficiency of the Berkeley automounter design.

  13. A novel inert crystal delivery medium for serial femtosecond crystallography

    SciTech Connect

    Conrad, Chelsie E.; Basu, Shibom; James, Daniel; Wang, Dingjie; Schaffer, Alexander; Roy-Chowdhury, Shatabdi; Zatsepin, Nadia A.; Aquila, Andrew; Coe, Jesse; Gati, Cornelius; Hunter, Mark S.; Koglin, Jason E.; Kupitz, Christopher; Nelson, Garrett; Subramanian, Ganesh; White, Thomas A.; Zhao, Yun; Zook, James; Boutet, Sébastien; Cherezov, Vadim; Spence, John C. H.; Fromme, Raimund; Weierstall, Uwe; Fromme, Petra

    2015-06-30

    Serial femtosecond crystallography (SFX) has opened a new era in crystallography by permitting nearly damage-free, room-temperature structure determination of challenging proteins such as membrane proteins. In SFX, femtosecond X-ray free-electron laser pulses produce diffraction snapshots from nanocrystals and microcrystals delivered in a liquid jet, which leads to high protein consumption. A slow-moving stream of agarose has been developed as a new crystal delivery medium for SFX. It has low background scattering, is compatible with both soluble and membrane proteins, and can deliver the protein crystals at a wide range of temperatures down to 4°C. Using this crystal-laden agarose stream, the structure of a multi-subunit complex, phycocyanin, was solved to 2.5Å resolution using 300µg of microcrystals embedded into the agarose medium post-crystallization. The agarose delivery method reduces protein consumption by at least 100-fold and has the potential to be used for a diverse population of proteins, including membrane protein complexes.

  14. High-pressure crystallography of periodic and aperiodic crystals

    PubMed Central

    Hejny, Clivia; Minkov, Vasily S.

    2015-01-01

    More than five decades have passed since the first single-crystal X-ray diffraction experiments at high pressure were performed. These studies were applied historically to geochemical processes occurring in the Earth and other planets, but high-pressure crystallography has spread across different fields of science including chemistry, physics, biology, materials science and pharmacy. With each passing year, high-pressure studies have become more precise and comprehensive because of the development of instrumentation and software, and the systems investigated have also become more complicated. Starting with crystals of simple minerals and inorganic compounds, the interests of researchers have shifted to complicated metal–organic frameworks, aperiodic crystals and quasicrystals, molecular crystals, and even proteins and viruses. Inspired by contributions to the microsymposium ‘High-Pressure Crystallography of Periodic and Aperiodic Crystals’ presented at the 23rd IUCr Congress and General Assembly, the authors have tried to summarize certain recent results of single-crystal studies of molecular and aperiodic structures under high pressure. While the selected contributions do not cover the whole spectrum of high-pressure research, they demonstrate the broad diversity of novel and fascinating results and may awaken the reader’s interest in this topic. PMID:25866659

  15. Proline: Mother Nature;s cryoprotectant applied to protein crystallography

    SciTech Connect

    Pemberton, Travis A.; Still, Brady R.; Christensen, Emily M.; Singh, Harkewal; Srivastava, Dhiraj; Tanner, John J.

    2012-09-05

    L-Proline is one of Mother Nature's cryoprotectants. Plants and yeast accumulate proline under freeze-induced stress and the use of proline in the cryopreservation of biological samples is well established. Here, it is shown that L-proline is also a useful cryoprotectant for protein crystallography. Proline was used to prepare crystals of lysozyme, xylose isomerase, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase for low-temperature data collection. The crystallization solutions in these test cases included the commonly used precipitants ammonium sulfate, sodium chloride and polyethylene glycol and spanned the pH range 4.6-8.5. Thus, proline is compatible with typical protein-crystallization formulations. The proline concentration needed for cryoprotection of these crystals is in the range 2.0-3.0 M. Complete data sets were collected from the proline-protected crystals. Proline performed as well as traditional cryoprotectants based on the diffraction resolution and data-quality statistics. The structures were refined to assess the binding of proline to these proteins. As observed with traditional cryoprotectants such as glycerol and ethylene glycol, the electron-density maps clearly showed the presence of proline molecules bound to the protein. In two cases, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase, proline binds in the active site. It is concluded that L-proline is an effective cryoprotectant for protein crystallography.

  16. Smarter Drugs: How Protein Crystallography Revolutionizes Drug Design

    SciTech Connect

    Smith, Clyde

    2005-04-26

    According to Smith, protein crystallography allows scientists to design drugs in a much more efficient way than the standard methods traditionally used by large drug companies, which can cost close to a billion dollars and take 10 to 15 years. 'A lot of the work can be compressed down,' Smith said. Protein crystallography enables researchers to learn the structure of molecules involved in disease and health. Seeing the loops, folds and placement of atoms in anything from a virus to a healthy cell membrane gives important information about how these things work - and how to encourage, sidestep or stop their functions. Drug design can be much faster when the relationship between structure and function tells you what area of a molecule to target. Smith will use a timeline to illustrate the traditional methods of drug development and the new ways it can be done now. 'It is very exciting work. There have been some failures, but many successes too.' A new drug to combat the flu was developed in a year or so. Smith will tell us how. He will also highlight drugs developed to combat HIV, Tuberculosis, hypertension and Anthrax.

  17. Serial crystallography on in vivo grown microcrystals using synchrotron radiation

    PubMed Central

    Gati, Cornelius; Bourenkov, Gleb; Klinge, Marco; Rehders, Dirk; Stellato, Francesco; Oberthür, Dominik; Yefanov, Oleksandr; Sommer, Benjamin P.; Mogk, Stefan; Duszenko, Michael; Betzel, Christian; Schneider, Thomas R.; Chapman, Henry N.; Redecke, Lars

    2014-01-01

    Crystal structure determinations of biological macromolecules are limited by the availability of sufficiently sized crystals and by the fact that crystal quality deteriorates during data collection owing to radiation damage. Exploiting a micrometre-sized X-ray beam, high-precision diffractometry and shutterless data acquisition with a pixel-array detector, a strategy for collecting data from many micrometre-sized crystals presented to an X-ray beam in a vitrified suspension is demonstrated. By combining diffraction data from 80 Trypanosoma brucei procathepsin B crystals with an average volume of 9 µm3, a complete data set to 3.0 Å resolution has been assembled. The data allowed the refinement of a structural model that is consistent with that previously obtained using free-electron laser radiation, providing mutual validation. Further improvements of the serial synchrotron crystallography technique and its combination with serial femtosecond crystallography are discussed that may allow the determination of high-resolution structures of micrometre-sized crystals. PMID:25075324

  18. A novel inert crystal delivery medium for serial femtosecond crystallography

    DOE PAGES

    Conrad, Chelsie E.; Basu, Shibom; James, Daniel; ...

    2015-06-30

    Serial femtosecond crystallography (SFX) has opened a new era in crystallography by permitting nearly damage-free, room-temperature structure determination of challenging proteins such as membrane proteins. In SFX, femtosecond X-ray free-electron laser pulses produce diffraction snapshots from nanocrystals and microcrystals delivered in a liquid jet, which leads to high protein consumption. A slow-moving stream of agarose has been developed as a new crystal delivery medium for SFX. It has low background scattering, is compatible with both soluble and membrane proteins, and can deliver the protein crystals at a wide range of temperatures down to 4°C. Using this crystal-laden agarose stream, themore » structure of a multi-subunit complex, phycocyanin, was solved to 2.5 Å resolution using 300 µg of microcrystals embedded into the agarose medium post-crystallization. The agarose delivery method reduces protein consumption by at least 100-fold and has the potential to be used for a diverse population of proteins, including membrane protein complexes.« less

  19. A novel inert crystal delivery medium for serial femtosecond crystallography

    SciTech Connect

    Conrad, Chelsie E.; Basu, Shibom; James, Daniel; Wang, Dingjie; Schaffer, Alexander; Roy-Chowdhury, Shatabdi; Zatsepin, Nadia A.; Aquila, Andrew; Coe, Jesse; Gati, Cornelius; Hunter, Mark S.; Koglin, Jason E.; Kupitz, Christopher; Nelson, Garrett; Subramanian, Ganesh; White, Thomas A.; Zhao, Yun; Zook, James; Boutet, Sébastien; Cherezov, Vadim; Spence, John C. H.; Fromme, Raimund; Weierstall, Uwe; Fromme, Petra

    2015-06-30

    Serial femtosecond crystallography (SFX) has opened a new era in crystallography by permitting nearly damage-free, room-temperature structure determination of challenging proteins such as membrane proteins. In SFX, femtosecond X-ray free-electron laser pulses produce diffraction snapshots from nanocrystals and microcrystals delivered in a liquid jet, which leads to high protein consumption. A slow-moving stream of agarose has been developed as a new crystal delivery medium for SFX. It has low background scattering, is compatible with both soluble and membrane proteins, and can deliver the protein crystals at a wide range of temperatures down to 4°C. Using this crystal-laden agarose stream, the structure of a multi-subunit complex, phycocyanin, was solved to 2.5 Å resolution using 300 µg of microcrystals embedded into the agarose medium post-crystallization. The agarose delivery method reduces protein consumption by at least 100-fold and has the potential to be used for a diverse population of proteins, including membrane protein complexes.

  20. Combining X-ray and neutron crystallography with spectroscopy

    PubMed Central

    Smith, Oliver

    2017-01-01

    X-ray protein crystallography has, through the determination of the three-dimensional structures of enzymes and their complexes, been essential to the understanding of biological chemistry. However, as X-rays are scattered by electrons, the technique has difficulty locating the presence and position of H atoms (and cannot locate H+ ions), knowledge of which is often crucially important for the understanding of enzyme mechanism. Furthermore, X-ray irradiation, through photoelectronic effects, will perturb the redox state in the crystal. By using single-crystal spectrophotometry, reactions taking place in the crystal can be monitored, either to trap intermediates or follow photoreduction during X-ray data collection. By using neutron crystallography, the positions of H atoms can be located, as it is the nuclei rather than the electrons that scatter neutrons, and the scattering length is not determined by the atomic number. Combining the two techniques allows much greater insight into both reaction mechanism and X-ray-induced photoreduction. PMID:28177310

  1. Proline: Mother Nature's cryoprotectant applied to protein crystallography.

    PubMed

    Pemberton, Travis A; Still, Brady R; Christensen, Emily M; Singh, Harkewal; Srivastava, Dhiraj; Tanner, John J

    2012-08-01

    L-Proline is one of Mother Nature's cryoprotectants. Plants and yeast accumulate proline under freeze-induced stress and the use of proline in the cryopreservation of biological samples is well established. Here, it is shown that L-proline is also a useful cryoprotectant for protein crystallography. Proline was used to prepare crystals of lysozyme, xylose isomerase, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase for low-temperature data collection. The crystallization solutions in these test cases included the commonly used precipitants ammonium sulfate, sodium chloride and polyethylene glycol and spanned the pH range 4.6-8.5. Thus, proline is compatible with typical protein-crystallization formulations. The proline concentration needed for cryoprotection of these crystals is in the range 2.0-3.0 M. Complete data sets were collected from the proline-protected crystals. Proline performed as well as traditional cryoprotectants based on the diffraction resolution and data-quality statistics. The structures were refined to assess the binding of proline to these proteins. As observed with traditional cryoprotectants such as glycerol and ethylene glycol, the electron-density maps clearly showed the presence of proline molecules bound to the protein. In two cases, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase, proline binds in the active site. It is concluded that L-proline is an effective cryoprotectant for protein crystallography.

