Updated on 2021/06/18

写真a

 
CHAVAS Leonard Michel Gabriel Henri
 
Organization
Synchrotron Radiation Research Center Division of Synchrotron Radiation Professor
Graduate School
Graduate School of Engineering
Title
Professor
Contact information
メールアドレス
Profile
Frenchy expatriated in Japan, passionate by synchrotron radiation and related methodologies to unravel the mysteries behind biology, loving father of 3

Degree 3

  1. 博士(理学) ( 2005.9   総合研究大学院大学 ) 

  2. M.S. Functional and Structural Biology ( 2002.6 ) 

  3. B.S. Biochemistry ( 2000.6 ) 

Research Interests 5

  1. Synchrotron radiation

  2. Structural biology

  3. Protein crystallography

  4. Integrated biology

  5. In vivo crystallography

Research Areas 5

  1. Life Science / Biophysics

  2. Informatics / Life, health and medical informatics  / Multi-scale imaging informatics

  3. Nanotechnology/Materials / Bio chemistry  / in vivo crystallography

  4. Life Science / Functional biochemistry  / Integrated biology

  5. Life Science / Structural biochemistry  / Protein crystallography

Research History 7

  1. Nagoya University   Department of Applied Physics, Synchrotron Radiation Research Center   Professor

    2021.3

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    Country:Japan

  2. Synchrotron SOLEIL (France)   Health and Biology section   Head of Biology Scientific section

    2017.1 - 2021.2

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    Country:France

  3. Synchrotron SOLEIL (France)   Beamline PROXIMA-1   Beamline manager & Group leader

    2015.6 - 2021.2

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    Country:France

  4. DESY (Germany)   Center for Free Electron Laser sciences   Scientist & Team leader

    2013.9 - 2015.5

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    Country:Germany

  5. High Energy Accelerator Research Organization   Structural Biology Research Center   Assistant Professor   Beamline manager & Principal investigator

    2009.4 - 2013.8

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    Country:Japan

  6. The University of Manchester (UK)   Department of Life Sciences   Research associate

    2007.9 - 2009.3

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    Country:United Kingdom

  7. High Accelerator Research Organization   Structural Biology Research Center   Post-doctoral fellow

    2005.10 - 2007

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    Country:Japan

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Education 1

  1. The Graduate University for Advanced Studies   Ph. D., Functional and Structural Biology

    2002 - 2005

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    Country: Japan

 

Papers 57

  1. Pr‐favoured variants of the bacteriophytochrome from the plant pathogen Xanthomonas campestris hint on light regulation of virulence‐associated mechanisms

    Valeria Conforte, Lisandro Horacio Otero, Laila Toum, Serena Sirigu, Giuliano Tomás Antelo, Jimena Rinaldi, Sabrina Foscaldi, Sebastián Klinke, Leonard Michel Gabriel Chavas, Adrián Alberto Vojnov, Fernando Alberto Goldbaum, Florencia Malamud, Hernán Ruy Bonomi

    The FEBS Journal     2021.5

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Wiley  

    DOI: 10.1111/febs.15883

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    Other Link: https://onlinelibrary.wiley.com/doi/full-xml/10.1111/febs.15883

  2. PROXIMA-1 beamline for macromolecular crystallography measurements at Synchrotron SOLEIL

    Leonard M. G. Chavas, Patrick Gourhant, Beatriz G. Guimaraes, Tatiana Isabet, Pierre Legrand, Robin Lener, Pierre Montaville, Serena Sirigu, Andrew Thompson

    Journal of Synchrotron Radiation   Vol. 28 ( 3 ) page: 970 - 976   2021.5

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:International Union of Crystallography (IUCr)  

    The undulator beamline PROXIMA-1 at Synchrotron SOLEIL scheduled its first users in March 2008. The endstation is dedicated to biomolecular crystallography experiments, with a layout designed to favour anomalous data recording and studies of crystals with large cell dimensions. In 12 years, the beamline has accommodated 4267 shifts of 8 h and more than 6300 visitors. By the end of 2020, it saw 1039 identified published scientific papers referring to 1415 coordinates deposited in the Protein Data Bank. The current paper describes the PROXIMA-1 beamline, including the recent specific implementations developed for the sample environment. The setup installed in the experimental station contains numerous beam-shaping equipment, a chi-geometry three-axis goniometer, a single-photon-counting pixel-array X-ray detector, combined with a medium-throughput sample exchange robot. As part of a standard experimental scheme, PROXIMA-1 can also be accessed via `mail-in' services or remotely.

    DOI: 10.1107/s1600577521002605

  3. The microfluidic laboratory at Synchrotron SOLEIL. International journal

    Igor Chaussavoine, Anthony Beauvois, Tiphaine Mateo, Ramakrishna Vasireddi, Nadine Douri, Jordan Priam, Youssef Liatimi, Stéphane Lefrançois, Hervé Tabuteau, Mélanie Davranche, Delphine Vantelon, Thomas Bizien, Leonard M G Chavas, Benedikt Lassalle-Kaiser

    Journal of synchrotron radiation   Vol. 27 ( Pt 1 ) page: 230 - 237   2020.1

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:INT UNION CRYSTALLOGRAPHY  

    A microfluidic laboratory recently opened at Synchrotron SOLEIL, dedicated to in-house research and external users. Its purpose is to provide the equipment and expertise that allow the development of microfluidic systems adapted to the beamlines of SOLEIL as well as other light sources. Such systems can be used to continuously deliver a liquid sample under a photon beam, keep a solid sample in a liquid environment or provide a means to track a chemical reaction in a time-resolved manner. The laboratory provides all the amenities required for the design and preparation of soft-lithography microfluidic chips compatible with synchrotron-based experiments. Three examples of microfluidic systems that were used on SOLEIL beamlines are presented, which allow the use of X-ray techniques to study physical, chemical or biological phenomena.

    DOI: 10.1107/S1600577519015042

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  4. Integrated biology at large research infrastructures in Europe Invited Reviewed

    イメージング時代の構造生命科学   Vol. 38 ( 5 ) page: 858 - 862   2020

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    Authorship:Lead author, Last author, Corresponding author   Language:Japanese   Publishing type:Part of collection (book)  

  5. Improvement of Production and Isolation of Human Neuraminidase-1 in Cellulo Crystals

    Kotaro Koiwai, Jun Tsukimoto, Tetsuya Higashi, Fumitaka Mafune, Ken Miyajima, Takanori Nakane, Naohiro Matsugaki, Ryuichi Kato, Serena Sirigu, Arjen Jakobi, Matthias Wilmanns, Michihiro Sugahara, Tomoyuki Tanaka, Kensuke Tono, Yasumasa Joti, Makina Yabashi, Osamu Nureki, Eiichi Mizohata, Toru Nakatsu, Eriko Nango, So Iwata, Leonard M. G. Chavas, Toshiya Senda, Kohji Itoh, Fumiaki Yumoto

    ACS APPLIED BIO MATERIALS   Vol. 2 ( 11 ) page: 4941 - 4952   2019.11

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER CHEMICAL SOC  

    In cellulo crystallization is a developing technique to provide crystals for protein structure determination, particularly for proteins that are difficult to prepare by in vitro crystallization. This method has a key advantage: it requires neither a protein purification step nor a crystallization step. However, there is still no systematic strategy for improving the technique of in cellulo crystallization because the process occurs spontaneously. Here we report a protocol to produce and extract in cellulo crystals of human lysosomal neuraminidase-1 (NEU1) in human cultured cells. Overexpression of NEU1 protein by the retransfection of cells pretransfected with neu1-overexpressing plasmid improved the efficiency of NEU1 crystallization. Microscopic analysis revealed that NEU1 proteins were not crystallized in the lysosome but in the endoplasmic reticulum (ER). Screening of the buffer conditions used to extract crystals from cells further improved the crystal yield. The optimal pH was 7.0, which corresponds to the pH in the ER. Use of a high-yield flask with a large surface area also yielded more crystals. These optimizations enabled us to execute a serial femtosecond crystallography experiment with a sufficient number of crystals to generate a complete data set. Optimization of the in cellulo crystallization method was thus shown to be possible.

