Robust error calibration for serial crystallography
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Mittan-Moreau, TBD↗
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Conformational flexibility of soybean lipoxygenase is coupled to crystal solvent content in serial crystallography
XFEL diffraction frames from batch-crystallized Soybean Lipoxygenase-1 (SLO-1), revealing two crystal polymorphs arising from differential hydration during sample delivery
Wolff, Wolff↗
Rapid and efficient room-temperature serial synchrotron crystallography using the CFEL TapeDrive
Serial crystallography at conventional synchrotron light sources (SSX) offers the possibility to routinely collect data at room temperature using micrometre-sized crystals of biological macromolecules. However, SSX data collection is not yet as routine and currently takes significantly longer than the standard rotation series cryo-crystallography. Thus, its use for high-throughput approaches, such as fragment-based drug screening, where the possibility to measure at physiological temperatures would be a great benefit, is impaired. On the way to highthroughput SSX using a conveyor belt based sample delivery system – the CFEL TapeDrive – with three different proteins of biological relevance (Klebsiella pneumoniae CTX-M-14 β-lactamase, Nectria haematococca xylanase GH11 and Aspergillus flavus urate oxidase), it is shown here that complete datasets can be collected in less than a minute and only minimal amounts of sample are required.
36 MATERIALS SCIENCE↗
A fixed-target platform for serial femtosecond crystallography in a hydrated environment
For serial femtosecond crystallography at X-ray free-electron lasers, which entails collection of single-pulse diffraction patterns from a constantly refreshed supply of microcrystalline sample, delivery of the sample into the X-ray beam path while maintaining low background remains a technical challenge for some experiments, especially where this methodology is applied to relatively low-ordered samples or those difficult to purify and crystallize in large quantities. This work demonstrates a scheme to encapsulate biological samples using polymer thin films and graphene to maintain sample hydration in vacuum conditions. The encapsulated sample is delivered into the X-ray beam on fixed targets for rapid scanning using the Roadrunner fixed-target system towards a long-term goal of low-background measurements on weakly diffracting samples. As a proof of principle, we used microcrystals of the 24 kDa rapid encystment protein (REP24) to provide a benchmark for polymer/graphene sandwich performance. The REP24 microcrystal unit cell obtained from our sandwiched in-vacuum sample was consistent with previously established unit-cell parameters and with those measured by us without encapsulation in humidified helium, indicating that the platform is robust against evaporative losses. While significant scattering from water was observed because of the sample-deposition method, the polymer/graphene sandwich itself was shown to contribute minimally to background scattering.
36 MATERIALS SCIENCE↗
Shock Damage Analysis in Serial Femtosecond Crystallography Data Collected at MHz X-ray Free-Electron Lasers
Serial femtosecond crystallography (SFX) data were recorded at the European X-ray free-electron laser facility (EuXFEL) with protein microcrystals delivered via a microscopic liquid jet. An XFEL beam striking such a jet may launch supersonic shock waves up the jet, compromising the oncoming sample. To investigate this efficiently, we employed a novel XFEL pulse pattern to nominally expose the sample to between zero and four shock waves before being probed. Analyzing hit rate, indexing rate, and resolution for diffraction data recorded at MHz pulse rates, we found no evidence of damage. Notably, however, this conclusion could only be drawn after careful identification and assimilation of numerous interrelated experimental factors, which we describe in detail. Failure to do so would have led to an erroneous conclusion. Femtosecond photography of the sample-carrying jet revealed critically different jet behavior from that of all homogeneous liquid jets studied to date in this manner.
36 MATERIALS SCIENCE↗
Radiation damage and dose limits in serial synchrotron crystallography at cryo- and room temperatures
Radiation damage limits the accuracy of macromolecular structures in X-ray crystallography. Cryogenic (cryo-) cooling reduces the global radiation damage rate and, therefore, became the method of choice over the past decades. The recent advent of serial crystallography, which spreads the absorbed energy over many crystals, thereby reducing damage, has rendered room temperature (RT) data collection more practical and also extendable to microcrystals, both enabling and requiring the study of specific and global radiation damage at RT. Here, we performed sequential serial raster-scanning crystallography using a microfocused synchrotron beam that allowed for the collection of two series of 40 and 90 full datasets at 2- and 1.9-Å resolution at a dose rate of 40.3 MGy/s on hen egg white lysozyme (HEWL) crystals at RT and cryotemperature, respectively. The diffraction intensity halved its initial value at average doses ( D 1/2 ) of 0.57 and 15.3 MGy at RT and 100 K, respectively. Specific radiation damage at RT was observed at disulfide bonds but not at acidic residues, increasing and then apparently reversing, a peculiar behavior that can be modeled by accounting for differential diffraction intensity decay due to the nonuniform illumination by the X-ray beam. Specific damage to disulfide bonds is evident early on at RT and proceeds at a fivefold higher rate than global damage. The decay modeling suggests it is advisable not to exceed a dose of 0.38 MGy per dataset in static and time-resolved synchrotron crystallography experiments at RT. This rough yardstick might change for proteins other than HEWL and at resolutions other than 2 Å.
