Serial crystallography, using either femtosecond X‐ray pulses from free‐electron laser sources or short synchrotron‐radiation exposures, has the potential to reveal metalloprotein structural details ...while minimizing damage processes. However, deriving a self‐consistent set of Bragg intensities from numerous still‐crystal exposures remains a difficult problem, with optimal protocols likely to be quite different from those well established for rotation photography. Here several data processing issues unique to serial crystallography are examined. It is found that the limiting resolution differs for each shot, an effect that is likely to be due to both the sample heterogeneity and pulse‐to‐pulse variation in experimental conditions. Shots with lower resolution limits produce lower‐quality models for predicting Bragg spot positions during the integration step. Also, still shots by their nature record only partial measurements of the Bragg intensity. An approximate model that corrects to the full‐spot equivalent (with the simplifying assumption that the X‐rays are monochromatic) brings the distribution of intensities closer to that expected from an ideal crystal, and improves the sharpness of anomalous difference Fourier peaks indicating metal positions.
The DIALS project is a collaboration between Diamond Light Source, Lawrence Berkeley National Laboratory and CCP4 to develop a new software suite for the analysis of crystallographic X‐ray ...diffraction data, initially encompassing spot finding, indexing, refinement and integration. The design, core algorithms and structure of the software are introduced, alongside results from the analysis of data from biological and chemical crystallography experiments.
A new X‐ray diffraction data‐analysis package is presented with a description of the algorithms and examples of its application to biological and chemical crystallography.
Synaptotagmin-1 and neuronal SNARE proteins have central roles in evoked synchronous neurotransmitter release; however, it is unknown how they cooperate to trigger synaptic vesicle fusion. Here we ...report atomic-resolution crystal structures of Ca(2+)- and Mg(2+)-bound complexes between synaptotagmin-1 and the neuronal SNARE complex, one of which was determined with diffraction data from an X-ray free-electron laser, leading to an atomic-resolution structure with accurate rotamer assignments for many side chains. The structures reveal several interfaces, including a large, specific, Ca(2+)-independent and conserved interface. Tests of this interface by mutagenesis suggest that it is essential for Ca(2+)-triggered neurotransmitter release in mouse hippocampal neuronal synapses and for Ca(2+)-triggered vesicle fusion in a reconstituted system. We propose that this interface forms before Ca(2+) triggering, moves en bloc as Ca(2+) influx promotes the interactions between synaptotagmin-1 and the plasma membrane, and consequently remodels the membrane to promote fusion, possibly in conjunction with other interfaces.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Inorganic-organic hybrid materials represent a large share of newly reported structures, owing to their simple synthetic routes and customizable properties
. This proliferation has led to a ...characterization bottleneck: many hybrid materials are obligate microcrystals with low symmetry and severe radiation sensitivity, interfering with the standard techniques of single-crystal X-ray diffraction
and electron microdiffraction
. Here we demonstrate small-molecule serial femtosecond X-ray crystallography (smSFX) for the determination of material crystal structures from microcrystals. We subjected microcrystalline suspensions to X-ray free-electron laser radiation
and obtained thousands of randomly oriented diffraction patterns. We determined unit cells by aggregating spot-finding results into high-resolution powder diffractograms. After indexing the sparse serial patterns by a graph theory approach
, the resulting datasets can be solved and refined using standard tools for single-crystal diffraction data
. We describe the ab initio structure solutions of mithrene (AgSePh)
, thiorene (AgSPh) and tethrene (AgTePh), of which the latter two were previously unknown structures. In thiorene, we identify a geometric change in the silver-silver bonding network that is linked to its divergent optoelectronic properties
. We demonstrate that smSFX can be applied as a general technique for structure determination of beam-sensitive microcrystalline materials at near-ambient temperature and pressure.
Oxidation states of individual metal atoms within a metalloprotein can be assigned by examining X‐ray absorption edges, which shift to higher energy for progressively more positive valence numbers. ...Indeed, X‐ray crystallography is well suited for such a measurement, owing to its ability to spatially resolve the scattering contributions of individual metal atoms that have distinct electronic environments contributing to protein function. However, as the magnitude of the shift is quite small, about +2 eV per valence state for iron, it has only been possible to measure the effect when performed with monochromated X‐ray sources at synchrotron facilities with energy resolutions in the range 2–3 × 10−4 (ΔE/E). This paper tests whether X‐ray free‐electron laser (XFEL) pulses, which have a broader bandpass (ΔE/E = 3 × 10−3) when used without a monochromator, might also be useful for such studies. The program nanoBragg is used to simulate serial femtosecond crystallography (SFX) diffraction images with sufficient granularity to model the XFEL spectrum, the crystal mosaicity and the wavelength‐dependent anomalous scattering factors contributed by two differently charged iron centers in the 110‐amino‐acid protein, ferredoxin. Bayesian methods are then used to deduce, from the simulated data, the most likely X‐ray absorption curves for each metal atom in the protein, which agree well with the curves chosen for the simulation. The data analysis relies critically on the ability to measure the incident spectrum for each pulse, and also on the nanoBragg simulator to predict the size, shape and intensity profile of Bragg spots based on an underlying physical model that includes the absorption curves, which are then modified to produce the best agreement with the simulated data. This inference methodology potentially enables the use of SFX diffraction for the study of metalloenzyme mechanisms and, in general, offers a more detailed approach to Bragg spot data reduction.
Electronic configurations at distinct metal centers within a metalloprotein may be characterized by inspecting the scattering factors at the X‐ray absorption edge. Such experiments may be feasible at XFEL sources, using Bayesian data analysis.