    DOI: 10.1021/acsabm.9b00686

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  6. Author Correction: Coherent diffractive imaging of microtubules using an X-ray laser. International journal

    Gisela Brändén, Greger Hammarin, Rajiv Harimoorthy, Alexander Johansson, David Arnlund, Erik Malmerberg, Anton Barty, Stefan Tångefjord, Peter Berntsen, Daniel P DePonte, Carolin Seuring, Thomas A White, Francesco Stellato, Richard Bean, Kenneth R Beyerlein, Leonard M G Chavas, Holger Fleckenstein, Cornelius Gati, Umesh Ghoshdastider, Lars Gumprecht, Dominik Oberthür, David Popp, Marvin Seibert, Thomas Tilp, Marc Messerschmidt, Garth J Williams, N Duane Loh, Henry N Chapman, Peter Zwart, Mengning Liang, Sébastien Boutet, Robert C Robinson, Richard Neutze

    Nature communications   Vol. 10 ( 1 ) page: 4101 - 4101   2019.9

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    Language:English   Publisher:NATURE PUBLISHING GROUP  

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

    DOI: 10.1038/s41467-019-12151-3

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  7. Coherent diffractive imaging of microtubules using an X-ray laser. International journal

    Gisela Brändén, Greger Hammarin, Rajiv Harimoorthy, Alexander Johansson, David Arnlund, Erik Malmerberg, Anton Barty, Stefan Tångefjord, Peter Berntsen, Daniel P DePonte, Carolin Seuring, Thomas A White, Francesco Stellato, Richard Bean, Kenneth R Beyerlein, Leonard M G Chavas, Holger Fleckenstein, Cornelius Gati, Umesh Ghoshdastider, Lars Gumprecht, Dominik Oberthür, David Popp, Marvin Seibert, Thomas Tilp, Marc Messerschmidt, Garth J Williams, N Duane Loh, Henry N Chapman, Peter Zwart, Mengning Liang, Sébastien Boutet, Robert C Robinson, Richard Neutze

    Nature communications   Vol. 10 ( 1 ) page: 2589 - 2589   2019.6

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:NATURE PUBLISHING GROUP  

    X-ray free electron lasers (XFELs) create new possibilities for structural studies of biological objects that extend beyond what is possible with synchrotron radiation. Serial femtosecond crystallography has allowed high-resolution structures to be determined from micro-meter sized crystals, whereas single particle coherent X-ray imaging requires development to extend the resolution beyond a few tens of nanometers. Here we describe an intermediate approach: the XFEL imaging of biological assemblies with helical symmetry. We collected X-ray scattering images from samples of microtubules injected across an XFEL beam using a liquid microjet, sorted these images into class averages, merged these data into a diffraction pattern extending to 2 nm resolution, and reconstructed these data into a projection image of the microtubule. Details such as the 4 nm tubulin monomer became visible in this reconstruction. These results illustrate the potential of single-molecule X-ray imaging of biological assembles with helical symmetry at room temperature.

    DOI: 10.1038/s41467-019-10448-x

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  8. MXCuBE2: the dawn of MXCuBE Collaboration. International journal

    Marcus Oscarsson, Antonia Beteva, David Flot, Elspeth Gordon, Matias Guijarro, Gordon Leonard, Sean McSweeney, Stephanie Monaco, Christoph Mueller-Dieckmann, Max Nanao, Didier Nurizzo, Alexander N Popov, David von Stetten, Olof Svensson, Vicente Rey-Bakaikoa, Idrissou Chado, Leonard M G Chavas, Laurent Gadea, Patrick Gourhant, Tatiana Isabet, Pierre Legrand, Martin Savko, Serena Sirigu, William Shepard, Andrew Thompson, Uwe Mueller, Jie Nan, Mikel Eguiraun, Fredrick Bolmsten, Alberto Nardella, Antonio Milàn-Otero, Marjolein Thunnissen, Michael Hellmig, Alexandra Kastner, Lukas Schmuckermaier, Martin Gerlach, Christian Feiler, Manfred S Weiss, Matthew W Bowler, Alexandre Gobbo, Gergely Papp, Jeremy Sinoir, Andrew A McCarthy, Ivars Karpics, Marina Nikolova, Gleb Bourenkov, Thomas Schneider, Jordi Andreu, Guifré Cuní, Judith Juanhuix, Roeland Boer, Rasmus Fogh, Peter Keller, Claus Flensburg, Wlodek Paciorek, Clemens Vonrhein, Gerard Bricogne, Daniele de Sanctis

    Journal of synchrotron radiation   Vol. 26 ( Pt 2 ) page: 393 - 405   2019.3

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:INT UNION CRYSTALLOGRAPHY  

    MXCuBE2 is the second-generation evolution of the MXCuBE beamline control software, initially developed and used at ESRF - the European Synchrotron. MXCuBE2 extends, in an intuitive graphical user interface (GUI), the functionalities and data collection methods available to users while keeping all previously available features and allowing for the straightforward incorporation of ongoing and future developments. MXCuBE2 introduces an extended abstraction layer that allows easy interfacing of any kind of macromolecular crystallography (MX) hardware component, whether this is a diffractometer, sample changer, detector or optical element. MXCuBE2 also works in strong synergy with the ISPyB Laboratory Information Management System, accessing the list of samples available for a particular experimental session and associating, either from instructions contained in ISPyB or from user input via the MXCuBE2 GUI, different data collection types to them. The development of MXCuBE2 forms the core of a fruitful collaboration which brings together several European synchrotrons and a software development factory and, as such, defines a new paradigm for the development of beamline control platforms for the European MX user community.

    DOI: 10.1107/S1600577519001267

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  9. Diffracted X-ray Blinking Tracks Single Protein Motions. International journal

    Hiroshi Sekiguchi, Masahiro Kuramochi, Keigo Ikezaki, Yu Okamura, Kazuki Yoshimura, Ken Matsubara, Jae-Won Chang, Noboru Ohta, Tai Kubo, Kazuhiro Mio, Yoshio Suzuki, Leonard M G Chavas, Yuji C Sasaki

    Scientific reports   Vol. 8 ( 1 ) page: 17090 - 17090   2018.11

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:NATURE PUBLISHING GROUP  

    Single molecule dynamics studies have begun to use quantum probes. Single particle analysis using cryo-transmission electron microscopy has dramatically improved the resolution when studying protein structures and is shifting towards molecular motion observations. X-ray free-electron lasers are also being explored as routes for determining single molecule structures of biological entities. Here, we propose a new X-ray single molecule technology that allows observation of molecular internal motion over long time scales, ranging from milliseconds up to 103 seconds. Our method uses both low-dose monochromatic X-rays and nanocrystal labelling technology. During monochromatic X-ray diffraction experiments, the intensity of X-ray diffraction from moving single nanocrystals appears to blink because of Brownian motion in aqueous solutions. X-ray diffraction spots from moving nanocrystals were observed to cycle in and out of the Bragg condition. Consequently, the internal motions of a protein molecule labelled with nanocrystals could be extracted from the time trajectory using this diffracted X-ray blinking (DXB) approach. Finally, we succeeded in distinguishing the degree of fluctuation motions of an individual acetylcholine-binding protein (AChBP) interacting with acetylcholine (ACh) using a laboratory X-ray source.

    DOI: 10.1038/s41598-018-35468-3

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  10. The New Era of Microcrystallography

    Sanchari Banerjee, Pierre Montaville, Leonard M. G. Chavas, S. Ramaswamy

    JOURNAL OF THE INDIAN INSTITUTE OF SCIENCE   Vol. 98 ( 3 ) page: 273 - 281   2018.9

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    Language:English   Publisher:SPRINGER  

    The function of a protein dictates its physical state in a cell. Evolution has imparted selection pressure on proteins to maximize their function and minimize cell death. Most of the proteins exist in their soluble form inside or outside the cells. However, a small fraction of proteins in the total protein pool crystallizes with functional consequence. These in vivo-grown protein crystals perform a diversity of functions, ranging from food storage to defense. Sometimes limited by the volume of the cells and the cellular concentration of proteins, these crystals are very small in size. Hence, it has been difficult to carry out conventional X-ray crystallography on these crystals. With the advent of microcrystallography, it is now possible to study the structures of these tiny crystals. In this review, some of the diverse examples of in vivo crystals and the new approaches towards microcrystallography are summarized.

    DOI: 10.1007/s41745-018-0086-0

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  11. Flow-aligned, single-shot fiber diffraction using a femtosecond X-ray free-electron laser. International journal

    David Popp, N Duane Loh, Habiba Zorgati, Umesh Ghoshdastider, Lu Ting Liow, Magdalena I Ivanova, Mårten Larsson, Daniel P DePonte, Richard Bean, Kenneth R Beyerlein, Cornelius Gati, Dominik Oberthuer, David Arnlund, Gisela Brändén, Peter Berntsen, Duilio Cascio, Leonard M G Chavas, Joe P J Chen, Ke Ding, Holger Fleckenstein, Lars Gumprecht, Rajiv Harimoorthy, Estelle Mossou, Michael R Sawaya, Aaron S Brewster, Johan Hattne, Nicholas K Sauter, Marvin Seibert, Carolin Seuring, Francesco Stellato, Thomas Tilp, David S Eisenberg, Marc Messerschmidt, Garth J Williams, Jason E Koglin, Lee Makowski, Rick P Millane, Trevor Forsyth, Sébastien Boutet, Thomas A White, Anton Barty, Henry Chapman, Swaine L Chen, Mengning Liang, Richard Neutze, Robert C Robinson

    Cytoskeleton (Hoboken, N.J.)   Vol. 74 ( 12 ) page: 472 - 481   2017.12

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY  

    A major goal for X-ray free-electron laser (XFEL) based science is to elucidate structures of biological molecules without the need for crystals. Filament systems may provide some of the first single macromolecular structures elucidated by XFEL radiation, since they contain one-dimensional translational symmetry and thereby occupy the diffraction intensity region between the extremes of crystals and single molecules. Here, we demonstrate flow alignment of as few as 100 filaments (Escherichia coli pili, F-actin, and amyloid fibrils), which when intersected by femtosecond X-ray pulses result in diffraction patterns similar to those obtained from classical fiber diffraction studies. We also determine that F-actin can be flow-aligned to a disorientation of approximately 5 degrees. Using this XFEL-based technique, we determine that gelsolin amyloids are comprised of stacked β-strands running perpendicular to the filament axis, and that a range of order from fibrillar to crystalline is discernable for individual α-synuclein amyloids.

