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The practice of serial X-ray crystallography (SX) depends on efficient, continuous delivery of hydrated protein crystals while minimizing background scattering. Of the two major types of sample delivery devices, fixed-target devices offer several advantages over widely adopted jet injectors, including: lower sample consumption, clog-free delivery, and the ability to control on-chip crystal density to improve hit rates. Here we present our development of versatile, inexpensive, and robust polymer microfluidic chips for routine and reliable room temperature serial measurements at both synchrotrons and X-ray free electron lasers (XFELs). Our design includes highly X-ray-transparent enclosing thin film layers tuned to minimize scatter background, adaptable sample flow layers tuned to match crystal size, and a large sample area compatible with both raster scanning and rotation based serial data collection. The optically transparent chips can be used both for in situ protein crystallization (to eliminate crystal handling) or crystal slurry loading, with prepared samples stable for weeks in a humidified environment and for several hours in ambient conditions. Serial oscillation crystallography, using a multi-crystal rotational data collection approach, at a microfocus synchrotron beamline (SSRL, beamline 12-1) was used to benchmark the performance of the chips. High-resolution structures (1.3-2.7 A) were collected from five different proteins - hen egg white lysozyme, thaumatin, bovine liver catalase, concanavalin-A (type VI), and SARS-CoV-2 nonstructural protein NSP5. Overall, our modular fabrication approach enables precise control over the cross-section of materials in the X-ray beam path and facilitates chip adaption to different sample and beamline requirements for user-friendly, straightforward diffraction measurements at room temperature.

The combination of oscillation data collection with fixed-target microchips for regular crystal dispersion is an efficient method for collecting serial crystallography data at synchrotrons. Background scatter from noncrystal substrates is especially minimized., A fixed-target approach to high-throughput room-temperature serial synchrotron crystallography with oscillation is described. Patterned silicon chips with microwells provide high crystal-loading density with an extremely high hit rate. The microfocus, undulator-fed beamline at CHESS, which has compound refractive optics and a fast-framing detector, was built and optimized for this experiment. The high-throughput oscillation method described here collects 1–5° of data per crystal at room temperature with fast (10° s−1) oscillation rates and translation times, giving a crystal-data collection rate of 2.5 Hz. Partial datasets collected by the oscillation method at a storage-ring source provide more complete data per crystal than still images, dramatically lowering the total number of crystals needed for a complete dataset suitable for structure solution and refinement – up to two orders of magnitude fewer being required. Thus, this method is particularly well suited to instances where crystal quantities are low. It is demonstrated, through comparison of first and last oscillation images of two systems, that dose and the effects of radiation damage can be minimized through fast rotation and low angular sweeps for each crystal.

In recent years, the success of serial femtosecond crystallography and the paucity of beamtime at X-ray free-electron lasers have motivated the development of serial microcrystallography experiments at storage-ring synchrotron sources. However, especially at storage-ring sources, if a crystal is too small it will have suffered significant radiation damage before diffracting a sufficient number of X-rays into Bragg peaks for peak-indexing software to determine the crystal orientation. As a consequence, the data frames of small crystals often cannot be indexed and are discarded. Introduced here is a method based on the expand–maximize–compress (EMC) algorithm to solve protein structures, specifically from data frames for which indexing methods fail because too few X-rays are diffracted into Bragg peaks. The method is demonstrated on a real serial microcrystallography data set whose signals are too weak to be indexed by conventional methods. In spite of the daunting background scatter from the sample-delivery medium, it was still possible to solve the protein structure at 2.1 Å resolution. The ability of the EMC algorithm to analyze weak data frames will help to reduce sample consumption. It will also allow serial microcrystallography to be performed with crystals that are otherwise too small to be feasibly analyzed at storage-ring sources.

To simulate the signal level of serial microcrystallography experiments at storage ring sources, data frames were collected from a large lysozyme crystal rotated about two orthogonal axes and illuminated by a dim X-ray source. Using the EMC algorithm, this study shows that three-dimensional intensity reconstruction is still feasible even without the knowledge of the crystal orientation in each data frame., Recently, there has been a growing interest in adapting serial microcrystallography (SMX) experiments to existing storage ring (SR) sources. For very small crystals, however, radiation damage occurs before sufficient numbers of photons are diffracted to determine the orientation of the crystal. The challenge is to merge data from a large number of such ‘sparse’ frames in order to measure the full reciprocal space intensity. To simulate sparse frames, a dataset was collected from a large lysozyme crystal illuminated by a dim X-ray source. The crystal was continuously rotated about two orthogonal axes to sample a subset of the rotation space. With the EMC algorithm [expand–maximize–compress; Loh & Elser (2009). Phys. Rev. E, 80, 026705], it is shown that the diffracted intensity of the crystal can still be reconstructed even without knowledge of the orientation of the crystal in any sparse frame. Moreover, parallel computation implementations were designed to considerably improve the time and memory scaling of the algorithm. The results show that EMC-based SMX experiments should be feasible at SR sources.