36 MATERIALS SCIENCE↗
Resolution extension by image summing in serial femtosecond crystallography of two-dimensional membrane-protein crystals
Previous proof-of-concept measurements on single-layer two-dimensional membrane-protein crystals performed at X-ray free-electron lasers (FELs) have demonstrated that the collection of meaningful diffraction patterns, which is not possible at synchrotrons because of radiation-damage issues, is feasible. Here, the results obtained from the analysis of a thousand single-shot, roomtemperature X-ray FEL diffraction images from two-dimensional crystals of a bacteriorhodopsin mutant are reported in detail. The high redundancy in the measurements boosts the intensity signal-to-noise ratio, so that the values of the diffracted intensities can be reliably determined down to the detector-edge resolution of 4 A ° . The results show that two-dimensional serial crystallography at X-ray FELs is a suitable method to study membrane proteins to near-atomic length scales at ambient temperature. The method presented here can be extended to pump–probe studies of optically triggered structural changes on submillisecond timescales in two-dimensional crystals, which allow functionally relevant large-scale motions that may be quenched in three-dimensional crystals.
serial crystallography, free-electron lasers, memb↗
Structure-factor amplitude reconstruction from serial femtosecond crystallography of two-dimensional membrane-protein crystals
Serial femtosecond crystallography of two-dimensional membrane-protein crystals at X-ray free-electron lasers has the potential to address the dynamics of functionally relevant large-scale motions, which can be sterically hindered in three-dimensional crystals and suppressed in cryocooled samples. In previous work, diffraction data limited to a two-dimensional reciprocal-space slice were evaluated and it was demonstrated that the low intensity of the diffraction signal can be overcome by collecting highly redundant data, thus enhancing the achievable resolution. Here, the application of a newly developed method to analyze diffraction data covering three reciprocal-space dimensions, extracting the reciprocal-space map of the structure-factor amplitudes, is presented. Despite the low resolution and completeness of the data set, it is shown by molecular replacement that the reconstructed amplitudes carry meaningful structural information. Therefore, it appears that these intrinsic limitations in resolution and completeness from two-dimensional crystal diffraction may be overcome by collecting highly redundant data along the three reciprocal-space axes, thus allowing the measurement of large-scale dynamics in pump–probe experiments.
59 BASIC BIOLOGICAL SCIENCES↗
Advanced manufacturing provides tailor-made solutions for crystallography with x-ray free-electron lasers
Serial crystallography at large facilities, such as x-ray free-electron lasers and synchrotrons, evolved as a powerful method for the high-resolution structural investigation of proteins that are critical for human health, thus advancing drug discovery and novel therapies. However, a critical barrier to successful serial crystallography experiments lies in the efficient handling of the protein microcrystals and solutions at microscales. Microfluidics are the obvious approach for any high-throughput, nano-to-microliter sample handling, that also requires design flexibility and rapid prototyping to deal with the variable shapes, sizes, and density of crystals. Here, we discuss recent advances in polymer 3D printing for microfluidics-based serial crystallography research and present a demonstration of emerging, large-scale, nano-3D printing approaches leading into the future of 3D sample environment and delivery device fabrication from liquid jet gas-dynamic virtual nozzles devices to fixed-target sample environment technology.
36 MATERIALS SCIENCE↗
Chemical Crystallography by Serial Femtosecond X-ray Diffraction
Please check the README file for more information about the dataset.