    DOI: 10.1002/cm.21378

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  12. Post-sample aperture for low background diffraction experiments at X-ray free-electron lasers. International journal

    Max O Wiedorn, Salah Awel, Andrew J Morgan, Miriam Barthelmess, Richard Bean, Kenneth R Beyerlein, Leonard M G Chavas, Niko Eckerskorn, Holger Fleckenstein, Michael Heymann, Daniel A Horke, Juraj Knoška, Valerio Mariani, Dominik Oberthür, Nils Roth, Oleksandr Yefanov, Anton Barty, Saša Bajt, Jochen Küpper, Andrei V Rode, Richard A Kirian, Henry N Chapman

    Journal of synchrotron radiation   Vol. 24 ( Pt 6 ) page: 1296 - 1298   2017.11

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:INT UNION CRYSTALLOGRAPHY  

    The success of diffraction experiments from weakly scattering samples strongly depends on achieving an optimal signal-to-noise ratio. This is particularly important in single-particle imaging experiments where diffraction signals are typically very weak and the experiments are often accompanied by significant background scattering. A simple way to tremendously reduce background scattering by placing an aperture downstream of the sample has been developed and its application in a single-particle X-ray imaging experiment at FLASH is demonstrated. Using the concept of a post-sample aperture it was possible to reduce the background scattering levels by two orders of magnitude.

    DOI: 10.1107/S1600577517011961

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  13. Crystallization via tubing microfluidics permits both in situ and ex situ X-ray diffraction International journal

    Charline J. J. Gerard, Gilles Ferry, Laurent M. Vuillard, Jean A. Boutin, Leonard M. G. Chavas, Tiphaine Huet, Nathalie Ferte, Romain Grossier, Nadine Candoni, Stéphane Veesler

    Acta Crystallographica Section F Structural Biology Communications   Vol. 73 ( 10 ) page: 574 - 578   2017.10

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:International Union of Crystallography ({IUCr})  

    A microfluidic platform was used to address the problems of obtaining diffraction-quality crystals and crystal handling during transfer to the X-ray diffractometer. Crystallization conditions of a protein of pharmaceutical interest were optimized and X-ray data were collected both in situ and ex situ.

    DOI: 10.1107/S2053230X17013826

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  14. The impact of the butterfly effect on human parainfluenza virus haemagglutinin-neuraminidase inhibitor design. International journal

    Larissa Dirr, Ibrahim M El-Deeb, Leonard M G Chavas, Patrice Guillon, Mark von Itzstein

    Scientific reports   Vol. 7 ( 1 ) page: 4507 - 4507   2017.7

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:NATURE PUBLISHING GROUP  

    Human parainfluenza viruses represent a leading cause of lower respiratory tract disease in children, with currently no available approved drug or vaccine. The viral surface glycoprotein haemagglutinin-neuraminidase (HN) represents an ideal antiviral target. Herein, we describe the first structure-based study on the rearrangement of key active site amino acid residues by an induced opening of the 216-loop, through the accommodation of appropriately functionalised neuraminic acid-based inhibitors. We discovered that the rearrangement is influenced by the degree of loop opening and is controlled by the neuraminic acid's C-4 substituent's size (large or small). In this study, we found that these rearrangements induce a butterfly effect of paramount importance in HN inhibitor design and define criteria for the ideal substituent size in two different categories of HN inhibitors and provide novel structural insight into the druggable viral HN protein.

    DOI: 10.1038/s41598-017-04656-y

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  15. Status of the crystallography beamlines at synchrotron SOLEIL⋆

    A. Coati, L. M. G. Chavas, P. Fontaine, N. Foos, B. Guimaraes, P. Gourhant, P. Legrand, J. -P. Itie, P. Fertey, W. Shepard, T. Isabet, S. Sirigu, P. -L. Solari, D. Thiaudiere, A. Thompson

    The European Physical Journal Plus   Vol. 132 ( 4 )   2017.4

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Springer Science and Business Media LLC  

    DOI: 10.1140/epjp/i2017-11403-3

    Other Link: http://link.springer.com/article/10.1140/epjp/i2017-11403-3/fulltext.html

  16. Role of Quinone Reductase 2 in the Antimalarial Properties of Indolone-Type Derivatives. International journal

    Laure-Estelle Cassagnes, Nambinina Rakotoarivelo, Serena Sirigu, Pierre Pério, Ennaji Najahi, Léonard M G Chavas, Andrew Thompson, Régis Gayon, Gilles Ferry, Jean A Boutin, Alexis Valentin, Karine Reybier, Françoise Nepveu

    Molecules (Basel, Switzerland)   Vol. 22 ( 2 )   2017.1

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:MDPI AG  

    Indolone-N-oxides have antiplasmodial properties against Plasmodium falciparum at the erythrocytic stage, with IC50 values in the nanomolar range. The mechanism of action of indolone derivatives involves the production of free radicals, which follows their bioreduction by an unknown mechanism. In this study, we hypothesized that human quinone reductase 2 (hQR2), known to act as a flavin redox switch upon binding to the broadly used antimalarial chloroquine, could be involved in the activity of the redox-active indolone derivatives. Therefore, we investigated the role of hQR2 in the reduction of indolone derivatives. We analyzed the interaction between hQR2 and several indolone-type derivatives by examining enzymatic kinetics, the substrate/protein complex structure with X-ray diffraction analysis, and the production of free radicals with electron paramagnetic resonance. The reduction of each compound in cells overexpressing hQR2 was compared to its reduction in naïve cells. This process could be inhibited by the specific hQR2 inhibitor, S29434. These results confirmed that the anti-malarial activity of indolone-type derivatives was linked to their ability to serve as hQR2 substrates and not as hQR2 inhibitors as reported for chloroquine, leading to the possibility that substrate of hQR2 could be considered as a new avenue for the design of new antimalarial compounds.

    DOI: 10.3390/molecules22020210

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  17. De novo in-vivo protein crystal structure: is experimental phasing required?

    Pierre Montaville, Leonard Chavas

    ACTA CRYSTALLOGRAPHICA A-FOUNDATION AND ADVANCES   Vol. 73   page: C878 - C878   2017

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    Language:English   Publisher:INT UNION CRYSTALLOGRAPHY  

    DOI: 10.1107/S205327331708696X

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  18. Structure of in vivo protein crystals from viviparous Diploptera punctata

    Sanchari Banerjee, Nathan P. Coussens, Francois-Xavier Gallat, Nitish Sathyanarayanan, Koichiro J. Yagi, James S. S. Gray, Stephen S. Tobe, Barbara Stay, Leonard M. G. Chavas, S. Ramaswamy

    ACTA CRYSTALLOGRAPHICA A-FOUNDATION AND ADVANCES   Vol. 73   page: C177 - C177   2017

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    DOI: 10.1107/S2053273317093962

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  19. Total chemical synthesis, refolding, and crystallographic structure of fully active immunophilin calstabin 2 (FKBP12.6). International journal

    Marine Bacchi, Magali Jullian, Serena Sirigu, Benjamin Fould, Tiphaine Huet, Lisa Bruyand, Mathias Antoine, Laurent Vuillard, Luisa Ronga, Leonard M G Chavas, Olivier Nosjean, Gilles Ferry, Karine Puget, Jean A Boutin

    Protein science : a publication of the Protein Society   Vol. 25 ( 12 ) page: 2225 - 2242   2016.12

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY-BLACKWELL  

    Synthetic biology (or chemical biology) is a growing field to which the chemical synthesis of proteins, particularly enzymes, makes a fundamental contribution. However, the chemical synthesis of catalytically active proteins (enzymes) remains poorly documented because it is difficult to obtain enough material for biochemical experiments. We chose calstabin, a 107-amino-acid proline isomerase, as a model. We synthesized the enzyme using the native chemical ligation approach and obtained several tens of milligrams. The polypeptide was refolded properly, and we characterized its biophysical properties, measured its catalytic activity, and then crystallized it in order to obtain its tridimensional structure after X-ray diffraction. The refolded enzyme was compared to the recombinant, wild-type enzyme. In addition, as a first step of validating the whole process, we incorporated exotic amino acids into the N-terminus. Surprisingly, none of the changes altered the catalytic activities of the corresponding mutants. Using this body of techniques, avenues are now open to further obtain enzymes modified with exotic amino acids in a way that is only barely accessible by molecular biology, obtaining detailed information on the structure-function relationship of enzymes reachable by complete chemical synthesis.