  2. Ab initio solution of macromolecular crystal structures without direct methods.

    PubMed

    McCoy, Airlie J; Oeffner, Robert D; Wrobel, Antoni G; Ojala, Juha R M; Tryggvason, Karl; Lohkamp, Bernhard; Read, Randy J

    2017-04-04

    The majority of macromolecular crystal structures are determined using the method of molecular replacement, in which known related structures are rotated and translated to provide an initial atomic model for the new structure. A theoretical understanding of the signal-to-noise ratio in likelihood-based molecular replacement searches has been developed to account for the influence of model quality and completeness, as well as the resolution of the diffraction data. Here we show that, contrary to current belief, molecular replacement need not be restricted to the use of models comprising a substantial fraction of the unknown structure. Instead, likelihood-based methods allow a continuum of applications depending predictably on the quality of the model and the resolution of the data. Unexpectedly, our understanding of the signal-to-noise ratio in molecular replacement leads to the finding that, with data to sufficiently high resolution, fragments as small as single atoms of elements usually found in proteins can yield ab initio solutions of macromolecular structures, including some that elude traditional direct methods.

  3. Production of macromolecular chloramines by chlorine-transfer reactions.

    PubMed

    Bedner, Mary; MacCrehan, William A; Helz, George R

    2004-03-15

    Chlorination of treated wastewaters is undertaken to prevent dispersal of human pathogens into the environment. Except in well-nitrified effluents, the primary agents in chlorination, Cl2(g) or NaOCl(aq), are short-lived and quickly transfer oxidative chlorine to secondary agents (N-chloramines), which then participate in the disinfection process. Maturation of residual chlorine resulting from chlorine-transfer reactions is still poorly characterized. Using gel permeation and reversed-phase liquid chromatography combined with a novel, oxidant-specific detector, unanticipated trends during the maturation of residual chlorine in wastewater are identified. Within 2 min after addition of NaOCl, and continuing for several hours at least, significant amounts of oxidative chlorine are transferred to secondary agents that are moderately to strongly hydrophobic and to agents that have high relative molecular masses (Mr 1300-25000). It is hypothesized that hydrophobic stabilization of organic chloramines (RNHCl(o)) thermodynamically drives these transfers, making macromolecular chloramines the ultimate oxidative chlorine carriers. Macromolecular chloramines are expected to be sluggish oxidants, as observed in their reduction by sulfite, and are expected to be poor disinfectants. If transfer of oxidative chlorine to high Mr components occurs widely at treatment plants, then this phenomenon offers a new, physicochemical explanation for the well-known impotency of organic chloramines in wastewater disinfection.

  4. Preparation of phenylboronate affinity rigid monolith with macromolecular porogen.

    PubMed

    Li, Xiang-Jie; Jia, Man; Zhao, Yong-Xin; Liu, Zhao-Sheng; Akber Aisa, Haji

    2016-03-18

    Boronate-affinity monolithic column was first prepared via polystyrene (PS) as porogen in this work. The monolithic polymer was synthetized using 4-vinylphenylboronic acid (4-VPBA) as functional monomer, ethylene glycol dimethacrylate (EDMA) as crosslinker monomer, and a mixture of PS solution in tetrahydrofuran, the linear macromolecular porogen, and toluene as porogen. Isoquercitrin (ISO) and hyperoside (HYP), isomer diol flavonoid glycosides, can be baseline separated on the poly(VPBA-co-EDMA) monolith. The effect of polymerization variables on the selectivity factor, e.g., the ratio of monomer to crosslinker (M/C), the amount of PS and the molecular weight of macromolecular porogen was investigated. The surface properties of the monolithic polymer were characterized by scanning electron microscopy and nitrogen adsorption. The best polymerization condition was the M/C ratio of 7:3, and the PS concentration of 40 mg/ml. The poly(VPBA-co-EDMA) polymer was also applied to extract cis-diol flavonoid glycosides from the crude extraction of cotton flower. After treated by poly(VPBA-co-EDMA) for solid phase extraction, high purity ISO and HYP (>99.96%) can be obtained with recovery of 83.7% and 78.6%, respectively.

  5. Enhancing Endosomal Escape for Intracellular Delivery of Macromolecular Biologic Therapeutics

    PubMed Central

    Lönn, Peter; Kacsinta, Apollo D.; Cui, Xian-Shu; Hamil, Alexander S.; Kaulich, Manuel; Gogoi, Khirud; Dowdy, Steven F.

    2016-01-01

    Bioactive macromolecular peptides and oligonucleotides have significant therapeutic potential. However, due to their size, they have no ability to enter the cytoplasm of cells. Peptide/Protein transduction domains (PTDs), also called cell-penetrating peptides (CPPs), can promote uptake of macromolecules via endocytosis. However, overcoming the rate-limiting step of endosomal escape into the cytoplasm remains a major challenge. Hydrophobic amino acid R groups are known to play a vital role in viral escape from endosomes. Here we utilize a real-time, quantitative live cell split-GFP fluorescence complementation phenotypic assay to systematically analyze and optimize a series of synthetic endosomal escape domains (EEDs). By conjugating EEDs to a TAT-PTD/CPP spilt-GFP peptide complementation assay, we were able to quantitatively measure endosomal escape into the cytoplasm of live cells via restoration of GFP fluorescence by intracellular molecular complementation. We found that EEDs containing two aromatic indole rings or one indole ring and two aromatic phenyl groups at a fixed distance of six polyethylene glycol (PEG) units from the TAT-PTD-cargo significantly enhanced cytoplasmic delivery in the absence of cytotoxicity. EEDs address the critical rate-limiting step of endosomal escape in delivery of macromolecular biologic peptide, protein and siRNA therapeutics into cells. PMID:27604151

  6. Enhancing Endosomal Escape for Intracellular Delivery of Macromolecular Biologic Therapeutics.

    PubMed

    Lönn, Peter; Kacsinta, Apollo D; Cui, Xian-Shu; Hamil, Alexander S; Kaulich, Manuel; Gogoi, Khirud; Dowdy, Steven F

    2016-09-08

    Bioactive macromolecular peptides and oligonucleotides have significant therapeutic potential. However, due to their size, they have no ability to enter the cytoplasm of cells. Peptide/Protein transduction domains (PTDs), also called cell-penetrating peptides (CPPs), can promote uptake of macromolecules via endocytosis. However, overcoming the rate-limiting step of endosomal escape into the cytoplasm remains a major challenge. Hydrophobic amino acid R groups are known to play a vital role in viral escape from endosomes. Here we utilize a real-time, quantitative live cell split-GFP fluorescence complementation phenotypic assay to systematically analyze and optimize a series of synthetic endosomal escape domains (EEDs). By conjugating EEDs to a TAT-PTD/CPP spilt-GFP peptide complementation assay, we were able to quantitatively measure endosomal escape into the cytoplasm of live cells via restoration of GFP fluorescence by intracellular molecular complementation. We found that EEDs containing two aromatic indole rings or one indole ring and two aromatic phenyl groups at a fixed distance of six polyethylene glycol (PEG) units from the TAT-PTD-cargo significantly enhanced cytoplasmic delivery in the absence of cytotoxicity. EEDs address the critical rate-limiting step of endosomal escape in delivery of macromolecular biologic peptide, protein and siRNA therapeutics into cells.

  7. On the calculation of absolute macromolecular binding free energies

    PubMed Central

    Luo, Hengbin; Sharp, Kim

    2002-01-01

    The standard framework for calculating the absolute binding free energy of a macromolecular association reaction A + B → AB with an association constant KAB is to equate chemical potentials of the species on the left- and right-hand sides of this reaction and evaluate the chemical potentials from theory. This theory involves (usually hidden) assumptions about what constitutes the bound species, AB, and where the contribution of the solvent appears. We present here an alternative derivation that can be traced back to Bjerrum, in which the expectation value of KAB is obtained directly through the statistical mechanical method of evaluating its ensemble (Boltzmann-weighted) average. The generalized Bjerrum approach more clearly delineates: (i) the different contributions to binding; (ii) the origin of the much-discussed and somewhat controversial association entropy term; and (iii) where the solvent contribution appears. This approach also allows approximations required for practical evaluation of the binding constant in complex macromolecular systems, to be introduced in a well defined way. We provide an example, with application to test cases that illustrate a range of binding behavior. PMID:12149474

  8. Preparation of macromolecular complexes for cryo-electron microscopy.

    PubMed

    Grassucci, Robert A; Taylor, Derek J; Frank, Joachim

    2007-01-01

    This protocol describes the preparation of frozen-hydrated single-particle specimens of macromolecular complexes. First, it describes how to create a grid surface coated with holey carbon by first inducing holes in a Formvar film to act as a template for the holey carbon that is stable under cryo-electron microscopy (cryo-EM) conditions and is sample-friendly. The protocol then describes the steps required to deposit the homogeneous sample on the grid and to plunge-freeze the grid into liquid ethane at the temperature of liquid nitrogen, so that it is suitable for cryo-EM visualization. It takes 4-5 h to make several hundred holey carbon grids and about 1 h to make the frozen-hydrated grids. The time required for sample purification varies from hours to days, depending on the sample and the specific procedure required. A companion protocol details how to collect cryo-EM data using an FEI Tecnai transmission electron microscope that can subsequently be processed to obtain a three-dimensional reconstruction of the macromolecular complex.

