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1.  Taking Snapshots of Photosynthetic Water Oxidation Using Femtosecond X-ray Diffraction and Spectroscopy 
Nature communications  2014;5:4371.
The dioxygen we breathe is formed from water by its light-induced oxidation in photosystem II. O2 formation takes place at a catalytic manganese cluster within milliseconds after the photosystem II reaction center is excited by three single-turnover flashes. Here we present combined X-ray emission spectra and diffraction data of 2 flash (2F) and 3 flash (3F) photosystem II samples, and of a transient 3F′ state (250 μs after the third flash), collected under functional conditions using an X-ray free electron laser. The spectra show that the initial O-O bond formation, coupled to Mn-reduction, does not yet occur within 250 μs after the third flash. Diffraction data of all states studied exhibit an anomalous scattering signal from Mn but show no significant structural changes at the present resolution of 4.5 Å. This study represents the initial frames in a molecular movie of the structural changes during the catalytic reaction in photosystem II.
doi:10.1038/ncomms5371
PMCID: PMC4151126  PMID: 25006873
2.  Fixed-target protein serial microcrystallography with an x-ray free electron laser 
Scientific Reports  2014;4:6026.
We present results from experiments at the Linac Coherent Light Source (LCLS) demonstrating that serial femtosecond crystallography (SFX) can be performed to high resolution (~2.5 Å) using protein microcrystals deposited on an ultra-thin silicon nitride membrane and embedded in a preservation medium at room temperature. Data can be acquired at a high acquisition rate using x-ray free electron laser sources to overcome radiation damage, while sample consumption is dramatically reduced compared to flowing jet methods. We achieved a peak data acquisition rate of 10 Hz with a hit rate of ~38%, indicating that a complete data set could be acquired in about one 12-hour LCLS shift using the setup described here, or in even less time using hardware optimized for fixed target SFX. This demonstration opens the door to ultra low sample consumption SFX using the technique of diffraction-before-destruction on proteins that exist in only small quantities and/or do not produce the copious quantities of microcrystals required for flowing jet methods.
doi:10.1038/srep06026
PMCID: PMC4129423  PMID: 25113598
3.  Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography 
Nature communications  2014;5:3309.
Lipidic cubic phase (LCP) crystallization has proven successful for high-resolution structure determination of challenging membrane proteins. Here we present a technique for extruding gel-like LCP with embedded membrane protein microcrystals, providing a continuously-renewed source of material for serial femtosecond crystallography. Data collected from sub-10 μm-sized crystals produced with less than 0.5 mg of purified protein yield structural insights regarding cyclopamine binding to the Smoothened receptor.
doi:10.1038/ncomms4309
PMCID: PMC4061911  PMID: 24525480
4.  Serial Femtosecond Crystallography of G Protein-Coupled Receptors 
Science (New York, N.Y.)  2013;342(6165):1521-1524.
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.
doi:10.1126/science.1244142
PMCID: PMC3902108  PMID: 24357322
5.  Single mimivirus particles intercepted and imaged with an X-ray laser 
Seibert, M. Marvin | Ekeberg, Tomas | Maia, Filipe R. N. C. | Svenda, Martin | Andreasson, Jakob | Jönsson, Olof | Odić, Duško | Iwan, Bianca | Rocker, Andrea | Westphal, Daniel | Hantke, Max | DePonte, Daniel P. | Barty, Anton | Schulz, Joachim | Gumprecht, Lars | Coppola, Nicola | Aquila, Andrew | Liang, Mengning | White, Thomas A. | Martin, Andrew | Caleman, Carl | Stern, Stephan | Abergel, Chantal | Seltzer, Virginie | Claverie, Jean-Michel | Bostedt, Christoph | Bozek, John D. | Boutet, Sébastien | Miahnahri, A. Alan | Messerschmidt, Marc | Krzywinski, Jacek | Williams, Garth | Hodgson, Keith O. | Bogan, Michael J. | Hampton, Christina Y. | Sierra, Raymond G. | Starodub, Dmitri | Andersson, Inger | Bajt, Saša | Barthelmess, Miriam | Spence, John C. H. | Fromme, Petra | Weierstall, Uwe | Kirian, Richard | Hunter, Mark | Doak, R. Bruce | Marchesini, Stefano | Hau-Riege, Stefan P. | Frank, Matthias | Shoeman, Robert L. | Lomb, Lukas | Epp, Sascha W. | Hartmann, Robert | Rolles, Daniel | Rudenko, Artem | Schmidt, Carlo | Foucar, Lutz | Kimmel, Nils | Holl, Peter | Rudek, Benedikt | Erk, Benjamin | Hömke, André | Reich, Christian | Pietschner, Daniel | Weidenspointner, Georg | Strüder, Lothar | Hauser, Günter | Gorke, Hubert | Ullrich, Joachim | Schlichting, Ilme | Herrmann, Sven | Schaller, Gerhard | Schopper, Florian | Soltau, Heike | Kühnel, Kai-Uwe | Andritschke, Robert | Schröter, Claus-Dieter | Krasniqi, Faton | Bott, Mario | Schorb, Sebastian | Rupp, Daniela | Adolph, Marcus | Gorkhover, Tais | Hirsemann, Helmut | Potdevin, Guillaume | Graafsma, Heinz | Nilsson, Björn | Chapman, Henry N. | Hajdu, Janos
Nature  2011;470(7332):78-81.