    DOI: 10.1002/pro.3051

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  20. Structure of a heterogeneous, glycosylated, lipid-bound, in vivo-grown protein crystal at atomic resolution from the viviparous cockroach Diploptera punctata. International journal

    Sanchari Banerjee, Nathan P Coussens, François-Xavier Gallat, Nitish Sathyanarayanan, Jandhyam Srikanth, Koichiro J Yagi, James S S Gray, Stephen S Tobe, Barbara Stay, Leonard M G Chavas, Subramanian Ramaswamy

    IUCrJ   Vol. 3 ( Pt 4 ) page: 282 - 93   2016.7

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:INT UNION CRYSTALLOGRAPHY  

    Macromolecular crystals for X-ray diffraction studies are typically grown in vitro from pure and homogeneous samples; however, there are examples of protein crystals that have been identified in vivo. Recent developments in micro-crystallography techniques and the advent of X-ray free-electron lasers have allowed the determination of several protein structures from crystals grown in cellulo. Here, an atomic resolution (1.2 Å) crystal structure is reported of heterogeneous milk proteins grown inside a living organism in their functional niche. These in vivo-grown crystals were isolated from the midgut of an embryo within the only known viviparous cockroach, Diploptera punctata. The milk proteins crystallized in space group P1, and a structure was determined by anomalous dispersion from the native S atoms. The data revealed glycosylated proteins that adopt a lipocalin fold, bind lipids and organize to form a tightly packed crystalline lattice. A single crystal is estimated to contain more than three times the energy of an equivalent mass of dairy milk. This unique storage form of nourishment for developing embryos allows access to a constant supply of complete nutrients. Notably, the crystalline cockroach-milk proteins are highly heterogeneous with respect to amino-acid sequence, glycosylation and bound fatty-acid composition. These data present a unique example of protein heterogeneity within a single in vivo-grown crystal of a natural protein in its native environment at atomic resolution.

    DOI: 10.1107/S2052252516008903

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  21. Industrial integration at the French national synchrotron facility SOLEIL

    Tiphaine Huet, Gilbert Bey, Laurent M. Vuillard, Gilles Ferry, Leonard M. G. Chavas, Andrew W. Thompson

    ACTA CRYSTALLOGRAPHICA A-FOUNDATION AND ADVANCES   Vol. 72   page: S206 - S206   2016

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    Language:English   Publisher:INT UNION CRYSTALLOGRAPHY  

    DOI: 10.1107/S205327331609690X

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

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

    Structural Dynamics   Vol. 2 ( 4 ) page: 041709 - 041709   2015.7

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    DOI: 10.1063/1.4921220

    Other Link: http://aip.scitation.org/doi/10.1063/1.4921220

  23. Simple convergent-nozzle aerosol injector for single-particle diffractive imaging with X-ray free-electron lasers

    R. A. Kirian, S. Awel, N. Eckerskorn, H. Fleckenstein, M. Wiedorn, L. Adriano, S. Bajt, M. Barthelmess, R. Bean, K. R. Beyerlein, L. M. G. Chavas, M. Domaracky, M. Heymann, D. A. Horke, J. Knoska, M. Metz, A. Morgan, D. Oberthuer, N. Roth, T. Sato, P. L. Xavier, O. Yefanov, A. V. Rode, J. Küpper, H. N. Chapman

    Structural Dynamics   Vol. 2 ( 4 ) page: 041717 - 041717   2015.7

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    DOI: 10.1063/1.4922648

    Other Link: http://aip.scitation.org/doi/10.1063/1.4922648

  24. The catalytic mechanism of human parainfluenza virus type 3 haemagglutinin-neuraminidase revealed. International journal

    Larissa Dirr, Ibrahim M El-Deeb, Patrice Guillon, Cindy J Carroux, Leonard M G Chavas, Mark von Itzstein

    Angewandte Chemie (International ed. in English)   Vol. 54 ( 10 ) page: 2936 - 40   2015.3

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    Human parainfluenza virus type 3 (hPIV-3) is one of the leading causes for lower respiratory tract disease in children, with neither an approved antiviral drug nor vaccine available to date. Understanding the catalytic mechanism of human parainfluenza virus haemagglutinin-neuraminidase (HN) protein is key to the design of specific inhibitors against this virus. Herein, we used (1) H NMR spectroscopy, X-ray crystallography, and virological assays to study the catalytic mechanism of the HN enzyme activity and have identified the conserved Tyr530 as a key amino acid involved in catalysis. A novel 2,3-difluorosialic acid derivative showed prolonged enzyme inhibition and was found to react and form a covalent bond with Tyr530. Furthermore, the novel derivative exhibited enhanced potency in virus blockade assays relative to its Neu2en analogue. These outcomes open the door for a new generation of potent inhibitors against hPIV-3 HN.

    DOI: 10.1002/anie.201412243

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  25. Serial femtosecond crystallography on in vivo grown crystals at SACLA - developments and results

    Leonard M. G. Chavas, Sasa Bajt, Lars Gumprecht, Henry N. Chapman

    ACTA CRYSTALLOGRAPHICA A-FOUNDATION AND ADVANCES   Vol. 71   page: S148 - S148   2015

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    DOI: 10.1107/S2053273315097831

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  26. AGIPD detector for Serial Femtosecond Crystallography Apparatus at European XFEL

    Stephan Stern, Leonard Chavas, Henry Chapman, Adrian Mancuso, Andrew Aquila, Klaus Giewekemeyer, Julian Becker, Heinz Graafsma, Bernd Schmitt

    Acta Crystallographica Section A Foundations and Advances   Vol. 70 ( a1 ) page: C694 - C694   2014.8

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    The European X-ray Free-Electron Laser (XFEL.EU) [1] will provide ultra-short, highly-intense, coherent x-ray pulses at an unprecedented repetition rate, transforming experiments in many scientific areas, including serial femtosecond crystallography (SFX). For the purpose of SFX experiments at the XFEL.EU, a dedicated endstation is being developed to be installed within the Single Particles, Clusters and Biomolecules (SPB) instrument [2]. The setup will refocus the beam spent by SPB into a second interaction region, thereby enabling two parallel experiments. In order to overcome various challenges in XFEL crystallography, and to optimize the output for SFX experiments at XFEL.EU, the Adaptive Gain Integrating Pixel Detector (AGIPD) [3] is currently under development and is to be implemented in the SPB instrument, including a 4 Megapixel version for the SFX apparatus. The AGIPD is a hybrid-pixel detector with pixels of 200 x 200 micron^2 each. The gain of each single pixel dynamically and independently adapts to the incoming signal. Thus, diffraction patterns of high dynamic range can be recorded, with the measured signal within a single data frame ranging from single photons and up to 1e+4 photons at 12 keV. Moreover, the AGIPD is designed to store over 350 data frames from successive pulses prior to digitization and read-out, thereby enabling operation at the European XFEL with its challenging repetition rate with 10 Hz pulse trains and a 4.5 MHz intra-train repetition rate. Furthermore, the incorporation of a veto system in AGIPD will allow one to potentially store only the frames that contain diffraction data from actual crystal hits, which ultimately increases the efficiency of the detector and DAQ systems dramatically. In the present work, we will review the design of the 4Mpix AGIPD for the SFX apparatus and discuss simulations and tests of its expected performance under the conditions foreseen for SFX experiments at the XFEL.EU.

