IUCrJ

Journal: IUCrJ

Abbreviation

IUCrJ

Publisher

International Union of Crystallography

ISSN

2052-2525

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Publications1 - 10 of 13

Serial snapshot crystallography for materials science with SwissFEL
Dejoie, Catherine; Smeets, Stef; Baerlocher, Christian; et al. (2015)
IUCrJ
New opportunities for studying (sub)microcrystalline materials with small unit cells, both organic and inorganic, will open up when the X-ray free electron laser (XFEL) presently being constructed in Switzerland (SwissFEL) comes online in 2017. Our synchrotron-based experiments mimicking the 4%-energy-bandpass mode of the SwissFEL beam show that it will be possible to record a diffraction pattern of up to 10 randomly oriented crystals in a single snapshot, to index the resulting reflections, and to extract their intensities reliably. The crystals are destroyed with each XFEL pulse, but by combining snapshots from several sets of crystals, a complete set of data can be assembled, and crystal structures of materials that are difficult to analyze otherwise will become accessible. Even with a single shot, at least a partial analysis of the crystal structure will be possible, and with 10–50 femtosecond pulses, this offers tantalizing possibilities for time-resolved studies.
Capturing the blue-light activated state of the Phot-LOV1 domain from Chlamydomonas reinhardtii using time-resolved serial synchrotron crystallography
Gotthard, Guillaume; Mous, Sandra; Weinert, Tobias; et al. (2024)
IUCrJ
Light-oxygen-voltage (LOV) domains are small photosensory flavoprotein modules that allow the conversion of external stimuli (sunlight) into intracellular signals responsible for various cell behaviors (e.g. phototropism and chloroplast relocation). This ability relies on the light-induced formation of a covalent thioether adduct between a flavin chromophore and a reactive cysteine from the protein environment, which triggers a cascade of structural changes that result in the activation of a serine/threonine (Ser/Thr) kinase. Recent developments in time-resolved crystallography may allow the activation cascade of the LOV domain to be observed in real time, which has been elusive. In this study, we report a robust protocol for the production and stable delivery of microcrystals of the LOV domain of phototropin Phot-1 from Chlamydomonas reinhardtii (CrPhotLOV1) with a high-viscosity injector for time-resolved serial synchrotron crystallography (TR-SSX). The detailed process covers all aspects, from sample optimization to data collection, which may serve as a guide for soluble protein preparation for TR-SSX. In addition, we show that the crystals obtained preserve the photoreactivity using infrared spectroscopy. Furthermore, the results of the TR-SSX experiment provide high-resolution insights into structural alterations of CrPhotLOV1 from Δt = 2.5 ms up to Δt = 95 ms postphotoactivation, including resolving the geometry of the thioether adduct and the C-terminal region implicated in the signal transduction process.
Refinement of cryo-EM 3D maps with a selfsupervised denoising model: crefDenoiser
Agarwal, Ishaant; Kaczmar-Michalska, Joanna; Nørrelykke, Simon F.; et al. (2024)
IUCrJ
Cryogenic electron microscopy (cryo-EM) is a pivotal technique for imaging macromolecular structures. However, despite extensive processing of large image sets collected in cryo-EM experiments to amplify the signal-to-noise ratio, the reconstructed 3D protein-density maps are often limited in quality due to residual noise, which in turn affects the accuracy of the macromolecular representation. Here, crefDenoiser is introduced, a denoising neural network model designed to enhance the signal in 3D cryo-EM maps produced with standard processing pipelines. The crefDenoiser model is trained without the need for 'clean' ground-truth target maps. Instead, a custom dataset is employed, composed of real noisy protein half-maps sourced from the Electron Microscopy Data Bank repository. Competing with the current state-of-the-art, crefDenoiser is designed to optimize for the theoretical noise-free map during self-supervised training. We demonstrate that our model successfully amplifies the signal across a wide variety of protein maps, outperforming a classic map denoiser and following a network-based sharpening model. Without biasing the map, the proposed denoising method leads to improved visibility of protein structural features, including protein domains, secondary structure elements and modest high-resolution feature restoration.
Lipidic cubic phase serial millisecond crystallography using synchrotron radiation
Nogly, Przemyslaw; James, Daniel; Wang, Dingjie; et al. (2015)
IUCrJ
Lipidic cubic phases (LCPs) have emerged as successful matrixes for the crystallization of membrane proteins. Moreover, the viscous LCP also provides a highly effective delivery medium for serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs). Here, the adaptation of this technology to perform serial millisecond crystallography (SMX) at more widely available synchrotron microfocus beamlines is described. Compared with conventional microcrystallography, LCP-SMX eliminates the need for difficult handling of individual crystals and allows for data collection at room temperature. The technology is demonstrated by solving a structure of the light-driven proton-pump bacteriorhodopsin (bR) at a resolution of 2.4 Å. The room-temperature structure of bR is very similar to previous cryogenic structures but shows small yet distinct differences in the retinal ligand and proton-transfer pathway.
Unraveling the magnetic softness in Fe-Ni-B-based nanocrystalline material by magnetic small-angle neutron scattering
Bersweiler, Mathias; Adams, Michael P.; Peral, Inma; et al. (2022)
IUCrJ