X-ray lasers offer new capabilities in understanding the structure of biological systems, complex materials and matter under extreme conditions1–4. Very short and extremely bright, coherent X-ray pulses can be used to outrun key damage processes and obtain a single diffraction pattern from a large macromolecule, a virus or a cell before the sample explodes and turns into plasma1. The continuous diffraction pattern of non-crystalline objects permits oversampling and direct phase retrieval2. Here we show that high-quality diffraction data can be obtained with a single X-ray pulse from a non-crystalline biological sample, a single mimivirus particle, which was injected into the pulsed beam of a hard-X-ray free-electron laser, the Linac Coherent Light Source5. Calculations indicate that the energy deposited into the virus by the pulse heated the particle to over 100,000 K after the pulse had left the sample. The reconstructed exit wavefront (image) yielded 32-nm full-period resolution in a single exposure and showed no measurable damage. The reconstruction indicates inhomogeneous arrangement of dense material inside the virion. We expect that significantly higher resolutions will be achieved in such experiments with shorter and brighter photon pulses focused to a smaller area. The resolution in such experiments can be further extended for samples available in multiple identical copies.
doi:10.1038/nature09748
PMCID: PMC4038304  PMID: 21293374
6.  Accurate macromolecular structures using minimal measurements from X-ray free-electron lasers 
Nature methods  2014;11(5):545-548.
X-ray free-electron laser (XFEL) sources enable the use of crystallography to solve three-dimensional macromolecular structures under native conditions and free from 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 statistically significant high-resolution signals from fewer diffraction measurements.
doi:10.1038/nmeth.2887
PMCID: PMC4008696  PMID: 24633409
7.  Femtosecond X-ray diffraction from two-dimensional protein crystals 
Iucrj  2014;1(Pt 2):95-100.
Bragg diffraction achieved from two-dimensional protein crystals using femtosecond X-ray laser snapshots is presented.
X-ray diffraction patterns from two-dimensional (2-D) protein crystals obtained using femtosecond X-ray pulses from an X-ray free-electron laser (XFEL) are presented. To date, it has not been possible to acquire transmission X-ray diffraction patterns from individual 2-D protein crystals due to radiation damage. However, the intense and ultrafast pulses generated by an XFEL permit a new method of collecting diffraction data before the sample is destroyed. Utilizing a diffract-before-destroy approach at the Linac Coherent Light Source, Bragg diffraction was acquired to better than 8.5 Å resolution for two different 2-D protein crystal samples each less than 10 nm thick and maintained at room temperature. These proof-of-principle results show promise for structural analysis of both soluble and membrane proteins arranged as 2-D crystals without requiring cryogenic conditions or the formation of three-dimensional crystals.
doi:10.1107/S2052252514001444
PMCID: PMC4062087  PMID: 25075325
two-dimensional protein crystal; femtosecond crystallography; single layer X-ray diffraction; membrane protein
8.  Nanoflow electrospinning serial femtosecond crystallography 
A low flow rate liquid microjet method for delivery of hydrated protein crystals to X-ray lasers is presented. Linac Coherent Light Source data demonstrates serial femtosecond protein crystallography with micrograms, a reduction of sample consumption by orders of magnitude.