  9. Critical review and perspective of macromolecularly imprinted polymers.

    PubMed

    Kryscio, David R; Peppas, Nicholas A

    2012-02-01

    Molecular recognition is a fundamental and ubiquitous process that is the driving force behind life. Natural recognition elements - including antibodies, enzymes, nucleic acids, and cells - exploit non-covalent interactions to bind to their targets with exceptionally strong affinities. Due to this unparalleled proficiency, scientists have long sought to mimic natural recognition pathways. One promising approach is molecularly imprinted polymers (MIPs), which are fully synthetic systems formed via the crosslinking of organic polymers in the presence of a template molecule, which results in stereo-specific binding sites for this analyte of interest. Macromolecularly imprinted polymers, those synthesized in the presence of macromolecule templates (>1500 Da), are of particular importance because they open up the field for a whole new set of robust diagnostic tools. Although the specific recognition of small-molecular-weight analytes is now considered routine, extension of these efficacious procedures to the protein regime has, thus far, proved challenging. This paper reviews the main approaches employed, highlights studies of interest with an emphasis on recent work, and offers suggestions for future success in the field of macromolecularly imprinted polymers.

  10. Protein stabilization by macromolecular crowding through enthalpy rather than entropy.

    PubMed

    Senske, Michael; Törk, Lisa; Born, Benjamin; Havenith, Martina; Herrmann, Christian; Ebbinghaus, Simon

    2014-06-25

    The interior of the cell is a densely crowded environment in which protein stability is affected differently than in dilute solution. Macromolecular crowding is commonly understood in terms of an entropic volume exclusion effect based on hardcore repulsions among the macromolecules. We studied the thermal unfolding of ubiquitin in the presence of different cosolutes (glucose, dextran, poly(ethylene glycol), KCl, urea). Our results show that for a correct dissection of the cosolute-induced changes of the free energy into its enthalpic and entropic contributions, the temperature dependence of the heat capacity change needs to be explicitly taken into account. In contrast to the prediction by the excluded volume theory, we observed an enthalpic stabilization and an entropic destabilization for glucose, dextran, and poly(ethylene glycol). The enthalpic stabilization mechanism induced by the macromolecular crowder dextran was similar to the enthalpic stabilization mechanism of its monomeric building block glucose. In the case of poly(ethylene glycol), entropy is dominating over enthalpy leading to an overall destabilization. We propose a new model to classify cosolute effects in terms of their enthalpic contributions to protein stability.

  11. SVP-like MADS-box protein from Carya cathayensis forms higher-order complexes.

    PubMed

    Wang, Jingjing; Hou, Chuanming; Huang, Jianqin; Wang, Zhengjia; Xu, Yingwu

    2015-03-01

    To properly regulate plant flowering time and construct floral pattern, MADS-domain containing transcription factors must form multimers including homo- and hetero-dimers. They are also active in forming hetero-higher-order complexes with three to five different molecules. However, it is not well known if a MADS-box protein can also form homo-higher-order complex. In this study a biochemical approach is utilized to provide insight into the complex formation for an SVP-like MADS-box protein cloned from hickory. The results indicated that the protein is a heterogeneous higher-order complex with the peak population containing over 20 monomers. Y2H verified the protein to form homo-complex in yeast cells. Western blot of the hickory floral bud sample revealed that the protein exists in higher-order polymers in native. Deletion assays indicated that the flexible C-terminal residues are mainly responsible for the higher-order polymer formation and the heterogeneity. Current results provide direct biochemical evidences for an active MADS-box protein to be a high order complex, much higher than a quartermeric polymer. Analysis suggests that a MADS-box subset may be able to self-assemble into large complexes, and thereby differentiate one subfamily from the other in a higher-order structural manner. Present result is a valuable supplement to the action of mechanism for MADS-box proteins in plant development.

  12. Identifying, studying and making good use of macromolecular crystals

    SciTech Connect

    Calero, Guillermo; Cohen, Aina E.; Luft, Joseph R.; Newman, Janet; Snell, Edward H.

    2014-07-25

    As technology advances, the crystal volume that can be used to collect useful X-ray diffraction data decreases. The technologies available to detect and study growing crystals beyond the optical resolution limit and methods to successfully place the crystal into the X-ray beam are discussed. Structural biology has contributed tremendous knowledge to the understanding of life on the molecular scale. The Protein Data Bank, a depository of this structural knowledge, currently contains over 100 000 protein structures, with the majority stemming from X-ray crystallography. As the name might suggest, crystallography requires crystals. As detectors become more sensitive and X-ray sources more intense, the notion of a crystal is gradually changing from one large enough to embellish expensive jewellery to objects that have external dimensions of the order of the wavelength of visible light. Identifying these crystals is a prerequisite to their study. This paper discusses developments in identifying these crystals during crystallization screening and distinguishing them from other potential outcomes. The practical aspects of ensuring that once a crystal is identified it can then be positioned in the X-ray beam for data collection are also addressed.

  13. Macromolecular crowding directs extracellular matrix organization and mesenchymal stem cell behavior.

    PubMed

    Zeiger, Adam S; Loe, Felicia C; Li, Ran; Raghunath, Michael; Van Vliet, Krystyn J

    2012-01-01

    Microenvironments of biological cells are dominated in vivo by macromolecular crowding and resultant excluded volume effects. This feature is absent in dilute in vitro cell culture. Here, we induced macromolecular crowding in vitro by using synthetic macromolecular globules of nm-scale radius at physiological levels of fractional volume occupancy. We quantified the impact of induced crowding on the extracellular and intracellular protein organization of human mesenchymal stem cells (MSCs) via immunocytochemistry, atomic force microscopy (AFM), and AFM-enabled nanoindentation. Macromolecular crowding in extracellular culture media directly induced supramolecular assembly and alignment of extracellular matrix proteins deposited by cells, which in turn increased alignment of the intracellular actin cytoskeleton. The resulting cell-matrix reciprocity further affected adhesion, proliferation, and migration behavior of MSCs. Macromolecular crowding can thus aid the design of more physiologically relevant in vitro studies and devices for MSCs and other cells, by increasing the fidelity between materials synthesized by cells in vivo and in vitro.

  14. Macromolecular Crowding Directs Extracellular Matrix Organization and Mesenchymal Stem Cell Behavior

    PubMed Central

    Zeiger, Adam S.; Loe, Felicia C.; Li, Ran; Raghunath, Michael; Van Vliet, Krystyn J.

    2012-01-01

    Microenvironments of biological cells are dominated in vivo by macromolecular crowding and resultant excluded volume effects. This feature is absent in dilute in vitro cell culture. Here, we induced macromolecular crowding in vitro by using synthetic macromolecular globules of nm-scale radius at physiological levels of fractional volume occupancy. We quantified the impact of induced crowding on the extracellular and intracellular protein organization of human mesenchymal stem cells (MSCs) via immunocytochemistry, atomic force microscopy (AFM), and AFM-enabled nanoindentation. Macromolecular crowding in extracellular culture media directly induced supramolecular assembly and alignment of extracellular matrix proteins deposited by cells, which in turn increased alignment of the intracellular actin cytoskeleton. The resulting cell-matrix reciprocity further affected adhesion, proliferation, and migration behavior of MSCs. Macromolecular crowding can thus aid the design of more physiologically relevant in vitro studies and devices for MSCs and other cells, by increasing the fidelity between materials synthesized by cells in vivo and in vitro. PMID:22649562

  15. Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

    SciTech Connect

    Wan, Qun; Parks, Jerry M.; Hanson, B. Leif; Fisher, Suzanne Zoe; Ostermann, Andreas; Schrader, Tobias E.; Graham, David E.; Coates, Leighton; Langan, Paul; Kovalevsky, Andrey

    2015-09-21

    Glycoside hydrolase (GH) enzymes apply acid/base chemistry to catalyze the decomposition of complex carbohydrates. These ubiquitous enzymes accept protons from solvent and donate them to substrates at close to neutral pH by modulating the pKa values of key side chains during catalysis. However, it is not known how the catalytic acid residue acquires a proton and transfers it efficiently to the substrate. To better understand GH chemistry, we used macromolecular neutron crystallography to directly determine protonation and ionization states of the active site residues of a family 11 GH at multiple pD (pD = pH + 0.4) values. The general acid glutamate (Glu) cycles between two conformations, upward and downward, but is protonated only in the downward orientation. We performed continuum electrostatics calculations to estimate the pKa values of the catalytic Glu residues in both the apo- and substrate-bound states of the enzyme. The calculated pKa of the Glu increases substantially when the side chain moves down. The energy barrier required to rotate the catalytic Glu residue back to the upward conformation, where it can protonate the glycosidic oxygen of the substrate, is 4.3 kcal/mol according to free energy simulations. Lastly, these findings shed light on the initial stage of the glycoside hydrolysis reaction in which molecular motion enables the general acid catalyst to obtain a proton from the bulk solvent and deliver it to the glycosidic oxygen.

  16. Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

    DOE PAGES

    Wan, Qun; Parks, Jerry M.; Hanson, B. Leif; ...

