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1.  The point-spread function of fiber-coupled area detectors 
Journal of Synchrotron Radiation  2012;19(Pt 6):1006-1011.
The point-spread function (PSF) of a powdered-phosphor fiber-optic taper-coupled CCD area detector was measured, and an analytic expression for its functional form proposed. The ‘tails’ of this PSF were shown to be due almost entirely to the fiber-optic taper, with no contribution from the phosphor itself.
The point-spread function (PSF) of a fiber-optic taper-coupled CCD area detector was measured over five decades of intensity using a 20 µm X-ray beam and ∼2000-fold averaging. The ‘tails’ of the PSF clearly revealed that it is neither Gaussian nor Lorentzian, but instead resembles the solid angle subtended by a pixel at a point source of light held a small distance (∼27 µm) above the pixel plane. This converges to an inverse cube law far from the beam impact point. Further analysis revealed that the tails are dominated by the fiber-optic taper, with negligible contribution from the phosphor, suggesting that the PSF of all fiber-coupled CCD-type detectors is best described as a Moffat function.
doi:10.1107/S0909049512035571
PMCID: PMC3480276  PMID: 23093762
protein crystallography X-ray detector; CCD; phosphor; fiber optic
2.  Radiation damage in protein serial femtosecond crystallography using an x-ray free-electron laser 
X-ray free-electron lasers deliver intense femtosecond pulses that promise to yield high resolution diffraction data of nanocrystals before the destruction of the sample by radiation damage. Diffraction intensities of lysozyme nanocrystals collected at the Linac Coherent Light Source using 2 keV photons were used for structure determination by molecular replacement and analyzed for radiation damage as a function of pulse length and fluence. Signatures of radiation damage are observed for pulses as short as 70 fs. Parametric scaling used in conventional crystallography does not account for the observed effects.
doi:10.1103/PhysRevB.84.214111
PMCID: PMC3786679  PMID: 24089594
3.  Crystallographic data processing for free-electron laser sources 
A processing pipeline for diffraction data acquired using the ‘serial crystallography’ methodology with a free-electron laser source is described with reference to the crystallographic analysis suite CrystFEL and the pre-processing program Cheetah.
A processing pipeline for diffraction data acquired using the ‘serial crystallography’ methodology with a free-electron laser source is described with reference to the crystallographic analysis suite CrystFEL and the pre-processing program Cheetah. A detailed analysis of the nature and impact of indexing ambiguities is presented. Simulations of the Monte Carlo integration scheme, which accounts for the partially recorded nature of the diffraction intensities, are presented and show that the integration of partial reflections could be made to converge more quickly if the bandwidth of the X-rays were to be increased by a small amount or if a slight convergence angle were introduced into the incident beam.
doi:10.1107/S0907444913013620
PMCID: PMC3689526  PMID: 23793149
data processing; free-electron lasers; serial crystallography; CrystFEL; Cheetah
4.  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
5.  Implementation and performance of SIBYLS: a dual endstation small-angle X-ray scattering and macromolecular crystallography beamline at the Advanced Light Source 
The SIBYLS beamline of the Advanced Light Source at Lawrence Berkeley National Laboratory is a dual endstation small-angle X-ray scattering and macromolecular crystallography beamline. Key features and capabilities are described along with implementation and performance.
The SIBYLS beamline (12.3.1) of the Advanced Light Source at Lawrence Berkeley National Laboratory, supported by the US Department of Energy and the National Institutes of Health, is optimized for both small-angle X-ray scattering (SAXS) and macromolecular crystallography (MX), making it unique among the world’s mostly SAXS or MX dedicated beamlines. Since SIBYLS was commissioned, assessments of the limitations and advantages of a combined SAXS and MX beamline have suggested new strategies for integration and optimal data collection methods and have led to additional hardware and software enhancements. Features described include a dual mode monochromator [containing both Si(111) crystals and Mo/B4C multilayer elements], rapid beamline optics conversion between SAXS and MX modes, active beam stabilization, sample-loading robotics, and mail-in and remote data collection. These features allow users to gain valuable insights from both dynamic solution scattering and high-resolution atomic diffraction experiments performed at a single synchrotron beamline. Key practical issues considered for data collection and analysis include radiation damage, structural ensembles, alternative conformers and flexibility. SIBYLS develops and applies efficient combined MX and SAXS methods that deliver high-impact results by providing robust cost-effective routes to connect structures to biology and by performing experiments that aid beamline designs for next generation light sources.
doi:10.1107/S0021889812048698
PMCID: PMC3547225  PMID: 23396808
small-angle X-ray scattering (SAXS); macromolecular crystallography (MX); synchrotron beamlines; SIBYLS
6.  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
7.  A beginner’s guide to radiation damage 
Journal of Synchrotron Radiation  2009;16(Pt 2):133-142.