Magnetic small-angle neutron scattering is employed to investigate the magnetic interactions in (Fe0.7Ni0.3)86B14 alloy, a HiB-NANOPERM-type soft magnetic nanocrystalline material, which exhibits an ultrafine microstructure with an average grain size below 10nm. The neutron data reveal a significant spin-misalignment scattering which is mainly related to the jump of the longitudinal magnetization at internal particle-matrix interfaces. The field dependence of the neutron data can be well described by micromagnetic small-angle neutron scattering theory. In particular, the theory explains the 'clover-leaf-type' angular anisotropy observed in the purely magnetic neutron scattering cross section. The presented neutron data analysis also provides access to the magnetic interaction parameters, such as the exchange-stiffness constant, which plays a crucial role towards the optimization of the magnetic softness of Fe-based nanocrystalline materials.
Fixed-target pump-probe SFX: eliminating the scourge of light contamination
Gotthard, Guillaume; Flores-Ibarra, Andrea; Carrillo, Melissa; et al. (2024)
IUCrJ
X-ray free-electron laser (XFEL) light sources have enabled the rapid growth of time-resolved structural experiments, which provide crucial information on the function of macromolecules and their mechanisms. Here, the aim was to commission the SwissMX fixed-target sample-delivery system at the SwissFEL Cristallina experimental station using the PSI-developed micro-structured polymer (MISP) chip for pump-probe time-resolved experiments. To characterize the system, crystals of the light-sensitive protein light-oxygen-voltage domain 1 (LOV1) from Chlamydomonas reinhardtii were used. Using different experimental settings, the accidental illumination, referred to as light contamination, of crystals mounted in wells adjacent to those illuminated by the pump laser was examined. It was crucial to control the light scattering from and through the solid supports otherwise significant contamination occurred. However, the results here show that the opaque MISP chips are suitable for defined pump-probe studies of a light-sensitive protein. The experiment also probed the sub-millisecond structural dynamics of LOV1 and indicated that at Δt = 10 ms a covalent thioether bond is established between reactive Cys57 and its flavin mononucleotide cofactor. This experiment validates the crystals to be suitable for in-depth follow-up studies of this still poorly understood signaltransduction mechanism. Importantly, the fixed-target delivery system also permitted a tenfold reduction in protein sample consumption compared with the more common high-viscosity extrusion-based delivery system. This development creates the prospect of an increase in XFEL project throughput for the field.
Intermolecular atom–atom bonds in crystals?
Dunitz, Jack D. (2015)
IUCrJ
Advances in long-wavelength native phasing at X-ray free-electron lasers
Nass, Karol; Cheng, Robert; Vera, Laura; et al. (2020)
IUCrJ
Long-wavelength pulses from the Swiss X-ray free-electron laser (XFEL) have been used for de novo protein structure determination by native single-wavelength anomalous diffraction (native-SAD) phasing of serial femtosecond crystallography (SFX) data. In this work, sensitive anomalous data-quality indicators and model proteins were used to quantify improvements in native-SAD at XFELs such as utilization of longer wavelengths, careful experimental geometry optimization, and better post-refinement and partiality correction. Compared with studies using shorter wavelengths at other XFELs and older software versions, up to one order of magnitude reduction in the required number of indexed images for native-SAD was achieved, hence lowering sample consumption and beam-time requirements significantly. Improved data quality and higher anomalous signal facilitate so-far underutilized de novo structure determination of challenging proteins at XFELs. Improvements presented in this work can be used in other types of SFX experiments that require accurate measurements of weak signals, for example time-resolved studies.
Considerations for three-dimensional image reconstruction from experimental data in coherent diffractive imaging
Lundholm, Ida V.; Sellberg, Jonas A.; Ekeberg, Tomas; et al. (2018)
IUCrJ
Low-dose in situ prelocation of protein microcrystals by 2D X-ray phase-contrast imaging for serial crystallography
Martiel, Isabelle; Huang, Chia-Ying; Villanueva-Perez, Pablo; et al. (2020)
IUCrJ
Serial protein crystallography has emerged as a powerful method of data collection on small crystals from challenging targets, such as membrane proteins. Multiple microcrystals need to be located on large and often flat mounts while exposing them to an X-ray dose that is as low as possible. A crystal-prelocation method is demonstrated here using low-dose 2D full-field propagation-based X-ray phase-contrast imaging at the X-ray imaging beamline TOMCAT at the Swiss Light Source (SLS). This imaging step provides microcrystal coordinates for automated serial data collection at a microfocus macromolecular crystallography beamline on samples with an essentially flat geometry. This prelocation method was applied to microcrystals of a soluble protein and a membrane protein, grown in a commonly used double-sandwich in situ crystallization plate. The inner sandwiches of thin plastic film enclosing the microcrystals in lipid cubic phase were flash cooled and imaged at TOMCAT. Based on the obtained crystal coordinates, both still and rotation wedge serial data were collected automatically at the SLS PXI beamline, yielding in both cases a high indexing rate. This workflow can be easily implemented at many synchrotron facilities using existing equipment, or potentially integrated as an online technique in the next-generation macromolecular crystallography beamline, and thus benefit a number of dose-sensitive challenging protein targets.

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