    2015-09-21

    Glycoside hydrolase (GH) enzymes apply acid/base chemistry to catalyze the decomposition of complex carbohydrates. These ubiquitous enzymes accept protons from solvent and donate them to substrates at close to neutral pH by modulating the pKa values of key side chains during catalysis. However, it is not known how the catalytic acid residue acquires a proton and transfers it efficiently to the substrate. To better understand GH chemistry, we used macromolecular neutron crystallography to directly determine protonation and ionization states of the active site residues of a family 11 GH at multiple pD (pD = pH + 0.4) values. The generalmore » acid glutamate (Glu) cycles between two conformations, upward and downward, but is protonated only in the downward orientation. We performed continuum electrostatics calculations to estimate the pKa values of the catalytic Glu residues in both the apo- and substrate-bound states of the enzyme. The calculated pKa of the Glu increases substantially when the side chain moves down. The energy barrier required to rotate the catalytic Glu residue back to the upward conformation, where it can protonate the glycosidic oxygen of the substrate, is 4.3 kcal/mol according to free energy simulations. Lastly, these findings shed light on the initial stage of the glycoside hydrolysis reaction in which molecular motion enables the general acid catalyst to obtain a proton from the bulk solvent and deliver it to the glycosidic oxygen.« less

  17. The encounter with God in myth and madness

    PubMed Central

    Doerr, Otto; Velásquez, Óscar

    2007-01-01

    Background It is well known how often psychiatric patients report religious experiences. These are especially frequent in schizophrenic and epileptic patients as the subject of their delusions. The question we pose is: are there differences between this kind of religious experiences and those we find in religious texts or in the mythological tradition? Results An overview on famous mythological narratives, such as The Aeneid, allows us to establish that the divinities become recognizable to the human being at the moment of their departure. Thus, Aeneas does not recognise his mother, Venus, when she appears to him in the middle of the forest at the coast of Africa. A dialogue between the two takes place, and only at the end of the encounter, when she is going away and already with her back to Aeneas, she shows her son the signs of her divinity: the rose-flush emanating from her neck, her hair perfume and the majesty of her gait. Something analogous can be observed in the encounter of Moses with Yahweh on Mount Sinai. Moses asks God: "Show me your glory, I beg you". And God replies, among other things: "you shall see the back of me, but my face is not to be seen". In the same sense, the Emmaus disciples do not recognise Jesus till the moment of his disappearance ("but he had vanished from their sight"), and Saul of Tars falls off his horse just in the moment when he feels the divine presence. In short, the direct encounter with the divinity seems not to occur in the realm of myth or in religious tradition. The realm of madness is exactly the opposite. Our research on religious experiences in schizophrenic and epileptic patients leads us to conclude that God appears to them face to face, and the patient describes God the father, Jesus or the Virgin Mary in intimate detail, always in an everyday setting. So, the divinity is seen in the garden, or in the bedroom, or maybe above the wardrobe, without any of its majesty. The nearness to God also tends to be so extreme

  18. MAD2L2 controls DNA repair at telomeres and DNA breaks by inhibiting 5′ end-resection

    PubMed Central

    Segura-Bayona, Sandra; Peuscher, Marieke H.; van der Torre, Jaco; Wevers, Brigitte A.; Orthwein, Alexandre; Durocher, Daniel; Jacobs, Jacqueline J.L.

    2015-01-01

    Appropriate repair of DNA lesions and the inhibition of DNA repair activities at telomeres are critical to prevent genomic instability. By fuelling the generation of genetic alterations and by compromising cell viability, genomic instability is a driving force in cancer and aging1, 2. Here we identify MAD2L2 (also known as MAD2B or REV7) through functional genetic screening as a novel factor controlling DNA repair activities at mammalian telomeres. We show that MAD2L2 accumulates at uncapped telomeres and promotes non-homologous end-joining (NHEJ)-mediated fusion of deprotected chromosome ends and genomic instability. MAD2L2 depletion causes elongated 3′ telomeric overhangs, implying that MAD2L2 inhibits 5′ end-resection. End-resection blocks NHEJ while committing to homology-directed repair (HDR) and is under control of 53BP1, RIF1 and PTIP3. Consistent with MAD2L2 promoting NHEJ-mediated telomere fusion by inhibiting 5′ end-resection, knockdown of the nucleases CTIP or EXO1 partially restores telomere-driven genomic instability in MAD2L2-depleted cells. Control of DNA repair by MAD2L2 is not limited to telomeres. MAD2L2 also accumulates and inhibits end-resection at irradiation (IR)-induced DNA double-strand breaks (DSBs) and promotes end-joining of DSBs in multiple settings, including during immunoglobulin class switch recombination (CSR). These activities of MAD2L2 depend on ATM kinase activity, RNF8, RNF168, 53BP1 and RIF1, but not on PTIP, REV1 and REV3, the latter two acting with MAD2L2 in translesion synthesis (TLS)4. Together our data establish MAD2L2 as a critical contributor to the control of DNA repair activity by 53BP1 that promotes NHEJ by inhibiting 5′ end-resection downstream of RIF1. PMID:25799990

  19. Crystallography and elasticity of individual GaN nanotubes

    NASA Astrophysics Data System (ADS)

    Liu, Baodan; Bando, Yoshio; Wang, Mingsheng; Tang, Chengchun; Mitome, Masanori; Golberg, Dmitri

    2009-05-01

    High-purity, crystalline [001]-oriented GaN nanotubes with outer diameters of 200 nm or more, rough surfaces and irregular internal channels were synthesized under epitaxial growth on [001]-oriented sapphire substrates. Elastic property measurements on free-standing individual GaN nanotubes, using the in situ transmission electron microscopy (TEM) electromechanical resonance technique, pointed at an average Young's modulus E of 37 GPa and minimum quality factor of 320. These numbers are notably lower than those for previously reported GaN nanowires. The crystallography and chemistry of the GaN nanotubes were analyzed using TEM and energy dispersion x-ray spectroscopy (EDS). It is suggested that the lowered Young's modulus and quality factor of the nanotubes are mainly due to the surface roughness and defectiveness.

  20. Towards time-resolved serial crystallography in a microfluidic device.

    PubMed

    Pawate, Ashtamurthy S; Šrajer, Vukica; Schieferstein, Jeremy; Guha, Sudipto; Henning, Robert; Kosheleva, Irina; Schmidt, Marius; Ren, Zhong; Kenis, Paul J A; Perry, Sarah L

    2015-07-01

    Serial methods for crystallography have the potential to enable dynamic structural studies of protein targets that have been resistant to single-crystal strategies. The use of serial data-collection strategies can circumvent challenges associated with radiation damage and repeated reaction initiation. This work utilizes a microfluidic crystallization platform for the serial time-resolved Laue diffraction analysis of macroscopic crystals of photoactive yellow protein (PYP). Reaction initiation was achieved via pulsed laser illumination, and the resultant electron-density difference maps clearly depict the expected pR(1)/pR(E46Q) and pR(2)/pR(CW) states at 10 µs and the pB1 intermediate at 1 ms. The strategies presented here have tremendous potential for extension to chemical triggering methods for reaction initiation and for extension to dynamic, multivariable analyses.

  1. Serial Femtosecond Crystallography of G Protein-Coupled Receptors

    PubMed Central

    Liu, Wei; Wacker, Daniel; Gati, Cornelius; Han, Gye Won; James, Daniel; Wang, Dingjie; Nelson, Garrett; Weierstall, Uwe; Katritch, Vsevolod; Barty, Anton; Zatsepin, Nadia A.; Li, Dianfan; Messerschmidt, Marc; Boutet, Sébastien; Williams, Garth J.; Koglin, Jason E.; Seibert, M. Marvin; Wang, Chong; Shah, Syed T.A.; Basu, Shibom; Fromme, Raimund; Kupitz, Christopher; Rendek, Kimberley N.; Grotjohann, Ingo; Fromme, Petra; Kirian, Richard A.; Beyerlein, Kenneth R.; White, Thomas A.; Chapman, Henry N.; Caffrey, Martin; Spence, John C.H.; Stevens, Raymond C.; Cherezov, Vadim

    2014-01-01

    X-ray crystallography of G protein-coupled receptors and other membrane proteins is hampered by difficulties associated with growing sufficiently large crystals that withstand radiation damage and yield high-resolution data at synchrotron sources. Here we used an x-ray free-electron laser (XFEL) with individual 50-fs duration x-ray pulses to minimize radiation damage and obtained a high-resolution room temperature structure of a human serotonin receptor using sub-10 µm microcrystals grown in a membrane mimetic matrix known as lipidic cubic phase. Compared to the structure solved by traditional microcrystallography from cryo-cooled crystals of about two orders of magnitude larger volume, the room temperature XFEL structure displays a distinct distribution of thermal motions and conformations of residues that likely more accurately represent the receptor structure and dynamics in a cellular environment. PMID:24357322

  2. Towards time-resolved serial crystallography in a microfluidic device

    PubMed Central

    Pawate, Ashtamurthy S.; Šrajer, Vukica; Schieferstein, Jeremy; Guha, Sudipto; Henning, Robert; Kosheleva, Irina; Schmidt, Marius; Ren, Zhong; Kenis, Paul J. A.; Perry, Sarah L.

    2015-01-01

    Serial methods for crystallography have the potential to enable dynamic structural studies of protein targets that have been resistant to single-crystal strategies. The use of serial data-collection strategies can circumvent challenges associated with radiation damage and repeated reaction initiation. This work utilizes a microfluidic crystallization platform for the serial time-resolved Laue diffraction analysis of macroscopic crystals of photoactive yellow protein (PYP). Reaction initiation was achieved via pulsed laser illumination, and the resultant electron-density difference maps clearly depict the expected pR1/pRE46Q and pR2/pRCW states at 10 µs and the pB1 intermediate at 1 ms. The strategies presented here have tremendous potential for extension to chemical triggering methods for reaction initiation and for extension to dynamic, multivariable analyses. PMID:26144226

  3. Serial femtosecond crystallography of soluble proteins in lipidic cubic phase

    DOE PAGES

    Fromme, Raimund; Ishchenko, Andrii; Metz, Markus; ...

    2015-08-04

    Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) enables high-resolution protein structure determination using micrometre-sized crystals at room temperature with minimal effects from radiation damage. SFX requires a steady supply of microcrystals intersecting the XFEL beam at random orientations. An LCP–SFX method has recently been introduced in which microcrystals of membrane proteins are grown and delivered for SFX data collection inside a gel-like membrane-mimetic matrix, known as lipidic cubic phase (LCP), using a special LCP microextrusion injector. Here, it is shown enabling a dramatic reduction in the amount of crystallized protein required for data collection compared with crystals deliveredmore » by liquid injectors. High-quality LCP–SFX data sets were collected for two soluble proteins, lysozyme and phycocyanin, using less than 0.1 mg of each protein.« less

  4. Serial femtosecond crystallography of soluble proteins in lipidic cubic phase

    SciTech Connect

    Fromme, Raimund; Ishchenko, Andrii; Metz, Markus; Chowdhury, Shatabdi Roy; Basu, Shibom; Boutet, Sébastien; Fromme, Petra; White, Thomas A.; Barty, Anton; Spence, John C. H.; Weierstall, Uwe; Liu, Wei; Cherezov, Vadim

    2015-08-04

    Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) enables high-resolution protein structure determination using micrometre-sized crystals at room temperature with minimal effects from radiation damage. SFX requires a steady supply of microcrystals intersecting the XFEL beam at random orientations. An LCP–SFX method has recently been introduced in which microcrystals of membrane proteins are grown and delivered for SFX data collection inside a gel-like membrane-mimetic matrix, known as lipidic cubic phase (LCP), using a special LCP microextrusion injector. Here, it is shown enabling a dramatic reduction in the amount of crystallized protein required for data collection compared with crystals delivered by liquid injectors. High-quality LCP–SFX data sets were collected for two soluble proteins, lysozyme and phycocyanin, using less than 0.1 mg of each protein.