    DOI: 10.1107/s205327331409305x

  27. Serial Femtosecond Crystallography user's consortium apparatus at European XFEL

    Marc Messerschmidt, Leonard Chavas, Sunil Ananthaneni, Hamidreza Dadgostar, Heinz Graafsma, Mengning Liang, Adrian Mancuso, Steffen Raabe, Stephan Stern, Patrik Vagovic, Henry Chapman

    Acta Crystallographica Section A Foundations and Advances   Vol. 70 ( a1 ) page: C1748 - C1748   2014.8

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    The Serial Femtosecond Crystallography (SFX) user's consortium apparatus is to be installed within the Single Particles, Clusters and Biomolecules (SPB) instrument of the European X-ray Free-Electron Laser facility (XFEL.EU) [1, 2]. The XFEL.EU will provide ultra-short, highly intense, coherent X-ray pulses at an unprecedented repetition rate. The experimental setup and methodological approaches of many scientific areas will be transformed, including structural biology that could potentially overcome common problems and bottlenecks encountered in crystallography, such as creating large crystals, dealing with radiation damage, or understanding sub-picosecond time-resolved phenomena. The key concept of the SFX method is based on the kinetic insertion of protein crystal samples in solution via a gas dynamic virtual nozzle jet and recording diffraction signals of individual, randomly oriented crystals passing through the XFEL beam, as first demonstrated by Chapman et al. [3]. The SFX-apparatus will refocus the beam spent by the SPB instrument into a second interaction region, in some cases enabling two parallel experiments. The planned photon energy range at the SPB instrument is from 3 to 16 keV. The Adaptive Gain Integrating Pixel Detector (AGIPD) is to be implemented in the SPB instrument, including a 4 Megapixel version for the SFX-apparatus. The AGIPD is designed to store over 350 data frames from successive pulses, and aims to collect more than 3,000 images per second. Together with the implementation of automated procedures for sample exchange and injection, high-throughput nanocrystallography experiments can be integrated at the SFX-apparatus. In this work, we review the overall design of the SFX-apparatus and discuss the main parameters and challenges

    DOI: 10.1107/s2053273314082515

  28. In vivo crystallography at X-ray free-electron lasers: the next generation of structural biology? International journal

    François-Xavier Gallat, Naohiro Matsugaki, Nathan P Coussens, Koichiro J Yagi, Marion Boudes, Tetsuya Higashi, Daisuke Tsuji, Yutaka Tatano, Mamoru Suzuki, Eiichi Mizohata, Kensuke Tono, Yasumasa Joti, Takashi Kameshima, Jaehyun Park, Changyong Song, Takaki Hatsui, Makina Yabashi, Eriko Nango, Kohji Itoh, Fasséli Coulibaly, Stephen Tobe, S Ramaswamy, Barbara Stay, So Iwata, Leonard M G Chavas

    Philosophical transactions of the Royal Society of London. Series B, Biological sciences   Vol. 369 ( 1647 ) page: 20130497 - 20130497   2014.7

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    The serendipitous discovery of the spontaneous growth of protein crystals inside cells has opened the field of crystallography to chemically unmodified samples directly available from their natural environment. On the one hand, through in vivo crystallography, protocols for protein crystal preparation can be highly simplified, although the technique suffers from difficulties in sampling, particularly in the extraction of the crystals from the cells partly due to their small sizes. On the other hand, the extremely intense X-ray pulses emerging from X-ray free-electron laser (XFEL) sources, along with the appearance of serial femtosecond crystallography (SFX) is a milestone for radiation damage-free protein structural studies but requires micrometre-size crystals. The combination of SFX with in vivo crystallography has the potential to boost the applicability of these techniques, eventually bringing the field to the point where in vitro sample manipulations will no longer be required, and direct imaging of the crystals from within the cells will be achievable. To fully appreciate the diverse aspects of sample characterization, handling and analysis, SFX experiments at the Japanese SPring-8 angstrom compact free-electron laser were scheduled on various types of in vivo grown crystals. The first experiments have demonstrated the feasibility of the approach and suggest that future in vivo crystallography applications at XFELs will be another alternative to nano-crystallography.

    DOI: 10.1098/rstb.2013.0497

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  29. Small Angle X-Ray and Neutron Scattering from Solutions of Biological Macromolecules. By Dmitri I. Svergun, Michel H. J. Koch, Peter A. Timmins and Roland P. May. IUCr Texts on Crystallography, No. 19. Oxford University Press, 2013. Pp. 358. Price (hardback) £49.99. ISBN: 978-0-19-963953-3.

    Leonard Michel Gabriel Chavas

    Acta Crystallographica Section D Biological Crystallography   Vol. 70 ( 4 ) page: 1175 - 1176   2014.4

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    DOI: 10.1107/s1399004714005719

  30. Tuning mechanism-based inactivators of neuraminidases: mechanistic and structural insights. International journal

    Sabrina Buchini, François-Xavier Gallat, Ian R Greig, Jin-Hyo Kim, Soichi Wakatsuki, Leonard M G Chavas, Stephen G Withers

    Angewandte Chemie (International ed. in English)   Vol. 53 ( 13 ) page: 3382 - 6   2014.3

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    3-Fluorosialosyl fluorides are inhibitors of sialidases that function by the formation of a long-lived covalent active-site adduct and have potential as therapeutics if made specific for the pathogen sialidase. Surprisingly, human Neu2 and the Trypanosoma cruzi trans-sialidase are inactivated more rapidly by the reagent with an equatorial fluorine at C3 than by its axial epimer, with reactivation following the same pattern. To explore a possible stereoelectronic basis for this, rate constants for spontaneous hydrolysis of the full series of four 3-fluorosialosyl fluorides were measured, and ground-state energies for each computed. The alpha (equatorial) anomeric fluorides hydrolyze more rapidly than their beta anomers, consistent with their higher ground-state energies. However ground-state energies do not explain the relative spontaneous reactivities of the 3-fluoro-epimers. The three-dimensional structures of the two 3-fluoro-sialosyl enzyme intermediates of human Neu2 were solved, revealing key stabilizing interactions between Arg21 and the equatorial, but not the axial, fluorine. Because of changes in geometry these interactions will increase at the transition state, likely explaining the difference in reaction rates.

    DOI: 10.1002/anie.201309675

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  31. Improvement of an automated protein crystal exchange system PAM for high-throughput data collection. International journal

    Masahiko Hiraki, Yusuke Yamada, Leonard M G Chavas, Soichi Wakatsuki, Naohiro Matsugaki

    Journal of synchrotron radiation   Vol. 20 ( Pt 6 ) page: 890 - 3   2013.11

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    Photon Factory Automated Mounting system (PAM) protein crystal exchange systems are available at the following Photon Factory macromolecular beamlines: BL-1A, BL-5A, BL-17A, AR-NW12A and AR-NE3A. The beamline AR-NE3A has been constructed for high-throughput macromolecular crystallography and is dedicated to structure-based drug design. The PAM liquid-nitrogen Dewar can store a maximum of three SSRL cassettes. Therefore, users have to interrupt their experiments and replace the cassettes when using four or more of them during their beam time. As a result of investigation, four or more cassettes were used in AR-NE3A alone. For continuous automated data collection, the size of the liquid-nitrogen Dewar for the AR-NE3A PAM was increased, doubling the capacity. In order to check the calibration with the new Dewar and the cassette stand, calibration experiments were repeatedly performed. Compared with the current system, the parameters of the novel system are shown to be stable.

    DOI: 10.1107/S0909049513021067

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  32. New methodologies at PF AR-NW12A: the implementation of high-pressure macromolecular crystallography. International journal

    Leonard Michel Gabriel Chavas, Tadayuki Nagae, Hiroyuki Yamada, Nobuhisa Watanabe, Yusuke Yamada, Masahiko Hiraki, Naohiro Matsugaki

    Journal of synchrotron radiation   Vol. 20 ( Pt 6 ) page: 838 - 42   2013.11

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    The macromolecular crystallography (MX) beamline AR-NW12A is evolving from its original design of high-throughput crystallography to a multi-purpose end-station. Among the various options to be implemented, great efforts were made in making available high-pressure MX (HPMX) at the beamline. High-pressure molecular biophysics is a developing field that attracts the interest of a constantly growing scientific community. A plethora of activities can benefit from high pressure, and investigations have been performed on its applicability to study multimeric complex assemblies, compressibility of proteins and their crystals, macromolecules originating from extremophiles, or even the trapping of higher-energy conformers for molecules of biological interest. Recent studies using HPMX showed structural hydrostatic-pressure-induced changes in proteins. The conformational modifications could explain the enzymatic mechanism differences between proteins of the same family, living at different environmental pressures, as well as the initial steps in the pressure-denaturation process that have been attributed to water penetration into the protein interior. To facilitate further HPMX, while allowing access to various individualized set-ups and experiments, the AR-NW12A sample environment has been revisited. Altogether, the newly added implementations will bring a fresh breath of life to AR-NW12A and allow the MX community to experiment in a larger set of fields related to structural biology.