Radiation damage considerations affecting data collection by more than a factor of two are summarized and damage avoidance strategies are suggested.
Many advances in the understanding of radiation damage to protein crystals, particularly at cryogenic temperatures, have been made in recent years, but with this comes an expanding literature, and, to the new breed of protein crystallographer who is not really interested in X-ray physics or radiation chemistry but just wants to solve a biologically relevant structure, the technical nature and breadth of this literature can be daunting. The purpose of this paper is to serve as a rough guide to radiation damage issues, and to provide references to the more exacting and detailed work. No attempt has been made to report precise numbers (a factor of two is considered satisfactory), and, since there are aspects of radiation damage that are demonstrably unpredictable, the ‘worst case scenario’ as well as the ‘average crystal’ are discussed in terms of the practicalities of data collection.
doi:10.1107/S0909049509004361
PMCID: PMC2651760  PMID: 19240325
radiation damage; minimum crystal size; protein macromolecular crystallography; dose doubling; radioprotectant; data collection strategy
8.  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
9.  Structure-factor analysis of femtosecond microdiffraction patterns from protein nanocrystals 
A complete set of structure factors has been extracted from hundreds of thousands of femtosecond X-ray diffraction patterns from randomly oriented Photosystem I membrane protein nanocrystals, using the Monte Carlo method of intensity integration. The data, collected at the Linac Coherent Light Source, are compared with conventional single-crystal data collected at a synchrotron source, and the quality of each data set was found to be similar.
A complete set of structure factors has been extracted from hundreds of thousands of femtosecond single-shot X-ray microdiffraction patterns taken from randomly oriented nanocrystals. The method of Monte Carlo integration over crystallite size and orientation was applied to experimental data from Photosystem I nanocrystals. This arrives at structure factors from many partial reflections without prior knowledge of the particle-size distribution. The data were collected at the Linac Coherent Light Source (the first hard-X-ray laser user facility), to which was fitted a hydrated protein nanocrystal injector jet, according to the method of serial crystallography. The data are single ‘still’ diffraction snapshots, each from a different nanocrystal with sizes ranging between 100 nm and 2 µm, so the angular width of Bragg peaks was dominated by crystal-size effects. These results were compared with single-crystal data recorded from large crystals of Photosystem I at the Advanced Light Source and the quality of the data was found to be similar. The implications for improving the efficiency of data collection by allowing the use of very small crystals, for radiation-damage reduction and for time-resolved diffraction studies at room temperature are discussed.
doi:10.1107/S0108767310050981
PMCID: PMC3066792  PMID: 21325716
nanocrystals; femtosecond diffraction; free-electron lasers; Monte Carlo methods; protein microdiffraction
10.  Structural Insight into KCNQ (Kv7) Channel Assembly and Channelopathy 
Neuron  2007;53(5):663-675.
Summary
Kv7.x (KCNQ) voltage-gated potassium channels form the cardiac and auditory IKs current and the neuronal M-current. The five Kv7 subtypes have distinct assembly preferences encoded by a C-terminal cytoplasmic assembly domain, the A-domain Tail. Here, we present the high-resolution structure of the Kv7.4 A-domain Tail together with biochemical experiments that show that the domain is a self-assembling, parallel, four-stranded coiled coil. Structural analysis and biochemical studies indicate conservation of the coiled coil in all Kv7 subtypes and that a limited set of interactions encode assembly specificity determinants. Kv7 mutations have prominent roles in arrhythmias, deafness, and epilepsy. The structure together with biochemical data indicate that A-domain Tail arrhythmia mutations cluster on the solvent-accessible surface of the subunit interface at a likely site of action for modulatory proteins. Together, the data provide a framework for understanding Kv7 assembly specificity and the molecular basis of a distinct set of Kv7 channelopathies.
doi:10.1016/j.neuron.2007.02.010
PMCID: PMC3011230  PMID: 17329207
11.  A unique E1–E2 interaction required for optimal conjugation of the ubiquitin-like protein NEDD8 
Ubiquitin-like proteins (UBLs), such as NEDD8, are transferred to their targets by distinct, parallel, multi-enzyme cascades that involve the sequential action of E1, E2, and E3 enzymes. How do enzymes within a particular UBL conjugation cascade interact with each other? We report here that the unique N-terminal sequence of NEDD8’s E2, Ubc12, selectively recruits NEDD8’s E1 to promote Ubc12~NEDD8 thioester formation. A peptide corresponding to Ubc12’s N-terminus (Ubc12N26) specifically binds and inhibits NEDD8’s E1, the heterodimeric APPBP1-UBA3 complex. The structure of APPBP1-UBA3-Ubc12N26 reveals conserved Ubc12 residues docking in a groove generated by loops conserved in UBA3s but not other E1s, explaining why this interaction is unique to the NEDD8 pathway. These studies define a novel mechanism for E1–E2 interaction and show how enzymes within a particular UBL conjugation cascade can be tethered together by unique protein-protein interactions emanating from their common structural scaffolds.
doi:10.1038/nsmb826
PMCID: PMC2862556  PMID: 15361859
12.  The minimum crystal size needed for a complete diffraction data set 
A formula for absolute scattering power is derived to include spot fading arising from radiation damage and the crystal volume needed to collect diffraction data to a given resolution is calculated.