  5. Lens-Coupled CCD Detector for X-ray Crystallography

    PubMed Central

    Madden, Timothy J.; McGuigan, William; Molitsky, Michael J.; Naday, Istvan; McArthur, Alan; Westbrook, Edwin M.

    2007-01-01

    An x-ray crystallography detector (Blue-1) has been built based upon a Fairchild 486 back-illuminated CCD and a custom lens system designed by Optics One Inc. The advantages of our Blue-1 lens system over more conventional fiber-optic tapers are: lower noise and higher efficiency; improved point spread function; negligible spatial distortion; and lack of “chicken-wire” patterns. Also, the engineering is simpler because the CCD is not bonded to the fiber-optic taper. A unique mechanical design has been employed to accurately focus the image on the CCD. The detector software is based on MATLAB and takes advantage of its powerful imaging and signal processing libraries. The CCD timing can be updated on the fly by using a “CCD controller language” to specify timing. PMID:18185837

  6. Nonequilibrium phase transitions in cuprates observed by ultrafast electron crystallography.

    PubMed

    Gedik, Nuh; Yang, Ding-Shyue; Logvenov, Gennady; Bozovic, Ivan; Zewail, Ahmed H

    2007-04-20

    Nonequilibrium phase transitions, which are defined by the formation of macroscopic transient domains, are optically dark and cannot be observed through conventional temperature- or pressure-change studies. We have directly determined the structural dynamics of such a nonequilibrium phase transition in a cuprate superconductor. Ultrafast electron crystallography with the use of a tilted optical geometry technique afforded the necessary atomic-scale spatial and temporal resolutions. The observed transient behavior displays a notable "structural isosbestic" point and a threshold effect for the dependence of c-axis expansion (Deltac) on fluence (F), with Deltac/F = 0.02 angstrom/(millijoule per square centimeter). This threshold for photon doping occurs at approximately 0.12 photons per copper site, which is unexpectedly close to the density (per site) of chemically doped carriers needed to induce superconductivity.

  7. Proline: Mother Nature’s cryoprotectant applied to protein crystallography

    SciTech Connect

    Pemberton, Travis A.; Still, Brady R.; Christensen, Emily M.; Singh, Harkewal; Srivastava, Dhiraj; Tanner, John J.

    2012-08-01

    The amino acid l-proline is shown to be a good cryoprotectant for protein crystals. Four examples are provided; the range of proline used for cryoprotection is 2.0–3.0 M. l-Proline is one of Mother Nature’s cryoprotectants. Plants and yeast accumulate proline under freeze-induced stress and the use of proline in the cryopreservation of biological samples is well established. Here, it is shown that l-proline is also a useful cryoprotectant for protein crystallography. Proline was used to prepare crystals of lysozyme, xylose isomerase, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase for low-temperature data collection. The crystallization solutions in these test cases included the commonly used precipitants ammonium sulfate, sodium chloride and polyethylene glycol and spanned the pH range 4.6–8.5. Thus, proline is compatible with typical protein-crystallization formulations. The proline concentration needed for cryoprotection of these crystals is in the range 2.0–3.0 M. Complete data sets were collected from the proline-protected crystals. Proline performed as well as traditional cryoprotectants based on the diffraction resolution and data-quality statistics. The structures were refined to assess the binding of proline to these proteins. As observed with traditional cryoprotectants such as glycerol and ethylene glycol, the electron-density maps clearly showed the presence of proline molecules bound to the protein. In two cases, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase, proline binds in the active site. It is concluded that l-proline is an effective cryoprotectant for protein crystallography.

  8. Does UV CETI Suffer from the MAD Syndrome?

    NASA Technical Reports Server (NTRS)

    Drake, Jeremy

    1999-01-01

    Photometric data have been analysed and searched for events of flaring and other variability. Some flaring has been detected, though probably not at a level that will hinder our continuing spectral analysis. X-ray diagnostics for the very hot coronal emission measure are under investigation in order to determine whether or not the very hot coronal plasma contributes significantly to the observed X-ray flux in the EUV. The key test of the MAD syndrome lies in whether or not the coronal lines indicate a depletion in met- als in the corona relative to the underlying photosphere. While some progress has been made in this direction, not as much work has been accomplished as expected due to the increasing commitments of the PI to the CXO project as it nears launch. A no-cost extension has been granted in order to further the analysis and carry out the next stages of the investigation: to construct an emission measure distribution with which to compute a synthetic continuum to compare with the observed continuum.

  9. Jung, spirits and madness: lessons for cultural psychiatry.

    PubMed

    Koss-Chioino, Joan D

    2003-06-01

    An understanding of the nature and meaning of 'dissociative,' 'altered' or unusual states ultimately turns on the meaning and definition of consciousness. The view of consciousness from the discipline of psychiatry is largely based on a biomedically endorsed, culturally specific perspective of 'normal' consciousness as an integrated pattern of quotidian relationships with the 'observable' physical world. This perspective underlies the nosology for mental disorders, particularly psychoses, suggesting irreconcilable difference in cognition and affect of persons with these diagnostic labels. This article reviews some theories of Carl Gustav Jung regarding the structure and content of human consciousness and their relationship to aspects of 'dementia praecox' or 'schizophrenia.' It traces the origin and development of these ideas in part to Jung's early contact with, and intense interest in spiritualists and spirits, to later influences comprised of his own altered states (dreams and fantasies) and his involvement with patients diagnosed as schizophrenic. Data on current Spiritist beliefs and healing practices focused on 'madness' (i.e. most often diagnosed as schizophrenia in mental health settings), are described to explore parallels with Jung's ideas on the structure and dynamics of the psyche. These parallels are of special interest because the experience of spirits is ubiquitous, not well explained and often rejected as meaningful by psychiatrists and clinical psychologists. Jung, however, offers a cogent explanation of spirit phenomena as manifestations of the unconscious. A concluding section suggests contributions to cultural psychiatry by Jung.

  10. The promise of macromolecular crystallization in microfluidic chips

    NASA Technical Reports Server (NTRS)

    van der Woerd, Mark; Ferree, Darren; Pusey, Marc

    2003-01-01

    Microfluidics, or lab-on-a-chip technology, is proving to be a powerful, rapid, and efficient approach to a wide variety of bioanalytical and microscale biopreparative needs. The low materials consumption, combined with the potential for packing a large number of experiments in a few cubic centimeters, makes it an attractive technique for both initial screening and subsequent optimization of macromolecular crystallization conditions. Screening operations, which require a macromolecule solution with a standard set of premixed solutions, are relatively straightforward and have been successfully demonstrated in a microfluidics platform. Optimization methods, in which crystallization solutions are independently formulated from a range of stock solutions, are considerably more complex and have yet to be demonstrated. To be competitive with either approach, a microfluidics system must offer ease of operation, be able to maintain a sealed environment over several weeks to months, and give ready access for the observation and harvesting of crystals as they are grown.

  11. Macromolecular Crystallization in Microfluidics for the International Space Station

    NASA Technical Reports Server (NTRS)

    Monaco, Lisa A.; Spearing, Scott

    2003-01-01

    At NASA's Marshall Space Flight Center, the Iterative Biological Crystallization (IBC) project has begun development on scientific hardware for macromolecular crystallization on the International Space Station (ISS). Currently ISS crystallization research is limited to solution recipes that were prepared on the ground prior to launch. The proposed hardware will conduct solution mixing and dispensing on board the ISS, be fully automated, and have imaging functions via remote commanding from the ground. Utilizing microfluidic technology, IBC will allow for on orbit iterations. The microfluidics LabChip(R) devices that have been developed, along with Caliper Technologies, will greatly benefit researchers by allowing for precise fluid handling of nano/pico liter sized volumes. IBC will maximize the amount of science return by utilizing the microfluidic approach and be a valuable tool to structural biologists investigating medically relevant projects.

  12. Detecting stoichiometry of macromolecular complexes in live cells using FRET

    PubMed Central

    Ben-Johny, Manu; Yue, Daniel N.; Yue, David T.

    2016-01-01

    The stoichiometry of macromolecular interactions is fundamental to cellular signalling yet challenging to detect from living cells. Fluorescence resonance energy transfer (FRET) is a powerful phenomenon for characterizing close-range interactions whereby a donor fluorophore transfers energy to a closely juxtaposed acceptor. Recognizing that FRET measured from the acceptor's perspective reports a related but distinct quantity versus the donor, we utilize the ratiometric comparison of the two to obtain the stoichiometry of a complex. Applying this principle to the long-standing controversy of calmodulin binding to ion channels, we find a surprising Ca2+-induced switch in calmodulin stoichiometry with Ca2+ channels—one calmodulin binds at basal cytosolic Ca2+ levels while two calmodulins interact following Ca2+ elevation. This feature is curiously absent for the related Na channels, also potently regulated by calmodulin. Overall, our assay adds to a burgeoning toolkit to pursue quantitative biochemistry of dynamic signalling complexes in living cells. PMID:27922011

  13. E-MSD: the European Bioinformatics Institute Macromolecular Structure Database

    PubMed Central

    Boutselakis, H.; Dimitropoulos, D.; Fillon, J.; Golovin, A.; Henrick, K.; Hussain, A.; Ionides, J.; John, M.; Keller, P. A.; Krissinel, E.; McNeil, P.; Naim, A.; Newman, R.; Oldfield, T.; Pineda, J.; Rachedi, A.; Copeland, J.; Sitnov, A.; Sobhany, S.; Suarez-Uruena, A.; Swaminathan, J.; Tagari, M.; Tate, J.; Tromm, S.; Velankar, S.; Vranken, W.