An electrospun liquid microjet has been developed that delivers protein microcrystal suspensions at flow rates of 0.14–3.1 µl min−1 to perform serial femtosecond crystallography (SFX) studies with X-ray lasers. Thermolysin microcrystals flowed at 0.17 µl min−1 and diffracted to beyond 4 Å resolution, producing 14 000 indexable diffraction patterns, or four per second, from 140 µg of protein. Nanoflow electrospinning extends SFX to biological samples that necessitate minimal sample consumption.
doi:10.1107/S0907444912038152
PMCID: PMC3478121  PMID: 23090408
serial femtosecond crystallography; nanoflow electrospinning
9.  High-Resolution Protein Structure Determination by Serial Femtosecond Crystallography 
Science (New York, N.Y.)  2012;337(6092):362-364.
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.
doi:10.1126/science.1217737
PMCID: PMC3788707  PMID: 22653729
10.  Natively Inhibited Trypanosoma brucei Cathepsin B Structure Determined by Using an X-ray Laser 
Science (New York, N.Y.)  2012;339(6116):227-230.
The Trypanosoma brucei cysteine protease cathepsin B (TbCatB), which is involved in host protein degradation, is a promising target to develop new treatments against sleeping sickness, a fatal disease caused by this protozoan parasite. The structure of the mature, active form of TbCatB has so far not provided sufficient information for the design of a safe and specific drug against T. brucei. By combining two recent innovations, in vivo crystallization and serial femtosecond crystallography, we obtained the room-temperature 2.1 angstrom resolution structure of the fully glycosylated precursor complex of TbCatB. The structure reveals the mechanism of native TbCatB inhibition and demonstrates that new biomolecular information can be obtained by the “diffraction-before-destruction” approach of x-ray free-electron lasers from hundreds of thousands of individual microcrystals.
doi:10.1126/science.1229663
PMCID: PMC3786669  PMID: 23196907
11.  Serial femtosecond X-ray diffraction of 30S ribosomal subunit microcrystals in liquid suspension at ambient temperature using an X-ray free-electron laser 
Serial femtosecond X-ray (SFX) diffraction extending beyond 6 Å resolution using T. thermophilus 30S ribosomal subunit crystals is reported.
High-resolution ribosome structures determined by X-ray crystallography have provided important insights into the mechanism of translation. Such studies have thus far relied on large ribosome crystals kept at cryogenic temperatures to reduce radiation damage. Here, the application of serial femtosecond X-ray crystallography (SFX) using an X-ray free-electron laser (XFEL) to obtain diffraction data from ribosome microcrystals in liquid suspension at ambient temperature is described. 30S ribosomal subunit microcrystals diffracted to beyond 6 Å resolution, demonstrating the feasibility of using SFX for ribosome structural studies. The ability to collect diffraction data at near-physiological temperatures promises to provide fundamental insights into the structural dynamics of the ribosome and its functional complexes.
doi:10.1107/S174430911302099X
PMCID: PMC3758164  PMID: 23989164
30S ribosomal subunit; serial femtosecond X-ray crystallography; X-ray free-electron laser; ribosome
12.  Simultaneous Femtosecond X-ray Spectroscopy and Diffraction of Photosystem II at Room Temperature 
Science (New York, N.Y.)  2013;340(6131):491-495.
Intense femtosecond X-ray pulses produced at the Linac Coherent Light Source (LCLS) were used for simultaneous X-ray diffraction (XRD) and X-ray emission spectroscopy (XES) of microcrystals of Photosystem II (PS II) at room temperature. This method probes the overall protein structure and the electronic structure of the Mn4CaO5 cluster in the oxygen-evolving complex of PS II. XRD data are presented from both the dark state (S1) and the first illuminated state (S2) of PS II. Our simultaneous XRD/XES study shows that the PS II crystals are intact during our measurements at the LCLS, not only with respect to the structure of PS II, but also with regard to the electronic structure of the highly radiation sensitive Mn4CaO5 cluster, opening new directions for future dynamics studies.