In this work, classic intensity formulae were united with an empirical spot-fading model in order to calculate the diameter of a spherical crystal that will scatter the required number of photons per spot at a desired resolution over the radiation-damage-limited lifetime. The influences of molecular weight, solvent content, Wilson B factor, X-ray wavelength and attenuation on scattering power and dose were all included. Taking the net photon count in a spot as the only source of noise, a complete data set with a signal-to-noise ratio of 2 at 2 Å resolution was predicted to be attainable from a perfect lysozyme crystal sphere 1.2 µm in diameter and two different models of photoelectron escape reduced this to 0.5 or 0.34 µm. These represent 15-fold to 700-fold less scattering power than the smallest experimentally determined crystal size to date, but the gap was shown to be consistent with the background scattering level of the relevant experiment. These results suggest that reduction of background photons and diffraction spot size on the detector are the principal paths to improving crystallographic data quality beyond current limits.
doi:10.1107/S0907444910007262
PMCID: PMC2852304  PMID: 20382993
radiation damage; minimum crystal size; protein macromolecular crystallography; scattering power
13.  Basis for a ubiquitin-like protein thioester switch toggling E1-E2 affinity 
Nature  2007;445(7126):394-398.
Ubiquitin-like proteins (Ubls) are conjugated by dynamic E1-E2-E3 enzyme cascades. E1s activate Ubls by catalyzing Ubl C-terminal adenylation, forming a covalent E1~Ubl thioester intermediate, and generating a thioester-linked E2~Ubl product, which must be released for subsequent reactions. We report structural analysis of a trapped Ubl activation complex containing NEDD8’s heterodimeric E1 (APPBP1-UBA3), two NEDD8s (one thioester-linked to E1, one noncovalently-associated for adenylation), a catalytically-inactive E2 (Ubc12), and MgATP. The results suggest that a thioester switch toggles E1-E2 affinities. Two E2 binding sites depend on NEDD8 being thioester-linked to E1. One is unmasked by a striking E1 conformational change. The other comes directly from the thioester-bound NEDD8. After NEDD8 transfer to E2, reversion to an alternate E1 conformation would facilitate release of the E2~NEDD8 thioester product. Thus, transferring the Ubl’s thioester linkage between successive conjugation enzymes can induce conformational changes and alter interaction networks to drive consecutive steps in Ubl cascades.
doi:10.1038/nature05490
PMCID: PMC2821831  PMID: 17220875
14.  XANES measurements of the rate of radiation damage to selenomethionine side chains 
Journal of synchrotron radiation  2006;14(Pt 1):51-72.
The radiation-induced disordering of selenomethionine (SeMet) side chains represents a significant impediment to protein structure solution. Not only does the increased B-factor of these sites result in a serious drop in phasing power, but some sites decay much faster than others in the same unit cell. These radiolabile SeMet side chains decay faster than high-order diffraction spots with dose, making it difficult to detect this kind of damage by inspection of the diffraction pattern. The selenium X-ray absorbance near-edge spectrum (XANES) from samples containing SeMet was found to change significantly after application of X-ray doses of 10–100 MGy. Most notably, the sharp ‘white line’ feature near the canonical Se edge disappears. The change was attributed to breakage of the Cγ—Se bond in SeMet. This spectral change was used as a probe to measure the decay rate of SeMet with X-ray dose in cryo-cooled samples. Two protein crystal types and 15 solutions containing free SeMet amino acid were examined. The damage rate was influenced by the chemical and physical condition of the sample, and the half-decaying dose for the selenium XANES signal ranged from 5 to 43 MGy. These decay rates were 34- to 3.8-fold higher than the rate at which the Se atoms interacted directly with X-ray photons, so the damage mechanism must be a secondary effect. Samples that cooled to a more crystalline state generally decayed faster than samples that cooled to an amorphous solid. The single exception was a protein crystal where a nanocrystalline cryoprotectant had a protective effect. Lowering the pH, especially with ascorbic or nitric acids, had a protective effect, and SeMet lifetime increased monotonically with decreasing sample temperature (down to 93 K). The SeMet lifetime in one protein crystal was the same as that of the free amino acid, and the longest SeMet lifetime measured was found in the other protein crystal type. This protection was found to arise from the folded structure of the protein molecule. A mechanism to explain observed decay rates involving the damaging species following the electric field lines around protein molecules is proposed.