    2003-01-01

    The E-MSD macromolecular structure relational database (http://www.ebi.ac.uk/msd) is designed to be a single access point for protein and nucleic acid structures and related information. The database is derived from Protein Data Bank (PDB) entries. Relational database technologies are used in a comprehensive cleaning procedure to ensure data uniformity across the whole archive. The search database contains an extensive set of derived properties, goodness-of-fit indicators, and links to other EBI databases including InterPro, GO, and SWISS-PROT, together with links to SCOP, CATH, PFAM and PROSITE. A generic search interface is available, coupled with a fast secondary structure domain search tool. PMID:12520052

  14. Macromolecularly "Caged" Carbon Nanoparticles for Intracellular Trafficking via Switchable Photoluminescence.

    PubMed

    Misra, Santosh K; Srivastava, Indrajit; Tripathi, Indu; Daza, Enrique; Ostadhossein, Fatemeh; Pan, Dipanjan

    2017-02-08

    Reversible switching of photoluminescence (PL) of carbon nanoparticles (CNP) can be achieved with counterionic macromolecular caging and decaging at the nanoscale. A negatively charged uncoated, "bare" CNP with high luminescence loses its PL when positively charged macromolecules are wrapped around its surface. Prepared caged carbons could regain their emission only through interaction with anionic surfactant molecules, representing anionic amphiphiles of endocytic membranes. This process could be verified by gel electrophoresis, spectroscopically and in vitro confocal imaging studies. Results indicated for the first time that luminescence switchable CNPs can be synthesized for efficient intracellular tracking. This study further supports the origin of photoluminescence in CNP as a surface phenomenon correlated a function of characteristic charged macromolecules.

  15. Enhancement of Biological Reactions on Cell Surfaces via Macromolecular Crowding

    PubMed Central

    Chapanian, Rafi; Kwan, David H.; Constantinescu, Iren; Shaikh, Fathima A.; Rossi, Nicholas A.A.; Withers, Stephen G.; Kizhakkedathu, Jayachandran N.

    2016-01-01

    The reaction of macromolecules such as enzymes and antibodies with cell surfaces is often an inefficient process, requiring large amounts of expensive reagent. Here we report a general method based on macromolecular crowding with a range of neutral polymers to enhance such reactions, using red blood cells (RBCs) as a model system. Rates of conversion of Type A and B red blood cells to universal O type by removal of antigenic carbohydrates with selective glycosidases are increased up to 400-fold in the presence of crowders. Similar enhancements are seen for antibody binding. We further explore the factors underlying these enhancements using confocal microscopy and fluorescent recovery after bleaching (FRAP) techniques with various fluorescent protein fusion partners. Increased cell-surface concentration due to volume exclusion, along with two-dimensionally confined diffusion of enzymes close to the cell surface, appear to be the major contributing factors. PMID:25140641

  16. Bulk-solvent correction in large macromolecular structures.

    PubMed

    Rees, Bernard; Jenner, Lasse; Yusupov, Marat

    2005-09-01

    The estimation of the bulk-solvent contribution to the diffraction of a macromolecular crystal makes use of a solvent mask which delimits the bulk-solvent regions in the crystal. It is shown that the way this mask is usually defined in CNS contains a bias which can lead to absurd results in the case of very large structures, where the calculations can only be made on relatively coarse grids. A modified procedure is described and applied to 70S ribosome data at 5.5 A resolution. The B factor affecting the bulk solvent is also discussed. Even in this case of very high and widely variable atomic B factors, it seems sufficient to consider a constant and isotropic B factor for the bulk solvent. This is initially set to the average value of the atomic B factor, but can be refined.

  17. Revealing the macromolecular targets of complex natural products

    NASA Astrophysics Data System (ADS)

    Reker, Daniel; Perna, Anna M.; Rodrigues, Tiago; Schneider, Petra; Reutlinger, Michael; Mönch, Bettina; Koeberle, Andreas; Lamers, Christina; Gabler, Matthias; Steinmetz, Heinrich; Müller, Rolf; Schubert-Zsilavecz, Manfred; Werz, Oliver; Schneider, Gisbert

    2014-12-01

    Natural products have long been a source of useful biological activity for the development of new drugs. Their macromolecular targets are, however, largely unknown, which hampers rational drug design and optimization. Here we present the development and experimental validation of a computational method for the discovery of such targets. The technique does not require three-dimensional target models and may be applied to structurally complex natural products. The algorithm dissects the natural products into fragments and infers potential pharmacological targets by comparing the fragments to synthetic reference drugs with known targets. We demonstrate that this approach results in confident predictions. In a prospective validation, we show that fragments of the potent antitumour agent archazolid A, a macrolide from the myxobacterium Archangium gephyra, contain relevant information regarding its polypharmacology. Biochemical and biophysical evaluation confirmed the predictions. The results obtained corroborate the practical applicability of the computational approach to natural product ‘de-orphaning’.

  18. Metabolomics reveals elevated macromolecular degradation in periodontal disease.

    PubMed

    Barnes, V M; Ciancio, S G; Shibly, O; Xu, T; Devizio, W; Trivedi, H M; Guo, L; Jönsson, T J

    2011-11-01

    Periodontitis is a chronic inflammatory disease characterized by tissue destruction. In the diseased oral environment, saliva has primarily been considered to act as a protectant by lubricating the tissue, mineralizing the bones, neutralizing the pH, and combating microbes. To understand the metabolic role that saliva plays in the diseased state, we performed untargeted metabolomic profiling of saliva from healthy and periodontitic individuals. Several classes of biochemicals, including dipeptide, amino acid, carbohydrate, lipids, and nucleotide metabolites, were altered, consistent with increased macromolecular degradation of proteins, triacylglycerol, glycerolphospholipids, polysaccharides, and polynucleotides in the individuals with periodontal disease. These changes partially reflected the enhanced host-bacterial interactions in the diseased state as supported by increased levels of bacterially modified amino acids and creatine metabolite. More importantly, the increased lipase, protease, and glycosidase activities associated with periodontitis generated a more favorable energy environment for oral bacteria, potentially exacerbating the disease state.

  19. Scientific Benchmarks for Guiding Macromolecular Energy Function Improvement

    PubMed Central

    Leaver-Fay, Andrew; O’Meara, Matthew J.; Tyka, Mike; Jacak, Ron; Song, Yifan; Kellogg, Elizabeth H.; Thompson, James; Davis, Ian W.; Pache, Roland A.; Lyskov, Sergey; Gray, Jeffrey J.; Kortemme, Tanja; Richardson, Jane S.; Havranek, James J.; Snoeyink, Jack; Baker, David; Kuhlman, Brian

    2013-01-01

    Accurate energy functions are critical to macromolecular modeling and design. We describe new tools for identifying inaccuracies in energy functions and guiding their improvement, and illustrate the application of these tools to improvement of the Rosetta energy function. The feature analysis tool identifies discrepancies between structures deposited in the PDB and low energy structures generated by Rosetta; these likely arise from inaccuracies in the energy function. The optE tool optimizes the weights on the different components of the energy function by maximizing the recapitulation of a wide range of experimental observations. We use the tools to examine three proposed modifications to the Rosetta energy function: improving the unfolded state energy model (reference energies), using bicubic spline interpolation to generate knowledge based torisonal potentials, and incorporating the recently developed Dunbrack 2010 rotamer library (Shapovalov and Dunbrack, 2011). PMID:23422428

  20. Size-exclusion chromatography system for macromolecular interaction analysis

    DOEpatents

    Stevens, Fred J.

    1988-01-01

    A low pressure, microcomputer controlled system employing high performance liquid chromatography (HPLC) allows for precise analysis of the interaction of two reversibly associating macromolecules such as proteins. Since a macromolecular complex migrates faster than its components during size-exclusion chromatography, the difference between the elution profile of a mixture of two macromolecules and the summation of the elution profiles of the two components provides a quantifiable indication of the degree of molecular interaction. This delta profile is used to qualitatively reveal the presence or absence of significant interaction or to rank the relative degree of interaction in comparing samples and, in combination with a computer simulation, is further used to quantify the magnitude of the interaction in an arrangement wherein a microcomputer is coupled to analytical instrumentation in a novel manner.

  1. Extracting trends from two decades of microgravity macromolecular crystallization history

    NASA Technical Reports Server (NTRS)

    Judge, Russell A.; Snell, Edward H.; van der Woerd, Mark J.

    2005-01-01

    Since the 1980s hundreds of macromolecular crystal growth experiments have been performed in the reduced acceleration environment of an orbiting spacecraft. Significant enhancements in structural knowledge have resulted from X-ray diffraction of the crystals grown. Similarly, many samples have shown no improvement or degradation in comparison to those grown on the ground. A complex series of interrelated factors affect these experiments and by building a comprehensive archive of the results it was aimed to identify factors that result in success and those that result in failure. Specifically, it was found that dedicated microgravity missions increase the chance of success when compared with those where crystallization took place as a parasitic aspect of the mission. It was also found that the chance of success could not be predicted based on any discernible property of the macromolecule available to us.

  2. Reciprocal Space Mapping of Macromolecular Crystals in the Home Laboratory

    NASA Technical Reports Server (NTRS)

    Snell, Edward H.; Fewster, P. F.; Andrew, Norman; Boggon, T. J.; Judge, Russell A.; Pusey, Marc A.

    1999-01-01

    Reciprocal space mapping techniques are used widely by the materials science community to provide physical information about their crystal samples. We have used similar methods at synchrotron sources to look at the quality of macromolecular crystals produced both on the ground and under microgravity conditions. The limited nature of synchrotron time has led us to explore the use of a high resolution materials research diffractometer to perform similar measurements in the home laboratory. Although the available intensity is much reduced due to the beam conditioning necessary for high reciprocal space resolution, lower resolution data can be collected in the same detail as the synchrotron source. Experiments can be optimized at home to make most benefit from the synchrotron time available. Preliminary results including information on the mosaicity and the internal strains from reciprocal space maps will be presented.

  3. Femtosecond light-induced macromolecular self-assembly

    NASA Astrophysics Data System (ADS)

    Rebane, Aleksander; Mikhaylov, Alexander

    2016-09-01

    We report femtosecond light-induced macromolecular self-assembly (FLIMSA), which is observed when a high peak intensity femtosecond laser beam propagates through aqueous solution of pseudoisocyanine iodide (PIC) J-aggregates and induces the formation of 0.1 - 1.0 mm-size tube-like structure surrounding the laser beam, while at the same time allowing the beam to continue propagating without obstruction or scattering. The FLIMSA material is morphologically heterogeneous and gel-like and is formed at the margins rather than at the center of the beam. As a potential explanation of this effect we assume that the FLIMSA is induced by the high photon flux gradient characteristic of the femtosecond laser beam periphery. This hypothesis is corroborated by control experiments, where J-aggregate samples were illuminated with nanosecond laser sources with a varying pulse duration, power- and beam shape characteristics, but where no FLIMSA formation was observed.