doi:10.1126/science.1234273
PMCID: PMC3732582  PMID: 23413188
13.  Time-resolved protein nanocrystallography using an X-ray free-electron laser 
Aquila, Andrew | Hunter, Mark S. | Doak, R. Bruce | Kirian, Richard A. | Fromme, Petra | White, Thomas A. | Andreasson, Jakob | Arnlund, David | Bajt, Saša | Barends, Thomas R. M. | Barthelmess, Miriam | Bogan, Michael J. | Bostedt, Christoph | Bottin, Hervé | Bozek, John D. | Caleman, Carl | Coppola, Nicola | Davidsson, Jan | DePonte, Daniel P. | Elser, Veit | Epp, Sascha W. | Erk, Benjamin | Fleckenstein, Holger | Foucar, Lutz | Frank, Matthias | Fromme, Raimund | Graafsma, Heinz | Grotjohann, Ingo | Gumprecht, Lars | Hajdu, Janos | Hampton, Christina Y. | Hartmann, Andreas | Hartmann, Robert | Hau-Riege, Stefan | Hauser, Günter | Hirsemann, Helmut | Holl, Peter | Holton, James M. | Hömke, André | Johansson, Linda | Kimmel, Nils | Kassemeyer, Stephan | Krasniqi, Faton | Kühnel, Kai-Uwe | Liang, Mengning | Lomb, Lukas | Malmerberg, Erik | Marchesini, Stefano | Martin, Andrew V. | Maia, Filipe R.N.C. | Messerschmidt, Marc | Nass, Karol | Reich, Christian | Neutze, Richard | Rolles, Daniel | Rudek, Benedikt | Rudenko, Artem | Schlichting, Ilme | Schmidt, Carlo | Schmidt, Kevin E. | Schulz, Joachim | Seibert, M. Marvin | Shoeman, Robert L. | Sierra, Raymond | Soltau, Heike | Starodub, Dmitri | Stellato, Francesco | Stern, Stephan | Strüder, Lothar | Timneanu, Nicusor | Ullrich, Joachim | Wang, Xiaoyu | Williams, Garth J. | Weidenspointner, Georg | Weierstall, Uwe | Wunderer, Cornelia | Barty, Anton | Spence, John C. H. | Chapman, Henry N.
Optics Express  2012;20(3):2706-2716.
We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to obtain X-ray diffraction snapshots from the photoactivated states of large membrane protein complexes in the form of nanocrystals flowing in a liquid jet. Light-induced changes of Photosystem I-Ferredoxin co-crystals were observed at time delays of 5 to 10 µs after excitation. The result correlates with the microsecond kinetics of electron transfer from Photosystem I to ferredoxin. The undocking process that follows the electron transfer leads to large rearrangements in the crystals that will terminally lead to the disintegration of the crystals. We describe the experimental setup and obtain the first time-resolved femtosecond serial X-ray crystallography results from an irreversible photo-chemical reaction at the Linac Coherent Light Source. This technique opens the door to time-resolved structural studies of reaction dynamics in biological systems.
doi:10.1364/OE.20.002706
PMCID: PMC3413412  PMID: 22330507
(170.7160) Ultrafast technology; (170.7440) X-ray imaging; (140.3450) Laser-induced chemistry; (140.7090) Ultrafast lasers; (170.0170) Medical optics and biotechnology
14.  Femtosecond X-ray protein nanocrystallography 
Chapman, Henry N. | Fromme, Petra | Barty, Anton | White, Thomas A. | Kirian, Richard A. | Aquila, Andrew | Hunter, Mark S. | Schulz, Joachim | DePonte, Daniel P. | Weierstall, Uwe | Doak, R. Bruce | Maia, Filipe R. N. C. | Martin, Andrew V. | Schlichting, Ilme | Lomb, Lukas | Coppola, Nicola | Shoeman, Robert L. | Epp, Sascha W. | Hartmann, Robert | Rolles, Daniel | Rudenko, Artem | Foucar, Lutz | Kimmel, Nils | Weidenspointner, Georg | Holl, Peter | Liang, Mengning | Barthelmess, Miriam | Caleman, Carl | Boutet, Sébastien | Bogan, Michael J. | Krzywinski, Jacek | Bostedt, Christoph | Bajt, Saša | Gumprecht, Lars | Rudek, Benedikt | Erk, Benjamin | Schmidt, Carlo | Hömke, André | Reich, Christian | Pietschner, Daniel | Strüder, Lothar | Hauser, Günter | Gorke, Hubert | Ullrich, Joachim | Herrmann, Sven | Schaller, Gerhard | Schopper, Florian | Soltau, Heike | Kühnel, Kai-Uwe | Messerschmidt, Marc | Bozek, John D. | Hau-Riege, Stefan P. | Frank, Matthias | Hampton, Christina Y. | Sierra, Raymond G. | Starodub, Dmitri | Williams, Garth J. | Hajdu, Janos | Timneanu, Nicusor | Seibert, M. Marvin | Andreasson, Jakob | Rocker, Andrea | Jönsson, Olof | Svenda, Martin | Stern, Stephan | Nass, Karol | Andritschke, Robert | Schröter, Claus-Dieter | Krasniqi, Faton | Bott, Mario | Schmidt, Kevin E. | Wang, Xiaoyu | Grotjohann, Ingo | Holton, James M. | Barends, Thomas R. M. | Neutze, Richard | Marchesini, Stefano | Fromme, Raimund | Schorb, Sebastian | Rupp, Daniela | Adolph, Marcus | Gorkhover, Tais | Andersson, Inger | Hirsemann, Helmut | Potdevin, Guillaume | Graafsma, Heinz | Nilsson, Björn | Spence, John C. H.