    DOI: 10.1107/S0909049513020797

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  33. Improvements toward highly accurate diffraction experiments at the macromolecular micro-crystallography beamline BL-17A. International journal

    Yusuke Yamada, Leonard M G Chavas, Noriyuki Igarashi, Masahiko Hiraki, Soichi Wakatsuki, Naohiro Matsugaki

    Journal of synchrotron radiation   Vol. 20 ( Pt 6 ) page: 938 - 42   2013.11

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    BL-17A is a macromolecular crystallography beamline dedicated to diffraction experiments conducted using micro-crystals and structure determination studies using a lower energy X-ray beam. In these experiments, highly accurate diffraction intensity measurements are definitively important. Since this beamline was constructed, the beamline apparatus has been improved in several ways to enable the collection of accurate diffraction data. The stability of the beam intensities at the sample position was recently improved by modifying the monochromator. The diffractometer has also been improved. A new detector table was installed to prevent distortions in the diffractometer's base during the repositioning of the diffractometer detector. A new pinhole system and an on-axis viewing system were installed to improve the X-ray beam profile at the sample position and the centering of tiny crystal samples.

    DOI: 10.1107/S0909049513022875

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  34. 10 years of protein crystallography at AR-NW12A beamline

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

    Journal of Physics: Conference Series   Vol. 425 ( 1 ) page: 012008 - 012008   2013.3

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    DOI: 10.1088/1742-6596/425/1/012008

    Other Link: http://stacks.iop.org/1742-6596/425/i=1/a=012008?key=crossref.51e8bce4e1903fbc177b2d861ae7b433

  35. Data Management System at the Photon Factory Macromolecular Crystallography Beamline

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

    Journal of Physics: Conference Series   Vol. 425 ( 1 ) page: 012017 - 012017   2013.3

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    DOI: 10.1088/1742-6596/425/1/012017

    Other Link: http://stacks.iop.org/1742-6596/425/i=1/a=012017?key=crossref.8bd049c81e4cc7cfc80e9995dc668a1f

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

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

    Journal of Physics: Conference Series   Vol. 425 ( 1 ) page: 012014 - 012014   2013.3

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    DOI: 10.1088/1742-6596/425/1/012014

    Other Link: http://stacks.iop.org/1742-6596/425/i=1/a=012014?key=crossref.309f9dfdd82d0d95f7053d1e18487a0c

  37. Macromolecular structures from nanocrystals

    Chavas L.M.G.H

    Energy Recover Linac - Conceptual Design Report     2012.10

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    File: ERL_CDR_full_text.pdf

  38. Beamline AR-NW12A: high-throughput beamline for macromolecular crystallography at the Photon Factory

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

    Journal of Synchrotron Radiation   Vol. 19 ( 3 ) page: 450 - 454   2012.5

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    DOI: 10.1107/s0909049512009727

  39. High-pressure-induced water penetration into 3-isopropylmalate dehydrogenase. International journal

    Takayuki Nagae, Takashi Kawamura, Leonard M G Chavas, Ken Niwa, Masashi Hasegawa, Chiaki Kato, Nobuhisa Watanabe

    Acta crystallographica. Section D, Biological crystallography   Vol. 68 ( Pt 3 ) page: 300 - 9   2012.3

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    Hydrostatic pressure induces structural changes in proteins, including denaturation, the mechanism of which has been attributed to water penetration into the protein interior. In this study, structures of 3-isopropylmalate dehydrogenase (IPMDH) from Shewanella oneidensis MR-1 were determined at about 2 Å resolution under pressures ranging from 0.1 to 650 MPa using a diamond anvil cell (DAC). Although most of the protein cavities are monotonically compressed as the pressure increases, the volume of one particular cavity at the dimer interface increases at pressures over 340 MPa. In parallel with this volume increase, water penetration into the cavity could be observed at pressures over 410 MPa. In addition, the generation of a new cleft on the molecular surface accompanied by water penetration could also be observed at pressures over 580 MPa. These water-penetration phenomena are considered to be initial steps in the pressure-denaturation process of IPMDH.

    DOI: 10.1107/S0907444912001862

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  40. Structural biology beamlines at the Photon Factory

    Yusuke Yamada, Naohiro Matsugaki, Leonard M. G. Chavas, Masahiko Hiraki, Nobutaka Shimizu, Noriyuki Igarashi, Soichi Wakatsuki

    ACTA CRYSTALLOGRAPHICA A-FOUNDATION AND ADVANCES   Vol. 68   page: S148 - S148   2012

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    DOI: 10.1107/S0108767312097152

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  41. UV LED lighting for automated crystal centring. International journal

    Leonard M G Chavas, Yusuke Yamada, Masahiko Hiraki, Noriyuki Igarashi, Naohiro Matsugaki, Soichi Wakatsuki

    Journal of synchrotron radiation   Vol. 18 ( 1 ) page: 11 - 5   2011.1

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    A direct outcome of the exponential growth of macromolecular crystallography is the continuously increasing demand for synchrotron beam time, both from academic and industrial users. As more and more projects entail screening a profusion of sample crystals, fully automated procedures at every level of the experiments are being implemented at all synchrotron facilities. One of the major obstacles to achieving such automation lies in the sample recognition and centring in the X-ray beam. The capacity of UV light to specifically react with aromatic residues present in proteins or with DNA base pairs is at the basis of UV-assisted crystal centring. Although very efficient, a well known side effect of illuminating biological samples with strong UV sources is the damage induced on the irradiated samples. In the present study the effectiveness of a softer UV light for crystal centring by taking advantage of low-power light-emitting diode (LED) sources has been investigated. The use of UV LEDs represents a low-cost solution for crystal centring with high specificity.

    DOI: 10.1107/S0909049510028670

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  42. Low energy SAD experiments performed at the photon factory BL-1A

    Naohiro Matsugaki, Yusuke Yamada, Leonard M. G. Chavas, Masahiko Hiraki, Masato Kawasaki, Ryuichi Kato, Noriyuki Igarashi, Soichi Wakatsuki

    ACTA CRYSTALLOGRAPHICA A-FOUNDATION AND ADVANCES   Vol. 67   page: C355 - C355   2011

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    DOI: 10.1107/S0108767311091057

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  43. Structure study of IPMDH from piezosensitive and piezophilic Shewanella species

    Takayuki Nagae, Takashi Kawamura, Leonard Chavas, Ken Niwa, Masashi Hasegawa, Chiaki Kato, Nobuhisa Watanabe

    ACTA CRYSTALLOGRAPHICA A-FOUNDATION AND ADVANCES   Vol. 67   page: C275 - C276   2011

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    DOI: 10.1107/S0108767311093111

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  44. Structural biology and SAXS beamlines at the photon factory

    Noriyuki Igarashi, Naohiro Matsugaki, Yusuke Yamada, Leonard G. M. Chavas, Masahiko Hiraki, Nobutaka Shimizu, Takeharu Mori, Soichi Wakatsuki

    ACTA CRYSTALLOGRAPHICA A-FOUNDATION AND ADVANCES   Vol. 67   page: C803 - C803   2011

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    DOI: 10.1107/S0108767311079669

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  45. Potential of UV in phasing and its implementation for crystal centering at PF

    Leonard M. G. Chavas, Yusuke Yamada, Masahiro Hiraki, Seiji Okazaki, Noriyuki Igarashi, Naohiro Matsugaki, Soichi Wakatsuki

    ACTA CRYSTALLOGRAPHICA A-FOUNDATION AND ADVANCES   Vol. 67   page: C656 - C656   2011

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    DOI: 10.1107/S0108767311083425

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  46. [Advancement of synchrotron radiation protein crystallography in the targeted protein research program: beamline developments at the photon factory].

    Soichi Wakatsuki, Yusuke Yamada, Leonard M G Chavas, Noriyuki Igarashi, Masato Kawasaki, Ryuichi Kato, Masahiko Hiraki, Naohiro Matsugaki

    Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan   Vol. 130 ( 5 ) page: 631 - 40   2010.5

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    The Targeted Protein Research Program (TPRP) started in 2007 as a sequel of the Protein 3000 Project which lasted from 2002 to 2007. In the new project, four cores, Protein Production, Structure Analysis, Control of Protein Functions with Compounds, and Informatics, have been established as focus of methodology developments critical for functional and structural studies by the target protein research teams. Within the "Analysis Core" synchrotron radiation plays a pivotal role providing X-ray beams for structural analyses of the target proteins. The two large Japanese synchrotron radiation facilities, SPring-8 and Photon Factory (PF), along with three protein crystallography groups from Hokkaido, Kyoto and Osaka Universities have teamed up to develop two complementary micro-beam beamlines, one on each synchrotron site, and associated technologies for cutting edge structural biology research. At the PF, there are 5 operational beamlines which are equipped with state-of-the-art instrumentation for high-throughput protein crystallography experiments. Within the TPRP framework, the PF is developing a micro-focus beamline optimized for a lower energy single anomalous diffraction (SAD) experiment. This will be particularly useful for structure determination of difficult protein targets for which heavy atom derivatives or selenomethionine substitution does not work and other standard phasing methods fail to give structure solutions. This will augment the capabilities of the PF structural biology beamlines with similar look-and-feel experimental environments.