The practice of serial X-ray crystallography (SX) depends on efficient, continuous delivery of hydrated protein crystals while minimizing background scattering. Of the two major types of sample delivery devices, fixed-target devices offer several advantages over widely adopted jet injectors, including: lower sample consumption, clog-free delivery, and the ability to control on-chip crystal density to improve hit rates. Here we present our development of versatile, inexpensive, and robust polymer microfluidic chips for routine and reliable room temperature serial measurements at both synchrotrons and X-ray free electron lasers (XFELs). Our design includes highly X-ray-transparent enclosing thin film layers tuned to minimize scatter background, adaptable sample flow layers tuned to match crystal size, and a large sample area compatible with both raster scanning and rotation based serial data collection. The optically transparent chips can be used both for in situ protein crystallization (to eliminate crystal handling) or crystal slurry loading, with prepared samples stable for weeks in a humidified environment and for several hours in ambient conditions. Serial oscillation crystallography, using a multi-crystal rotational data collection approach, at a microfocus synchrotron beamline (SSRL, beamline 12-1) was used to benchmark the performance of the chips. High-resolution structures (1.3-2.7 A) were collected from five different proteins - hen egg white lysozyme, thaumatin, bovine liver catalase, concanavalin-A (type VI), and SARS-CoV-2 nonstructural protein NSP5. Overall, our modular fabrication approach enables precise control over the cross-section of materials in the X-ray beam path and facilitates chip adaption to different sample and beamline requirements for user-friendly, straightforward diffraction measurements at room temperature.

Using the EMC algorithm, the three-dimensional intensity was successfully reconstructed from millions of non-oriented, sparse data frames collected from a hen egg-white lysozyme crystal rotating about a single axis. The protein structure was solved from the reconstructed intensity. This result is encouraging for the development of synchrotron-based serial microcrystallography., X-ray free-electron lasers (XFELs) have inspired the development of serial femtosecond crystallography (SFX) as a method to solve the structure of proteins. SFX datasets are collected from a sequence of protein microcrystals injected across ultrashort X-ray pulses. The idea behind SFX is that diffraction from the intense, ultrashort X-ray pulses leaves the crystal before the crystal is obliterated by the effects of the X-ray pulse. The success of SFX at XFELs has catalyzed interest in analogous experiments at synchrotron-radiation (SR) sources, where data are collected from many small crystals and the ultrashort pulses are replaced by exposure times that are kept short enough to avoid significant crystal damage. The diffraction signal from each short exposure is so ‘sparse’ in recorded photons that the process of recording the crystal intensity is itself a reconstruction problem. Using the EMC algorithm, a successful reconstruction is demonstrated here in a sparsity regime where there are no Bragg peaks that conventionally would serve to determine the orientation of the crystal in each exposure. In this proof-of-principle experiment, a hen egg-white lysozyme (HEWL) crystal rotating about a single axis was illuminated by an X-ray beam from an X-ray generator to simulate the diffraction patterns of microcrystals from synchrotron radiation. Millions of these sparse frames, typically containing only ∼200 photons per frame, were recorded using a fast-framing detector. It is shown that reconstruction of three-dimensional diffraction intensity is possible using the EMC algorithm, even with these extremely sparse frames and without knowledge of the rotation angle. Further, the reconstructed intensity can be phased and refined to solve the protein structure using traditional crystallographic software. This suggests that synchrotron-based serial crystallography of micrometre-sized crystals can be practical with the aid of the EMC algorithm even in cases where the data are sparse.

The overall signal-to-noise ratio per unit dose for X-ray diffraction data from protein crystals can be improved by reducing the mass and density of all material surrounding the crystals. This article demonstrates a path towards the practical ultimate in background reduction by use of atomically thin graphene sheets as a crystal mounting platform for protein crystals. The results show the potential for graphene in protein crystallography and other cases where X-ray scatter from the mounting material must be reduced and specimen dehydration prevented, such as in coherent X-ray diffraction imaging of microscopic objects.

A figure in the article by Lan, Wierman, Tate, Philipp, Elser & Gruner [J. Appl. Cryst. (2017), 50, 985–993] is corrected.

High-pressure cryocooling has been developed as an alternative method for cryopreservation of macromolecular crystals and successfully applied for various technical and scientific studies. The method requires the preservation of crystal hydration as the crystal is pressurized with dry helium gas. Previously, crystal hydration was maintained either by coating crystals with a mineral oil or by enclosing crystals in a capillary which was filled with crystallization mother liquor. These methods are not well suited to weakly diffracting crystals because of the relatively high background scattering from the hydrating materials. Here, an alternative method of crystal hydration, called capillary shielding, is described. The specimen is kept hydratedviavapor diffusion in a shielding capillary while it is being pressure cryocooled. After cryocooling, the shielding capillary is removed to reduce background X-ray scattering. It is shown that, compared to previous crystal-hydration methods, the new hydration method produces superior crystal diffraction with little sign of crystal damage. Using the new method, a weakly diffracting protein crystal may be properly pressure cryocooled with little or no addition of external cryoprotectants, and significantly reduced background scattering can be observed from the resulting sample. Beyond the applications for macromolecular crystallography, it is shown that the method has great potential for the preparation of noncrystalline hydrated biological samples for coherent diffraction imaging with future X-ray sources.