  4. Studying macromolecular complex stoichiometries by peptide‐based mass spectrometry

    PubMed Central

    Wohlgemuth, Ingo; Lenz, Christof

    2015-01-01

    A majority of cellular functions are carried out by macromolecular complexes. A host of biochemical and spectroscopic methods exists to characterize especially protein/protein complexes, however there has been a lack of a universal method to determine protein stoichiometries. Peptide‐based MS, especially as a complementary method to the MS analysis of intact protein complexes, has now been developed to a point where it can be employed to assay protein stoichiometries in a routine manner. While the experimental demands are still significant, peptide‐based MS has been successfully applied to analyze stoichiometries for a variety of protein complexes from very different biological backgrounds. In this review, we discuss the requirements especially for targeted MS acquisition strategies to be used in this context, with a special focus on the interconnected experimental aspects of sample preparation, protein digestion, and peptide stability. In addition, different strategies for the introduction of quantitative peptide standards and their suitability for different scenarios are compared. PMID:25546807

  5. Detecting stoichiometry of macromolecular complexes in live cells using FRET.

    PubMed

    Ben-Johny, Manu; Yue, Daniel N; Yue, David T

    2016-12-06

    The stoichiometry of macromolecular interactions is fundamental to cellular signalling yet challenging to detect from living cells. Fluorescence resonance energy transfer (FRET) is a powerful phenomenon for characterizing close-range interactions whereby a donor fluorophore transfers energy to a closely juxtaposed acceptor. Recognizing that FRET measured from the acceptor's perspective reports a related but distinct quantity versus the donor, we utilize the ratiometric comparison of the two to obtain the stoichiometry of a complex. Applying this principle to the long-standing controversy of calmodulin binding to ion channels, we find a surprising Ca(2+)-induced switch in calmodulin stoichiometry with Ca(2+) channels-one calmodulin binds at basal cytosolic Ca(2+) levels while two calmodulins interact following Ca(2+) elevation. This feature is curiously absent for the related Na channels, also potently regulated by calmodulin. Overall, our assay adds to a burgeoning toolkit to pursue quantitative biochemistry of dynamic signalling complexes in living cells.

  6. Conformational States of macromolecular assemblies explored by integrative structure calculation.

    PubMed

    Thalassinos, Konstantinos; Pandurangan, Arun Prasad; Xu, Min; Alber, Frank; Topf, Maya

    2013-09-03

    A detailed description of macromolecular assemblies in multiple conformational states can be very valuable for understanding cellular processes. At present, structural determination of most assemblies in different biologically relevant conformations cannot be achieved by a single technique and thus requires an integrative approach that combines information from multiple sources. Different techniques require different computational methods to allow efficient and accurate data processing and analysis. Here, we summarize the latest advances and future challenges in computational methods that help the interpretation of data from two techniques-mass spectrometry and three-dimensional cryo-electron microscopy (with focus on alignment and classification of heterogeneous subtomograms from cryo-electron tomography). We evaluate how new developments in these two broad fields will lead to further integration with atomic structures to broaden our picture of the dynamic behavior of assemblies in their native environment.

  7. Spontaneous and specific activation of chemical bonds in macromolecular fluids.

    PubMed

    Park, Insun; Shirvanyants, David; Nese, Alper; Matyjaszewski, Krzysztof; Rubinstein, Michael; Sheiko, Sergei S

    2010-09-08

    Mechanical activation of chemical bonds typically involves the application of external forces, which implies a broad distribution of bond tensions. We demonstrate that controlling the flow profile of a macromolecular fluid generates and delineates mechanical force concentration, enabling a hierarchical activation of chemical bonds on different length scales from the macroscopic to the molecular. Bond tension is spontaneously generated within brushlike macromolecules as they spread on a solid substrate. The molecular architecture creates an uneven distribution of tension in the covalent bonds, leading to spatially controlled bond scission. By controlling the flow rate and the gradient of the film pressure, one can sever the flowing macromolecules with high precision. Specific chemical bonds are activated within distinct macromolecules located in a defined area of a thin film. Furthermore, the flow-controlled loading rate enables quantitative analysis of the bond activation parameters.

  8. Macromolecular Crystallization with Microfluidic Free-Interface Diffusion

    SciTech Connect

    Segelke, B

    2005-02-24

    Fluidigm released the Topaz 1.96 and 4.96 crystallization chips in the fall of 2004. Topaz 1.96 and 4.96 are the latest evolution of Fluidigm's microfluidics crystallization technologies that enable ultra low volume rapid screening for macromolecular crystallization. Topaz 1.96 and 4.96 are similar to each other but represent a major redesign of the Topaz system and have of substantially improved ease of automation and ease of use, improved efficiency and even further reduced amount of material needed. With the release of the new Topaz system, Fluidigm continues to set the standard in low volume crystallization screening which is having an increasing impact in the field of structural genomics, and structural biology more generally. In to the future we are likely to see further optimization and increased utility of the Topaz crystallization system, but we are also likely to see further innovation and the emergence of competing technologies.

  9. Macromolecular structure analysis and effective liquefaction pretreatment. Final report

    SciTech Connect

    Suuberg, E.M.; Yun, Y.; Lilly, W.D.; Leung, K.; Gates, T.; Otake, Y.; Deevi, S.C.

    1994-07-01

    This project was concerned with characterizing the changes in coal macromolecular structure, that are of significance for liquefaction pretreatments of coal. The macromolecular structure of the insoluble portion of coal is difficult to characterize. Techniques that do so indirectly (based upon, for example, NMR and FTIR characterizations of atomic linkages) are not particularly sensitive for this purpose. Techniques that characterize the elastic structure (such as solvent swelling) are much more sensitive to subtle changes in the network structure. It is for this reason that we focused upon these techniques. The overall objective involved identifying pretreatments that reduce the crosslinking (physical or chemical) of the network structure, and thus lead to materials that can be handled to a greater extent by traditional liquid-phase processing techniques. These techniques tend to be inherently more efficient at producing desirable products. This report is divided into seven chapters. Chapter II summarizes the main experimental approaches used throughout the project, and summarizes the main findings on the Argonne Premium coal samples. Chapter III considers synergistic effects of solvent pairs. It is divided into two subsections. The first is concerned with mixtures of CS{sub 2} with electron donor solvents. The second subsection is concerned with aromatic hydrocarbon - alcohol or hydrocarbon - alcohol mixtures, as might be of interest for preliquefaction delivery of catalysts into bituminous coals. Chapter IV deals with questions of how oxidation might influence the results that are obtained. Chapter V briefly details what conclusions may be drawn concerning the elastic behavior of coals, and the effects of thermal treatments on this behavior. Chapter VI is concerned with theories to describe the action of solvents that are capable of dissociating non-covalent crosslinks. Finally, Chapter VII discusses the practical implications of the study.

  10. Macromolecular crystal growth experiments on International Microgravity Laboratory--1.

    PubMed Central

    Day, J.; McPherson, A.

    1992-01-01

    Macromolecular crystal growth experiments, using satellite tobacco mosaic virus (STMV) and canavalin from jack beans as samples, were conducted on a US Space Shuttle mission designated International Microgravity Laboratory--1 (IML-1), flown January 22-29, 1992. Parallel experiments using identical samples were carried out in both a vapor diffusion-based device (PCG) and a liquid-liquid diffusion-based instrument (CRYOSTAT). The experiments in each device were run at 20-22 degrees C and at colder temperatures. Crystals were grown in virtually every trial, but the characteristics of the crystals were highly dependent on the crystallization technique employed and the temperature experience of the sample. In general, very good results, based on visual inspection of the crystals, were obtained in both PCG and CRYOSTAT. Unusually impressive results were, however, achieved for STMV in the CRYOSTAT instrument. STMV crystals grown in microgravity by liquid-liquid diffusion were more than 10-fold greater in total volume than any STMV crystals previously grown in the laboratory. X-ray diffraction data collected from eight STMV crystals grown in CRYOSTAT demonstrated a substantial improvement in diffraction quality over the entire resolution range when compared to data from crystals grown on Earth. In addition, the extent of the diffraction pattern for the STMV crystals grown in space extended to 1.8 A resolution, whereas the best crystals that were ever grown under conditions of Earth's gravity produced data limited to 2.3 A resolution. Other observations indicate that the growth of macromolecular crystals is indeed influenced by the presence or absence of gravity. These observations further suggest, consistent with earlier results, that the elimination of gravity provides a more favorable environment for such processes. PMID:1303744

  11. Radiation damage to protein specimens from electron beam imaging and diffraction: a mini-review of anti-damage approaches, with special reference to synchrotron X-ray crystallography.

    PubMed

    Massover, William H

    2007-01-01

    Recent research progress using X-ray cryo-crystallography with the photon beams from third-generation synchrotron sources has resulted in recognition that this intense radiation commonly damages protein samples even when they are held at 100 K. Other structural biologists examining thin protein crystals or single particle specimens encounter similar radiation damage problems during electron diffraction and imaging, but have developed some effective countermeasures. The aim of this concise review is to examine whether analogous approaches can be utilized to alleviate the X-ray radiation damage problem in synchrotron macromolecular crystallography. The critical discussion of this question is preceded by presentation of background material on modern technical procedures with electron beam instruments using 300-400 kV accelerating voltage, low-dose exposures for data recording, and protection of protein specimens by cryogenic cooling; these practical approaches to dealing with electron radiation damage currently permit best resolution levels of 6 A (0.6 nm) for single particle specimens, and of 1.9 A for two-dimensional membrane protein crystals. Final determination of the potential effectiveness and practical value of using such new or unconventional ideas will necessitate showing, by experimental testing, that these produce significantly improved protection of three-dimensional protein crystals during synchrotron X-ray diffraction.