    DOI: 10.1248/yakushi.130.631

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  47. Complexity in influenza virus targeted drug design: interaction with human sialidases. International journal

    Leonard M G Chavas, Ryuichi Kato, Nobuhiro Suzuki, Mark von Itzstein, Maretta C Mann, Robin J Thomson, Jeffrey C Dyason, Jennifer McKimm-Breschkin, Paola Fusi, Cristina Tringali, Bruno Venerando, Guido Tettamanti, Eugenio Monti, Soichi Wakatsuki

    Journal of medicinal chemistry   Vol. 53 ( 7 ) page: 2998 - 3002   2010.4

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    With the global spread of the pandemic H1N1 and the ongoing pandemic potential of the H5N1 subtype, the influenza virus represents one of the most alarming viruses spreading worldwide. The influenza virus sialidase is an effective drug target, and a number of inhibitors are clinically effective against the virus (zanamivir, oseltamivir, peramivir). Here we report structural and biochemical studies of the human cytosolic sialidase Neu2 with influenza virus sialidase-targeting drugs and related compounds.

    DOI: 10.1021/jm100078r

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  48. Biophysical and X-ray crystallographic analysis of Mps1 kinase inhibitor complexes. International journal

    Matthew L H Chu, Zhaolei Lang, Leonard M G Chavas, João Neres, Olga S Fedorova, Lydia Tabernero, Mike Cherry, David H Williams, Kenneth T Douglas, Patrick A Eyers

    Biochemistry   Vol. 49 ( 8 ) page: 1689 - 701   2010.3

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    The dual-specificity protein kinase monopolar spindle 1 (Mps1) is a central component of the mitotic spindle assembly checkpoint (SAC), a sensing mechanism that prevents anaphase until all chromosomes are bioriented on the metaphase plate. Partial depletion of Mps1 protein levels sensitizes transformed, but not untransformed, human cells to therapeutic doses of the anticancer agent Taxol, making it an attractive novel therapeutic cancer target. We have previously determined the X-ray structure of the catalytic domain of human Mps1 in complex with the anthrapyrazolone kinase inhibitor SP600125. In order to validate distinct inhibitors that target this enzyme and improve our understanding of nucleotide binding site architecture, we now report a biophysical and structural evaluation of the Mps1 catalytic domain in the presence of ATP and the aspecific model kinase inhibitor staurosporine. Collective in silico, enzymatic, and fluorescent screens also identified several new lead quinazoline Mps1 inhibitors, including a low-affinity compound termed Compound 4 (Cpd 4), whose interaction with the Mps1 kinase domain was further characterized by X-ray crystallography. A novel biophysical analysis demonstrated that the intrinsic fluorescence of SP600125 changed markedly upon Mps1 binding, allowing spectrophotometric displacement analysis and determination of dissociation constants for ATP-competitive Mps1 inhibitors. By illuminating the structure of the Mps1 ATP-binding site our results provide novel biophysical insights into Mps1-ligand interactions that will be useful for the development of specific Mps1 inhibitors, including those employing a therapeutically validated quinazoline template.

    DOI: 10.1021/bi901970c

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  49. Fully Automated Data Collection Using PAM and the Development of PAM/SPACE Reversible Cassettes

    Masahiko Hiraki, Shokei Watanabe, Leonard M. G. Chavas, Yusuke Yamada, Naohiro Matsugaki, Noriyuki Igarashi, Soichi Wakatsuki, Masahiro Fujihashi, Kunio Miki, Seiki Baba, Go Ueno, Masaki Yamamoto, Mamoru Suzuki, Atsushi Nakagawa, Nobuhisa Watanabe, Isao Tanaka

    SRI 2009: THE 10TH INTERNATIONAL CONFERENCE ON SYNCHROTRON RADIATION INSTRUMENTATION   Vol. 1234   page: 673 - +   2010

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    Language:English   Publishing type:Research paper (international conference proceedings)   Publisher:AMER INST PHYSICS  

    To remotely control and automatically collect data in high-throughput X-ray data collection experiments, the Structural Biology Research Center at the Photon Factory (PF) developed and installed sample exchange robots PAM (PF Automated Mounting system) at PF macromolecular crystallography beamlines; BL-5A, BL-17A, AR-NW12A and AR-NE3A. We developed and installed software that manages the flow of the automated X-ray experiments; sample exchanges, loop-centering and X-ray diffraction data collection. The fully automated data collection function has been available since February 2009. To identify sample cassettes, PAM employs a two-dimensional bar code reader. New beamlines, BL-1A at the Photon Factory and BL32XU at SPring-8, are currently under construction as part of Targeted Proteins Research Program (TPRP) by the Ministry of Education, Culture, Sports, Science and Technology of Japan. However, different robots, PAM and SPACE (SPring-8 Precise Automatic Cryo-sample Exchanger), will be installed at BL-1A and BL32XU, respectively. For the convenience of the users of both facilities, pins and cassettes for PAM and SPACE are developed as part of the TPRP.

    DOI: 10.1063/1.3463297

    Web of Science

    Scopus

  50. Elucidation of Rab27 recruitment by its effectors: structure of Rab27a bound to Exophilin4/Slp2-a. International journal

    Leonard M G Chavas, Kentaro Ihara, Masato Kawasaki, Seiji Torii, Tamami Uejima, Ryuichi Kato, Tetsuro Izumi, Soichi Wakatsuki

    Structure (London, England : 1993)   Vol. 16 ( 10 ) page: 1468 - 77   2008.10

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:CELL PRESS  

    Rab GTPases coordinate vesicular trafficking within eukaryotic cells by collaborating with a set of effector proteins. Rab27a regulates numerous exocytotic pathways, and its dysfunction causes the Griscelli syndrome human immunodeficiency. Exophilin4/Slp2-a localizes on phosphatidylserine-enriched plasma membrane, and its N-terminal Rab27-binding domain (RBD27) specifically recognizes Rab27 on the surfaces of melanosomes and secretory granules prior to docking and fusion. To characterize the selective binding of Rab27 to 11 various effectors, we have determined the 1.8 A resolution structure of Rab27a in complex with Exophilin4 RBD27. The effector packs against the switch and interswitch elements of Rab27a, and specific affinity toward Rab27a is modulated by a shift in the orientation of the effector structural motif (S/T)(G/L)xW(F/Y)(2). The observed structural complementation between the interacting surfaces of Rab27a and Exophilin4 sheds light on the disparities among the Rab27 effectors and outlines a general mechanism for their recruitment.

    DOI: 10.1016/j.str.2008.07.015

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    PubMed

  51. Crystal structure of the catalytic domain of the mitotic checkpoint kinase Mps1 in complex with SP600125. International journal

    Matthew L H Chu, Leonard M G Chavas, Kenneth T Douglas, Patrick A Eyers, Lydia Tabernero

    The Journal of biological chemistry   Vol. 283 ( 31 ) page: 21495 - 500   2008.8

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Chromosomal instability can result from defective control of checkpoints and is associated with malignant cell growth. Monopolar spindle 1 (Mps1) is a dual-specificity protein kinase that has important roles in the prevention of aneuploidy during the cell cycle and might therefore be a potential target for new therapeutic agents in the treatment of cancer. To gain insights into the molecular mechanism of Mps1 inhibition by small molecules, we determined the x-ray structure of Mps1, both alone and in complex with the ATP-competitive inhibitor SP600125. Mps1 adopts a classic protein kinase fold, with the inhibitor sitting in the ATP-binding site where it is stabilized by hydrophobic interactions. We identified a secondary pocket, not utilized by SP600125, which might be exploited for the rational design of specific Mps1 inhibitors. These structures provide important insights into the interaction of this protein kinase with small molecules and suggest potential mechanisms for Mps1 regulation.

    DOI: 10.1074/jbc.M803026200

    Web of Science

    PubMed

  52. Purification, crystallization and preliminary X-ray crystallographic analysis of Rab27a GTPase in complex with exophilin4/Slp2-a effector. International journal

    Leonard M G Chavas, Kentaro Ihara, Masato Kawasaki, Ryuichi Kato, Tetsuro Izumi, Soichi Wakatsuki

    Acta crystallographica. Section F, Structural biology and crystallization communications   Vol. 64 ( Pt 7 ) page: 599 - 601   2008.7

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:INT UNION CRYSTALLOGRAPHY  

    By switching between GTP-active and GDP-inactive conformations, small Ras GTPases partly regulate membrane trafficking, cell growth and cytoskeleton dynamics. Among Rab GTPases, the Rab27 subfamily, which comprises Rab27a and Rab27b, controls the proper targeting of secretory vesicles to the plasma membrane. GppNHp-bound Rab27a in complex with the Rab27-binding domain of exophilin4/Slp2-a effector has been purified and crystallized for structural studies. The crystals belong to space group P2(1)2(1)2(1) and a complete data set was collected to a resolution of 1.8 A. Eventually, the structural characterization of the Rab27a-exophilin4/Slp2-a complex will clarify Rab27 recognition by its effectors prior to vesicle tethering and docking.