doi:10.1107/S0909049506048898
PMCID: PMC2806430  PMID: 17211072
X-ray dose; protective solutes; pH and temperature effects; protein crystal structure; mechanism of radiation damage
15.  Molecular functions of the histone acetyltransferase chaperone complex Rtt109-Vps75 
Histone acetylation and nucleosome remodeling regulate DNA damage repair, replication and transcription. Rtt109, a recently discovered histone acetyltransferase (HAT) from Saccharomyces cerevisiae, functions with the histone chaperone Asf1 to acetylate lysine K56 on histone H3 (H3K56), a modification associated with newly synthesized histones. In vitro analysis of Rtt109 revealed that Vps75, a Nap1 family histone chaperone, could also stimulate Rtt109-dependent acetylation of H3K56. However, the molecular function of the Rtt109-Vps75 complex remains elusive. Here we have probed the molecular functions of Vps75 and the Rtt109-Vps75 complex through biochemical, structural and genetic means. We find that Vps75 stimulates the kcat of histone acetylation by ∼100-fold relative to Rtt109 alone and enhances acetylation of K9 in the H3 histone tail. Consistent with the In vitro evidence, cells lacking Vps75 showed a substantial reduction (60%) in H3K9 acetylation during S phase. X-ray structural, biochemical and genetic analyses of Vps75 indicate a unique, structurally dynamic Nap1-like fold that suggests a potential mechanism of Vps75-dependent activation of Rtt109. Together, these data provide evidence for a multifunctional HAT-chaperone complex that acetylates histone H3 and deposits H3-H4 onto DNA, linking histone modification and nucleosome assembly.
PMCID: PMC2678805  PMID: 19172748
16.  Determination of X-ray flux using silicon pin diodes 
Journal of Synchrotron Radiation  2009;16(Pt 2):143-151.
Accurate measurement of photon flux from an X-ray source is a parameter required to calculate the dose absorbed by a sample. The development of a model for determining the photon flux incident on pin diodes, and experiments to test this model, are described for incident energies between 4 and 18 keV used in macromolecular crystallography.
Accurate measurement of photon flux from an X-ray source, a parameter required to calculate the dose absorbed by the sample, is not yet routinely available at macromolecular crystallography beamlines. The development of a model for determining the photon flux incident on pin diodes is described here, and has been tested on the macromolecular crystallography beamlines at both the Swiss Light Source, Villigen, Switzerland, and the Advanced Light Source, Berkeley, USA, at energies between 4 and 18 keV. These experiments have shown that a simple model based on energy deposition in silicon is sufficient for determining the flux incident on high-quality silicon pin diodes. The derivation and validation of this model is presented, and a web-based tool for the use of the macromolecular crystallography and wider synchrotron community is introduced.
doi:10.1107/S0909049508040429
PMCID: PMC2651761  PMID: 19240326
macromolecular crystallography; flux determination; silicon pin diode; absorbed dose
17.  The R-factor gap in macromolecular crystallography: an untapped potential for insights on accurate structures 
The Febs Journal  2014;281(18):4046-4060.
In macromolecular crystallography, the agreement between observed and predicted structure factors (Rcryst and Rfree) is seldom better than 20%. This is much larger than the estimate of experimental error (Rmerge). The difference between Rcryst and Rmerge is the R-factor gap. There is no such gap in small-molecule crystallography, for which calculated structure factors are generally considered more accurate than the experimental measurements. Perhaps the true noise level of macromolecular data is higher than expected? Or is the gap caused by inaccurate phases that trap refined models in local minima? By generating simulated diffraction patterns using the program MLFSOM, and including every conceivable source of experimental error, we show that neither is the case. Processing our simulated data yielded values that were indistinguishable from those of real data for all crystallographic statistics except the final Rcryst and Rfree. These values decreased to 3.8% and 5.5% for simulated data, suggesting that the reason for high R-factors in macromolecular crystallography is neither experimental error nor phase bias, but rather an underlying inadequacy in the models used to explain our observations. The present inability to accurately represent the entire macromolecule with both its flexibility and its protein-solvent interface may be improved by synergies between small-angle X-ray scattering, computational chemistry and crystallography. The exciting implication of our finding is that macromolecular data contain substantial hidden and untapped potential to resolve ambiguities in the true nature of the nanoscale, a task that the second century of crystallography promises to fulfill.
Database
Coordinates and structure factors for the real data have been submitted to the Protein Data Bank under accession 4tws.
doi:10.1111/febs.12922
PMCID: PMC4282448  PMID: 25040949
crystallography; R-factor; R-value; simulation; theoretical

Results 1-17 (17)