AgBeh powder, mithrene powder, mithrene crystals, ↗
3D printed droplet generation devices for serial femtosecond crystallography enabled by surface coating
The role of surface wetting properties and their impact on the performance of 3D printed microfluidic droplet generation devices for serial femtosecond crystallography (SFX) are reported herein. SFX is a novel crystallography method enabling structure determination of proteins at room temperature with atomic resolution using X-ray free-electron lasers (XFELs). In SFX, protein crystals in their mother liquor are delivered and intersected with a pulsed X-ray beam using a liquid jet injector. Owing to the pulsed nature of the X-ray beam, liquid jets tend to waste the vast majority of injected crystals, which this work aims to overcome with the delivery of aqueous protein crystal suspension droplets segmented by an oil phase. For this purpose, 3D printed droplet generators that can be easily customized for a variety of XFEL measurements have been developed. The surface properties, in particular the wetting properties of the resist materials compatible with the employed two-photon printing technology, have so far not been characterized extensively, but are crucial for stable droplet generation. This work investigates experimentally the effectiveness and the long-term stability of three different surface treatments on photoresist films and glass as models for our 3D printed droplet generator and the fused silica capillaries employed in the other fluidic components of an SFX experiment. Finally, the droplet generation performance of an assembly consisting of the 3D printed device and fused silica capillaries is examined. Stable and reproducible droplet generation was achieved with a fluorinated surface coating which also allowed for robust downstream droplet delivery. Experimental XFEL diffraction data of crystals formed from the large membrane protein complex photosystem I demonstrate the full compatibility of the new injection method with very fragile membrane protein crystals and show that successful droplet generation of crystal-laden aqueous droplets intersected by an oil phase correlates with increased crystal hit rates.
47 OTHER INSTRUMENTATION↗
Chemical crystallography by serial femtosecond X-ray diffraction
Abstract Inorganic–organic hybrid materials represent a large share of newly reported structures, owing to their simple synthetic routes and customizable properties 1 . 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 2,3 and electron microdiffraction 4–11 . 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 12,13 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 14 , the resulting datasets can be solved and refined using standard tools for single-crystal diffraction data 15–17 . We describe the ab initio structure solutions of mithrene (AgSePh) 18–20 , 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 20 . 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.
36 MATERIALS SCIENCE↗
MyD88 TIR domain higher-order assembly interactions revealed by microcrystal electron diffraction and serial femtosecond crystallography
MyD88 and MAL are Toll-like receptor (TLR) adaptors that signal to induce pro-inflammatory cytokine production. We previously observed that the TIR domain of MAL (MAL TIR ) forms filaments in vitro and induces formation of crystalline higher-order assemblies of the MyD88 TIR domain (MyD88 TIR ). These crystals are too small for conventional X-ray crystallography, but are ideally suited to structure determination by microcrystal electron diffraction (MicroED) and serial femtosecond crystallography (SFX). Here, we present MicroED and SFX structures of the MyD88 TIR assembly, which reveal a two-stranded higher-order assembly arrangement of TIR domains analogous to that seen previously for MAL TIR . We demonstrate via mutagenesis that the MyD88 TIR assembly interfaces are critical for TLR4 signaling in vivo, and we show that MAL promotes unidirectional assembly of MyD88 TIR . Collectively, our studies provide structural and mechanistic insight into TLR signal transduction and allow a direct comparison of the MicroED and SFX techniques.
59 BASIC BIOLOGICAL SCIENCES↗
Serial femtosecond and serial synchrotron crystallography can yield data of equivalent quality: A systematic comparison
For the two proteins myoglobin and fluoroacetate dehalogenase, we present a systematic comparison of crystallographic diffraction data collected by serial femtosecond (SFX) and serial synchrotron crystallography (SSX). To maximize comparability, we used the same batch of micron-sized crystals, the same sample delivery device, and the same data analysis software. Overall figures of merit indicate that the data of both radiation sources are of equivalent quality. For both proteins, reasonable data statistics can be obtained with approximately 5000 room-temperature diffraction images irrespective of the radiation source. The direct comparability of SSX and SFX data indicates that the quality of diffraction data obtained from these samples is linked to the properties of the crystals rather than to the radiation source. Therefore, for other systems with similar properties, time-resolved experiments can be conducted at the radiation source that best matches the desired time resolution.
59 BASIC BIOLOGICAL SCIENCES↗
Nylon mesh-based sample holder for fixed-target serial femtosecond crystallography
Explore the source record for details and available documents.