    DOI: 10.1107/S1744309108009251

    Web of Science

    PubMed

  53. Atomic model of Rab27a: Exophilin4/Slp2-a complex: Structural studies on vesicular transport

    Leonard M. G. Chavas, Kentaro Ihara, Masato Kawasaki, Ryuichi Kato, Tetsuro Izumi, Soichi Wakatsuki

    ACTA CRYSTALLOGRAPHICA A-FOUNDATION AND ADVANCES   Vol. 64   page: C333 - C333   2008

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    Language:English   Publisher:INT UNION CRYSTALLOGRAPHY  

    DOI: 10.1107/S0108767308089344

    Web of Science

  54. Structure of the small GTPase Rab27b shows an unexpected swapped dimer

    Leonard M. G. Chavas, Seiji Torii, Hironari Kamikubo, Masato Kawasaki, Kentaro Ihara, Ryuichi Kato, Mikio Kataoka, Tetsuro Izumi, Soichi Wakatsuki

    ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY   Vol. 63   page: 769 - 779   2007.7

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:INT UNION CRYSTALLOGRAPHY  

    Members of the Rab family of small GTPases regulate membrane traffic within the cell by recruiting their specific effectors in a nucleotide-dependent manner. The Rab27 subfamily consists of Rab27a and Rab27b, which share 70% sequence identity. By interacting with a large set of effector proteins such as melanophilin and granuphilin, both Rab27a and Rab27b regulate the exocytosis of secretory lysosomes. Here, the crystal structures of mouse Rab27b in complex with GDP have been determined in three distinct crystal lattices. Surprisingly, Rab27b-GDP exists in an open conformation with protruding switch and interswitch regions, which are stabilized through dimerization by means of domain-swapping in the crystals. In contrast, small-angle X-ray scattering measurements showed an extended monomer form of Rab27b in solution. The observed dimer formation of Rab27b-GDP in the crystals would restrain the highly flexible switch regions. Possible biological implications of this atypical structure of Rab27b and its plausible influence in effector interaction are discussed.

    DOI: 10.1107/S0907444907019725

    Web of Science

  55. Structure of the small GTPase Rab27b shows an unexpected swapped dimer. International journal

    Leonard M G Chavas, Seiji Torii, Hironari Kamikubo, Masato Kawasaki, Kentaro Ihara, Ryuichi Kato, Mikio Kataoka, Tetsuro Izumi, Soichi Wakatsuki

    Acta crystallographica. Section D, Biological crystallography   Vol. 63 ( Pt 7 ) page: 769 - 79   2007.7

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    Language:English   Publishing type:Research paper (scientific journal)  

    Members of the Rab family of small GTPases regulate membrane traffic within the cell by recruiting their specific effectors in a nucleotide-dependent manner. The Rab27 subfamily consists of Rab27a and Rab27b, which share 70% sequence identity. By interacting with a large set of effector proteins such as melanophilin and granuphilin, both Rab27a and Rab27b regulate the exocytosis of secretory lysosomes. Here, the crystal structures of mouse Rab27b in complex with GDP have been determined in three distinct crystal lattices. Surprisingly, Rab27b-GDP exists in an open conformation with protruding switch and interswitch regions, which are stabilized through dimerization by means of domain-swapping in the crystals. In contrast, small-angle X-ray scattering measurements showed an extended monomer form of Rab27b in solution. The observed dimer formation of Rab27b-GDP in the crystals would restrain the highly flexible switch regions. Possible biological implications of this atypical structure of Rab27b and its plausible influence in effector interaction are discussed.

    PubMed

  56. Development of an automated large-scale protein-crystallization and monitoring system for high-throughput protein-structure analyses. International journal

    Masahiko Hiraki, Ryuichi Kato, Minoru Nagai, Tadashi Satoh, Satoshi Hirano, Kentaro Ihara, Norio Kudo, Masamichi Nagae, Masanori Kobayashi, Michio Inoue, Tamami Uejima, Shunichiro Oda, Leonard M G Chavas, Masato Akutsu, Yusuke Yamada, Masato Kawasaki, Naohiro Matsugaki, Noriyuki Igarashi, Mamoru Suzuki, Soichi Wakatsuki

    Acta crystallographica. Section D, Biological crystallography   Vol. 62 ( Pt 9 ) page: 1058 - 65   2006.9

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:BLACKWELL PUBLISHING  

    Protein crystallization remains one of the bottlenecks in crystallographic analysis of macromolecules. An automated large-scale protein-crystallization system named PXS has been developed consisting of the following subsystems, which proceed in parallel under unified control software: dispensing precipitants and protein solutions, sealing crystallization plates, carrying robot, incubators, observation system and image-storage server. A sitting-drop crystallization plate specialized for PXS has also been designed and developed. PXS can set up 7680 drops for vapour diffusion per hour, which includes time for replenishing supplies such as disposable tips and crystallization plates. Images of the crystallization drops are automatically recorded according to a preprogrammed schedule and can be viewed by users remotely using web-based browser software. A number of protein crystals were successfully produced and several protein structures could be determined directly from crystals grown by PXS. In other cases, X-ray quality crystals were obtained by further optimization by manual screening based on the conditions found by PXS.

    DOI: 10.1107/S0907444906023821

    Web of Science

    PubMed

  57. Crystal structure of the human cytosolic sialidase Neu2. Evidence for the dynamic nature of substrate recognition. International journal

    Leonard M G Chavas, Cristina Tringali, Paola Fusi, Bruno Venerando, Guido Tettamanti, Ryuichi Kato, Eugenio Monti, Soichi Wakatsuki

    The Journal of biological chemistry   Vol. 280 ( 1 ) page: 469 - 75   2005.1

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    Language:English   Publishing type:Research paper (scientific journal)  

    Gangliosides play key roles in cell differentiation, cell-cell interactions, and transmembrane signaling. Sialidases hydrolyze sialic acids to produce asialo compounds, which is the first step of degradation processes of glycoproteins and gangliosides. Sialidase involvement has been implicated in some lysosomal storage disorders such as sialidosis and galactosialidosis. Neu2 is a recently identified human cytosolic sialidase. Here we report the first high resolution x-ray structures of mammalian sialidase, human Neu2, in its apo form and in complex with an inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA). The structure shows the canonical six-blade beta-propeller observed in viral and bacterial sialidases with its active site in a shallow crevice. In the complex structure, the inhibitor lies in the catalytic crevice surrounded by ten amino acids. In particular, the arginine triad, conserved among sialidases, aids in the proper positioning of the carboxylate group of DANA within the active site region. The tyrosine residue, Tyr(334), conserved among mammalian and bacterial sialidases as well as in viral neuraminidases, facilitates the enzymatic reaction by stabilizing a putative carbonium ion in the transition state. The loops containing Glu(111) and the catalytic aspartate Asp(46) are disordered in the apo form but upon binding of DANA become ordered to adopt two short alpha-helices to cover the inhibitor, illustrating the dynamic nature of substrate recognition. The N-acetyl and glycerol moieties of DANA are recognized by Neu2 residues not shared by bacterial sialidases and viral neuraminidases, which can be regarded as a key structural difference for potential drug design against bacteria, influenza, and other viruses.

    PubMed

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Presentations 2

  1. VISION FOR AN INTEGRATED STRUCTURAL BIOLOGY AND METHODOLOGY

    Chavas Leo

    2021.3.30 

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    Event date: 2021.3

    Language:English   Presentation type:Oral presentation (general)  

    Venue:Nagoya University   Country:Japan  

    DOI: 10.5281/zenodo.4646448

  2. VISION FOR AN INTEGRATED STRUCTURAL BIOLOGY AND METHODOLOGY

    Chavas Leo

    2021.3.30 

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    Language:English   Presentation type:Oral presentation (general)  

    Venue:Nagoya University   Country:Japan  

    Other Link: https://doi.org/10.5281/zenodo.4646448

KAKENHI (Grants-in-Aid for Scientific Research) 1

  1. Study of UV-light applied to macromolecular crystallography

    Grant number:22770161  2010 - 2011

    CHAVAS Leonard

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    Authorship:Principal investigator 

    Grant amount:\3900000 ( Direct Cost: \3000000 、 Indirect Cost:\900000 )

    The goal of this study is to take advantage of UV-radiation for structure determination of biological macromolecule crystals. The concrete effects of UV-radiation on protein models were investigated, the aim being to develop a general methodology for UV-radiation induced phasing(UV-RIP). Exposing the crystals to an intense UV-light caused significant damages, resulting in poorer electron densities. A general methodology for UV-RIP and a semi-automated data analysis software were implemented for taking full advantage of the specific damages observed in irradiated crystals for structure determination.