Structural dynamics of water and its hydrogen-bonding networks play an important role in enzyme function via the transport of protons, ions, and substrates. To gain insights into these mechanisms in ...the water oxidation reaction in Photosystem II (PS II), we have performed crystalline molecular dynamics (MD) simulations of the dark-stable S1 state. Our MD model consists of a full unit cell with 8 PS II monomers in explicit solvent (861 894 atoms), enabling us to compute the simulated crystalline electron density and to compare it directly with the experimental density from serial femtosecond X-ray crystallography under physiological temperature collected at X-ray free electron lasers (XFELs). The MD density reproduced the experimental density and water positions with high fidelity. The detailed dynamics in the simulations provided insights into the mobility of water molecules in the channels beyond what can be interpreted from experimental B-factors and electron densities alone. In particular, the simulations revealed fast, coordinated exchange of waters at sites where the density is strong, and water transport across the bottleneck region of the channels where the density is weak. By computing MD hydrogen and oxygen maps separately, we developed a novel Map-based Acceptor–Donor Identification (MADI) technique that yields information which helps to infer hydrogen-bond directionality and strength. The MADI analysis revealed a series of hydrogen-bond wires emanating from the Mn cluster through the Cl1 and O4 channels; such wires might provide pathways for proton transfer during the reaction cycle of PS II. Our simulations provide an atomistic picture of the dynamics of water and hydrogen-bonding networks in PS II, with implications for the specific role of each channel in the water oxidation reaction.
New Python-based methods for data processing Sauter, Nicholas K.; Hattne, Johan; Grosse-Kunstleve, Ralf W. ...
Acta crystallographica. Section D, Biological crystallography.,
July 2013, Letnik:
69, Številka:
7
Journal Article
Recenzirano
Odprti dostop
Current pixel‐array detectors produce diffraction images at extreme data rates (of up to 2 TB h−1) that make severe demands on computational resources. New multiprocessing frameworks are required to ...achieve rapid data analysis, as it is important to be able to inspect the data quickly in order to guide the experiment in real time. By utilizing readily available web‐serving tools that interact with the Python scripting language, it was possible to implement a high‐throughput Bragg‐spot analyzer (cctbx.spotfinder) that is presently in use at numerous synchrotron‐radiation beamlines. Similarly, Python interoperability enabled the production of a new data‐reduction package (cctbx.xfel) for serial femtosecond crystallography experiments at the Linac Coherent Light Source (LCLS). Future data‐reduction efforts will need to focus on specialized problems such as the treatment of diffraction spots on interleaved lattices arising from multi‐crystal specimens. In these challenging cases, accurate modeling of close‐lying Bragg spots could benefit from the high‐performance computing capabilities of graphics‐processing units.
A new tool is introduced for screening macromolecular X‐ray crystallography diffraction images produced at an X‐ray free‐electron laser light source. Based on a data‐driven deep learning approach, ...the proposed tool executes a convolutional neural network to detect Bragg spots. Automatic image processing algorithms described can enable the classification of large data sets, acquired under realistic conditions consisting of noisy data with experimental artifacts. Outcomes are compared for different data regimes, including samples from multiple instruments and differing amounts of training data for neural network optimization.
Deep learning provides one possible avenue to reduce the data stream generated by serial macromolecular X‐ray crystallography. Convolutional neural networks can be trained to recognize the presence or absence of Bragg spots, forming a criterion to veto events prior to downstream data processing.
There is considerable potential for X-ray free electron lasers (XFELs) to enable determination of macromolecular crystal structures that are difficult to solve using current synchrotron sources. ...Prior XFEL studies often involved the collection of thousands to millions of diffraction images, in part due to limitations of data processing methods. We implemented a data processing system based on classical post-refinement techniques, adapted to specific properties of XFEL diffraction data. When applied to XFEL data from three different proteins collected using various sample delivery systems and XFEL beam parameters, our method improved the quality of the diffraction data as well as the resulting refined atomic models and electron density maps. Moreover, the number of observations for a reflection necessary to assemble an accurate data set could be reduced to a few observations. These developments will help expand the applicability of XFEL crystallography to challenging biological systems, including cases where sample is limited.
Research towards using X‐ray free‐electron laser (XFEL) data to solve structures using experimental phasing methods such as sulfur single‐wavelength anomalous dispersion (SAD) has been hampered by ...shortcomings in the diffraction models for X‐ray diffraction from FELs. Owing to errors in the orientation matrix and overly simple partiality models, researchers have required large numbers of images to converge to reliable estimates for the structure‐factor amplitudes, which may not be feasible for all biological systems. Here, data for cytoplasmic polyhedrosis virus type 17 (CPV17) collected at 1.3 Å wavelength at the Linac Coherent Light Source (LCLS) are revisited. A previously published definition of a partiality model for reflections illuminated by self‐amplified spontaneous emission (SASE) pulses is built upon, which defines a fraction between 0 and 1 based on the intersection of a reflection with a spread of Ewald spheres modelled by a super‐Gaussian wavelength distribution in the X‐ray beam. A method of post‐refinement to refine the parameters of this model is suggested. This has generated a merged data set with an overall discrepancy (by calculating the Rsplit value) of 3.15% to 1.46 Å resolution from a 7225‐image data set. The atomic numbers of C, N and O atoms in the structure are distinguishable in the electron‐density map. There are 13 S atoms within the 237 residues of CPV17, excluding the initial disordered methionine. These only possess 0.42 anomalous scattering electrons each at 1.3 Å wavelength, but the 12 that have single predominant positions are easily detectable in the anomalous difference Fourier map. It is hoped that these improvements will lead towards XFEL experimental phase determination and structure determination by sulfur SAD and will generally increase the utility of the method for difficult cases.
PDF