Lysozyme↗
Megahertz pulse trains enable multi-hit serial femtosecond crystallography experiments at X-ray free electron lasers
The European X-ray Free Electron Laser (XFEL) and Linac Coherent Light Source (LCLS) II are extremely intense sources of X-rays capable of generating Serial Femtosecond Crystallography (SFX) data at megahertz (MHz) repetition rates. Previous work has shown that it is possible to use consecutive X-ray pulses to collect diffraction patterns from individual crystals. Here, we exploit the MHz pulse structure of the European XFEL to obtain two complete datasets from the same lysozyme crystal, first hit and the second hit, before it exits the beam. The two datasets, separated by <1 µs, yield up to 2.1 Å resolution structures. Comparisons between the two structures reveal no indications of radiation damage or significant changes within the active site, consistent with the calculated dose estimates. This demonstrates MHz SFX can be used as a tool for tracking sub-microsecond structural changes in individual single crystals, a technique we refer to as multi-hit SFX.
38 RADIATION CHEMISTRY, RADIOCHEMISTRY, AND NUCLEA↗
Low-pass spectral analysis of time-resolved serial femtosecond crystallography data
Low-pass spectral analysis (LPSA) is a recently developed dynamics retrieval algorithm showing excellent retrieval properties when applied to model data affected by extreme incompleteness and stochastic weighting. In this work, we apply LPSA to an experimental time-resolved serial femtosecond crystallography (TR-SFX) dataset from the membrane protein bacteriorhodopsin (bR) and analyze its parametric sensitivity. While most dynamical modes are contaminated by nonphysical high-frequency features, we identify two dominant modes, which are little affected by spurious frequencies. The dynamics retrieved using these modes shows an isomerization signal compatible with previous findings. We employ synthetic data with increasing timing uncertainty, increasing incompleteness level, pixel-dependent incompleteness, and photon counting errors to investigate the root cause of the high-frequency contamination of our TR-SFX modes. By testing a range of methods, we show that timing errors comparable to the dynamical periods to be retrieved produce a smearing of dynamical features, hampering dynamics retrieval, but with no introduction of spurious components in the solution, when convergence criteria are met. Using model data, we are able to attribute the high-frequency contamination of low-order dynamical modes to the high levels of noise present in the data. Finally, we propose a method to handle missing observations that produces a substantial dynamics retrieval improvement from synthetic data with a significant static component. Reprocessing of the bR TR-SFX data using the improved method yields dynamical movies with strong isomerization signals compatible with previous findings.
59 BASIC BIOLOGICAL SCIENCES↗
Fixed-target serial femtosecond crystallography using in cellulo grown microcrystals
The crystallization of recombinant proteins in living cells is an exciting new approach in structural biology. Recent success has highlighted the need for fast and efficient diffraction data collection, optimally directly exposing intact crystal-containing cells to the X-ray beam, thus protecting the in cellulo crystals from environmental challenges. Serial femtosecond crystallography (SFX) at free-electron lasers (XFELs) allows the collection of detectable diffraction even from tiny protein crystals, but requires very fast sample exchange to utilize each XFEL pulse. Here, an efficient approach is presented for high-resolution structure elucidation using serial femtosecond in cellulo diffraction of micometre-sized crystals of the protein HEX-1 from the fungus Neurospora crassa on a fixed target. Employing the fast and highly accurate Roadrunner II translation-stage system allowed efficient raster scanning of the pores of micro-patterned, single-crystalline silicon chips loaded with living, crystal-containing insect cells. Compared with liquid-jet and LCP injection systems, the increased hit rates of up to 30% and reduced background scattering enabled elucidation of the HEX-1 structure. Using diffraction data from only a single chip collected within 12 min at the Linac Coherent Light Source, a 1.8 Å resolution structure was obtained with significantly reduced sample consumption compared with previous SFX experiments using liquid-jet injection. This HEX-1 structure is almost superimposable with that previously determined using synchrotron radiation from single HEX-1 crystals grown by sitting-drop vapour diffusion, validating the approach. This study demonstrates that fixed-target SFX using micro-patterned silicon chips is ideally suited for efficient in cellulo diffraction data collection using living, crystal-containing cells, and offers huge potential for the straightforward structure elucidation of proteins that form intracellular crystals at both XFELs and synchrotron sources.
Lahey-Rudolph, J. Mia (ORCID:0000000152797267)↗