Nature  2011;470(7332):73-77.
X-ray crystallography provides the vast majority of macromolecular structures, but the success of the method relies on growing crystals of sufficient size. In conventional measurements, the necessary increase in X-ray dose to record data from crystals that are too small leads to extensive damage before a diffraction signal can be recorded1-3. It is particularly challenging to obtain large, well-diffracting crystals of membrane proteins, for which fewer than 300 unique structures have been determined despite their importance in all living cells. Here we present a method for structure determination where single-crystal X-ray diffraction ‘snapshots’ are collected from a fully hydrated stream of nanocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the Linac Coherent Light Source4. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes5. More than 3,000,000 diffraction patterns were collected in this study, and a three-dimensional data set was assembled from individual photosystem I nanocrystals (~200 nm to 2 μm in size). We mitigate the problem of radiation damage in crystallography by using pulses briefer than the timescale of most damage processes6. This offers a new approach to structure determination of macromolecules that do not yield crystals of sufficient size for studies using conventional radiation sources or are particularly sensitive to radiation damage.
doi:10.1038/nature09750
PMCID: PMC3429598  PMID: 21293373
15.  In vivo protein crystallization opens new routes in structural biology 
Nature methods  2012;9(3):259-262.
Protein crystallization in cells has been observed several times in nature. However, owing to their small size these crystals have not yet been used for X-ray crystallographic analysis. We prepared nano-sized in vivo–grown crystals of Trypanosoma brucei enzymes and applied the emerging method of free-electron laser-based serial femtosecond crystallography to record interpretable diffraction data. This combined approach will open new opportunities in structural systems biology.
doi:10.1038/nmeth.1859
PMCID: PMC3429599  PMID: 22286384
16.  Time-resolved protein nanocrystallography using an X-ray free-electron laser 
Aquila, Andrew | Hunter, Mark S | Bruce Doak, R. | Kirian, Richard A. | Fromme, Petra | White, Thomas A. | Andreasson, Jakob | Arnlund, David | Bajt, Saša | Barends, Thomas R. M. | Barthelmess, Miriam | Bogan, Michael J. | Bostedt, Christoph | Bottin, Hervé | Bozek, John D. | Caleman, Carl | Coppola, Nicola | Davidsson, Jan | DePonte, Daniel P. | Elser, Veit | Epp, Sascha W. | Erk, Benjamin | Fleckenstein, Holger | Foucar, Lutz | Frank, Matthias | Fromme, Raimund | Graafsma, Heinz | Grotjohann, Ingo | Gumprecht, Lars | Hajdu, Janos | Hampton, Christina Y. | Hartmann, Andreas | Hartmann, Robert | Hau-Riege, Stefan | Hauser, Günter | Hirsemann, Helmut | Holl, Peter | Holton, James M. | Hömke, André | Johansson, Linda | Kimmel, Nils | Kassemeyer, Stephan | Krasniqi, Faton | Kühnel, Kai-Uwe | Liang, Mengning | Lomb, Lukas | Malmerberg, Erik | Marchesini, Stefano | Martin, Andrew V. | Maia, Filipe R.N.C. | Messerschmidt, Marc | Nass, Karol | Reich, Christian | Neutze, Richard | Rolles, Daniel | Rudek, Benedikt | Rudenko, Artem | Schlichting, Ilme | Schmidt, Carlo | Schmidt, Kevin E. | Schulz, Joachim | Seibert, M. Marvin | Shoeman, Robert L. | Sierra, Raymond | Soltau, Heike | Starodub, Dmitri | Stellato, Francesco | Stern, Stephan | Strüder, Lothar | Timneanu, Nicusor | Ullrich, Joachim | Wang, Xiaoyu | Williams, Garth J. | Weidenspointner, Georg | Weierstall, Uwe | Wunderer, Cornelia | Barty, Anton | Spence, John C. H | Chapman, Henry N.
Optics express  2012;20(3):2706-2716.