  12. Structure–Biological Function Relationship Extended to Mitotic Arrest-Deficient 2-Like Protein Mad2 Native and Mutants-New Opportunity for Genetic Disorder Control

    PubMed Central

    Avram, Speranta; Milac, Adina; Mernea, Maria; Mihailescu, Dan; Putz, Mihai V.; Buiu, Catalin

    2014-01-01

    Overexpression of mitotic arrest-deficient proteins Mad1 and Mad2, two components of spindle assembly checkpoint, is a risk factor for chromosomal instability (CIN) and a trigger of many genetic disorders. Mad2 transition from inactive open (O-Mad2) to active closed (C-Mad2) conformations or Mad2 binding to specific partners (cell-division cycle protein 20 (Cdc20) or Mad1) were targets of previous pharmacogenomics studies. Here, Mad2 binding to Cdc20 and the interconversion rate from open to closed Mad2 were predicted and the molecular features with a critical contribution to these processes were determined by extending the quantitative structure-activity relationship (QSAR) method to large-size proteins such as Mad2. QSAR models were built based on available published data on 23 Mad2 mutants inducing CIN-related functional changes. The most relevant descriptors identified for predicting Mad2 native and mutants action mechanism and their involvement in genetic disorders are the steric (van der Waals area and solvent accessible area and their subdivided) and energetic van der Waals energy descriptors. The reliability of our QSAR models is indicated by significant values of statistical coefficients: Cross-validated correlation q2 (0.53–0.65) and fitted correlation r2 (0.82–0.90). Moreover, based on established QSAR equations, we rationally design and analyze nine de novo Mad2 mutants as possible promoters of CIN. PMID:25411801

  13. Towards an efficient compression of 3D coordinates of macromolecular structures

    PubMed Central

    Valasatava, Yana; Bradley, Anthony R.; Rose, Alexander S.; Duarte, Jose M.; Prlić, Andreas

    2017-01-01

    The size and complexity of 3D macromolecular structures available in the Protein Data Bank is constantly growing. Current tools and file formats have reached limits of scalability. New compression approaches are required to support the visualization of large molecular complexes and enable new and scalable means for data analysis. We evaluated a series of compression techniques for coordinates of 3D macromolecular structures and identified the best performing approaches. By balancing compression efficiency in terms of the decompression speed and compression ratio, and code complexity, our results provide the foundation for a novel standard to represent macromolecular coordinates in a compact and useful file format. PMID:28362865

  14. Towards an efficient compression of 3D coordinates of macromolecular structures.

    PubMed

    Valasatava, Yana; Bradley, Anthony R; Rose, Alexander S; Duarte, Jose M; Prlić, Andreas; Rose, Peter W

    2017-01-01

    The size and complexity of 3D macromolecular structures available in the Protein Data Bank is constantly growing. Current tools and file formats have reached limits of scalability. New compression approaches are required to support the visualization of large molecular complexes and enable new and scalable means for data analysis. We evaluated a series of compression techniques for coordinates of 3D macromolecular structures and identified the best performing approaches. By balancing compression efficiency in terms of the decompression speed and compression ratio, and code complexity, our results provide the foundation for a novel standard to represent macromolecular coordinates in a compact and useful file format.

  15. Adaptive evolution in the Arabidopsis MADS-box gene family inferred from its complete resolved phylogeny

    PubMed Central

    Martínez-Castilla, León Patricio; Alvarez-Buylla, Elena R.

    2003-01-01

    Gene duplication is a substrate of evolution. However, the relative importance of positive selection versus relaxation of constraints in the functional divergence of gene copies is still under debate. Plant MADS-box genes encode transcriptional regulators key in various aspects of development and have undergone extensive duplications to form a large family. We recovered 104 MADS sequences from the Arabidopsis genome. Bayesian phylogenetic trees recover type II lineage as a monophyletic group and resolve a branching sequence of monophyletic groups within this lineage. The type I lineage is comprised of several divergent groups. However, contrasting gene structure and patterns of chromosomal distribution between type I and II sequences suggest that they had different evolutionary histories and support the placement of the root of the gene family between these two groups. Site-specific and site-branch analyses of positive Darwinian selection (PDS) suggest that different selection regimes could have affected the evolution of these lineages. We found evidence for PDS along the branch leading to flowering time genes that have a direct impact on plant fitness. Sites with high probabilities of having been under PDS were found in the MADS and K domains, suggesting that these played important roles in the acquisition of novel functions during MADS-box diversification. Detected sites are targets for further experimental analyses. We argue that adaptive changes in MADS-domain protein sequences have been important for their functional divergence, suggesting that changes within coding regions of transcriptional regulators have influenced phenotypic evolution of plants. PMID:14597714

  16. Science with ESO's Multi-conjugate Adaptive-optics Demonstrator - MAD

    NASA Astrophysics Data System (ADS)

    Melnick, Jorge; Marchetti, Enrico; Amico, Paola

    2012-07-01

    ESO's Multi-conjugate Adaptive-optics Demonstrator (MAD) was a prototype designed and built to demonstrate wide-field adaptive optics science on large telescopes. The outstanding results obtained during commissioning and guaranteed time observations (GTO) prompted ESO to issue and open call to the community for 23 science demonstration (SD) observing nights distributed in three runs (in order to provide access to the summer an winter skies). Thus, in total MAD was used for science for 33 nights including the 10 nights of GTO time. date, 19 articles in refereed journals (including one in Nature) have been published based fully or partially o MAD data. To the best of our knowledge, these are not only the first, but also the only scientific publication from MCAO instruments world-wide to date (at least in Astronomy). The scientific impact of these publication, as measured by the h-index, is comparable to that of other AO instruments on the VLT, although over the years these instruments have been allocated many more nights than MAD. In this contribution we present an overview of the scientific results from MAD and a more detailed discussion of the most cited papers.

  17. Functional conservation of MIKC*-Type MADS box genes in Arabidopsis and rice pollen maturation.

    PubMed

    Liu, Yuan; Cui, Shaojie; Wu, Feng; Yan, Shuo; Lin, Xuelei; Du, Xiaoqiu; Chong, Kang; Schilling, Susanne; Theißen, Günter; Meng, Zheng

    2013-04-01

    There are two groups of MADS intervening keratin-like and C-terminal (MIKC)-type MADS box genes, MIKC(C) type and MIKC* type. In seed plants, the MIKC(C) type shows considerable diversity, but the MIKC* type has only two subgroups, P- and S-clade, which show conserved expression in the gametophyte. To examine the functional conservation of MIKC*-type genes, we characterized all three rice (Oryza sativa) MIKC*-type genes. All three genes are specifically expressed late in pollen development. The single knockdown or knockout lines, respectively, of the S-clade MADS62 and MADS63 did not show a mutant phenotype, but lines in which both S-clade genes were affected showed severe defects in pollen maturation and germination, as did knockdown lines of MADS68, the only P-clade gene in rice. The rice MIKC*-type proteins form strong heterodimeric complexes solely with partners from the other subclade; these complexes specifically bind to N10-type C-A-rich-G-boxes in vitro and regulate downstream gene expression by binding to N10-type promoter motifs. The rice MIKC* genes have a much lower degree of functional redundancy than the Arabidopsis thaliana MIKC* genes. Nevertheless, our data indicate that the function of heterodimeric MIKC*-type protein complexes in pollen development has been conserved since the divergence of monocots and eudicots, roughly 150 million years ago.

  18. Agave tequilana MADS genes show novel expression patterns in meristems, developing bulbils and floral organs.

    PubMed

    Delgado Sandoval, Silvia del Carmen; Abraham Juárez, María Jazmín; Simpson, June

    2012-03-01

    Agave tequilana is a monocarpic perennial species that flowers after 5-8 years of vegetative growth signaling the end of the plant's life cycle. When fertilization is unsuccessful, vegetative bulbils are induced on the umbels of the inflorescence near the bracteoles from newly formed meristems. Although the regulation of inflorescence and flower development has been described in detail for monocarpic annuals and polycarpic species, little is known at the molecular level for these processes in monocarpic perennials, and few studies have been carried out on bulbils. Histological samples revealed the early induction of umbel meristems soon after the initiation of the vegetative to inflorescence transition in A. tequilana. To identify candidate genes involved in the regulation of floral induction, a search for MADS-box transcription factor ESTs was conducted using an A. tequilana transcriptome database. Seven different MIKC MADS genes classified into 6 different types were identified based on previously characterized A. thaliana and O. sativa MADS genes and sequences from non-grass monocotyledons. Quantitative real-time PCR analysis of the seven candidate MADS genes in vegetative, inflorescence, bulbil and floral tissues uncovered novel patterns of expression for some of the genes in comparison with orthologous genes characterized in other species. In situ hybridization studies using two different genes showed expression in specific tissues of vegetative meristems and floral buds. Distinct MADS gene regulatory patterns in A. tequilana may be related to the specific reproductive strategies employed by this species.

  19. Anomalous Diffraction at Ultra-High Energy for Protein Crystallography

    SciTech Connect

    Jakoncic,J.; Di Michiel, M.; Zhong, Z.; Honkimaki, V.; Jouanneau, Y.; Stojanoff, V.

    2006-01-01

    Single-wavelength anomalous diffraction (SAD), multiwavelength anomalous diffraction (MAD) and single isomorphous replacement with anomalous scattering (SIRAS) phasing at ultra-high X-ray energy, 55 keV, are used successfully to determine a high-quality and high-resolution experimental electronic density map of hen egg-white lysozyme, a model protein. Several combinations, between single- and three-wavelength, with native data were exploited to demonstrate that standard phasing procedures with standard equipment and software can successfully be applied to three-dimensional crystal structure determination of a macromolecule, even at these very short wavelengths. For the first time, a high-quality three-dimensional molecular structure is reported from SAD phasing with ultra-high-energy X-rays. The quality of the crystallographic data and the experimental electron density maps meet current standards. The 2.7% anomalous signal from three Ho atoms, at the Ho K edge, was sufficient to obtain a remarkable electron density and build the first lanthanide structure for HEWL in its entirety.

  20. Hydraulic analysis, Mad River at State Highway 41, Springfield, Ohio

    USGS Publications Warehouse

    Mayo, Ronald I.

    1977-01-01

    A hydraulic analysis of the lad River in a reach at Springfield, Ohio was made to determine the effects of relocating State Highway 41 in 1S76. The main channel was cleaned by dredging in the vicinity cf the new highway bridge and at the Detroit, Toledo and Ironton Railway bridge upstream. The new highway was placed on a high fill with relief structures for flood plain drainage consisting of a 12-foot corrugated metal pipe culvert and a bridge opening to accommodate the Detroit, Toledo and Ironton Railway and a property access road. The effect of the new highway embankment on drainage from the flood plain was requested. Also requested was the effect that might be expected on the elevation of flood waters above the new highway embankment if the access road through the new highway embankment were raised.The study indicates that the improvement in the capacity of the main channel to carry water was such that, up to a discharge equivalent to a 25-year frequency flood, the water-surface elevation in the reach upstream from the Detroit, Toledo and Ironton Railway bridge would be about 0.6 foot lower than under conditions prior to the construction on State Highway 41. Diversion through the Mad River left bank levee break above the Detroit, Toledo and Ironton Railway bridge to the flood Flain would be decreased about one-half in terms of rate of discharge in cubic feet per second. The maximum difference in elevation cf the flood water between the upstream and downstream side of the new State Highway 41 embankment would be about 0.2 foot, with an additional 0.4 foot to be expected if the access road were raised 1.5 feet.