We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to obtain X-ray diffraction snapshots from the photoactivated states of large membrane protein complexes in the form of nanocrystals flowing in a liquid jet. Light-induced changes of Photosystem I-Ferredoxin co-crystals were observed at time delays of 5 to 10 μs after excitation. The result correlates with the microsecond kinetics of electron transfer from Photosystem I to ferredoxin. The undocking process that follows the electron transfer leads to large rearrangements in the crystals that will terminally lead to the disintegration of the crystals. We describe the experimental setup and obtain the first time-resolved femtosecond serial X-ray crystallography results from an irreversible photo-chemical reaction at the Linac Coherent Light Source. This technique opens the door to time-resolved structural studies of reaction dynamics in biological systems.
PMCID: PMC3413412  PMID: 22330507
17.  Optimizing the accuracy and precision of the single-pulse Laue technique for synchrotron photo-crystallography 
Journal of Synchrotron Radiation  2010;17(Pt 4):479-485.
The accuracy achieved in single-pulse pump-probe Laue experiments at beamline 14-ID at APS is estimated to be 3–4%.
The accuracy that can be achieved in single-pulse pump-probe Laue experiments is discussed. It is shown that with careful tuning of the experimental conditions a reproducibility of the intensity ratios of equivalent intensities obtained in different measurements of 3–4% can be achieved. The single-pulse experiments maximize the time resolution that can be achieved and, unlike stroboscopic techniques in which the pump-probe cycle is rapidly repeated, minimize the temperature increase due to the laser exposure of the sample.
doi:10.1107/S0909049510019710
PMCID: PMC2891491  PMID: 20567080
single-pulse diffraction; accuracy; Laue method; RATIO method; photo-crystallography
18.  Time-resolved synchrotron diffraction and theoretical studies of very short-lived photo-induced molecular species 
Excited-state geometries determined by time-resolved synchrotron diffraction are summarized with emphasis on their comparison with a series of theoretical results. The relative merits of monochromatic and polychromatic (Laue) techniques are discussed.
Definitive experimental results on the geometry of fleeting species are at the time of writing still limited to monochromatic data collection, but methods for modifications of the polychromatic Laue data to increase their accuracy and their suitability for pump–probe experiments have been implemented and are reviewed. In the monochromatic experiments summarized, excited-state conversion percentages are small when neat crystals are used, but are higher when photoactive species are embedded in an inert framework in supramolecular crystals. With polychromatic techniques and increasing source brightness, smaller samples down to tenths of a micrometre or less can be used, increasing homogeneity of exposure and the fractional population of the excited species. Experiments described include a series of transition metal complexes and a fully organic example involving excimer formation. In the final section, experimental findings are compared with those from theoretical calculations on the isolated species. Qualitative agreement is generally obtained, but the theoretical results are strongly dependent on the details of the calculation, indicating the need for further systematic analysis.
doi:10.1107/S0108767309055342
PMCID: PMC2824528  PMID: 20164641
pump–probe experiments; time-resolved diffraction; excited-state molecular geometries; excimers
19.  The RATIO method for time-resolved Laue crystallography 
Journal of Synchrotron Radiation  2009;16(Pt 2):226-230.
A RATIO method for analysis of intensity changes in time-resolved pump–probe Laue diffraction experiments is described.
A RATIO method for analysis of intensity changes in time-resolved pump–probe Laue diffraction experiments is described. The method eliminates the need for scaling the data with a wavelength curve representing the spectral distribution of the source and removes the effect of possible anisotropic absorption. It does not require relative scaling of series of frames and removes errors due to all but very short term fluctuations in the synchrotron beam.
doi:10.1107/S0909049508040892
PMCID: PMC2651764  PMID: 19240334
Laue diffraction; time-resolved diffraction; ratio method; data reduction
20.  Structure of a photosynthetic reaction centre determined by serial femtosecond crystallography 
Nature Communications  2013;4:2911.
Serial femtosecond crystallography is an X-ray free-electron-laser-based method with considerable potential to have an impact on challenging problems in structural biology. Here we present X-ray diffraction data recorded from microcrystals of the Blastochloris viridis photosynthetic reaction centre to 2.8 Å resolution and determine its serial femtosecond crystallography structure to 3.5 Å resolution. Although every microcrystal is exposed to a dose of 33 MGy, no signs of X-ray-induced radiation damage are visible in this integral membrane protein structure.
Serial femtosecond crystallography is an X-ray free-electron-laser-based method that uses X-ray bursts to determine protein structures. Here the authors present the structure of a photosynthetic reaction centre, an integral membrane protein, achieved with no sign of X-ray-induced radiation damage.
doi:10.1038/ncomms3911
PMCID: PMC3905732  PMID: 24352554

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