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Abstract: Efficient sample delivery is an essential aspect of serial crystallography at both synchrotrons and X-ray free-electron lasers. Rastering fixed target chips through the X-ray beam is an efficient method for serial delivery from the perspectives of both sample consumption and beam time usage. Here, an approach for loading fixed targets using acoustic drop ejection is presented that does not compromise crystal quality, can reduce sample consumption by more than an order of magnitude and allows serial diffraction to be collected from a larger proportion of the crystals in the slurry.

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Abstract: An approach is demonstrated to obtain, in a sample- and time-efficient manner, multiple dose-resolved crystal structures from room-temperature protein microcrystals using identical fixed-target supports at both synchrotrons and X-ray free-electron lasers (XFELs). This approach allows direct comparison of dose-resolved serial synchrotron and damage-free XFEL serial femtosecond crystallography structures of radiation-sensitive proteins. Specifically, serial synchrotron structures of a heme peroxidase enzyme reveal that X-ray induced changes occur at far lower doses than those at which diffraction quality is compromised (the Garman limit), consistent with previous studies on the reduction of heme proteins by low X-ray doses. In these structures, a functionally relevant bond length is shown to vary rapidly as a function of absorbed dose, with all room-temperature synchrotron structures exhibiting linear deformation of the active site compared with the XFEL structure. It is demonstrated that extrapolation of dose-dependent synchrotron structures to zero dose can closely approximate the damage-free XFEL structure. This approach is widely applicable to any protein where the crystal structure is altered by the synchrotron X-ray beam and provides a solution to the urgent requirement to determine intact structures of such proteins in a high-throughput and accessible manner.

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Abstract: The tangential curvature of actively bent X-ray mirrors at synchrotron radiation and X-ray free-electron laser (XFEL) facilities is typically only changed every few hours or even days. This operation can take tens of minutes for active optics with multiple bending actuators and often requires expert guidance using in situ monitoring devices. Hence, the dynamic performance of active X-ray optics for synchrotron beamlines has historically not been exploited. This is in stark contrast to many other scientific fields. However, many areas of synchrotron radiation and XFEL science, including macromolecular crystallography, could greatly benefit from the ability to change the size and shape of the X-ray beam rapidly and continuously. The advantages of this innovative approach are twofold: a large reduction in the dead time required to change the size of the X-ray beam for different-sized samples and the possibility of making multiple changes to the beam during the measurement of a single sample. In the preceding paper [Part I; Alcock, Nistea, Signorato & Sawhney (2019[Alcock, S. G., Nistea, I.-T., Signorato, R. & Sawhney, K. (2019). J. Synchrotron Rad. 26, this issue.]), J. Synchrotron Rad. 26, this issue], which accompanies this article, high-speed visible-light Fizeau interferometry was used to identify the factors which influence the dynamic bending behaviour of piezoelectric bimorph deformable X-ray mirrors. Building upon this ex situ metrology study, provided here is the first synchrotron radiation beamline implementation of high-speed adaptive X-ray optics using two bimorphs operating as a Kirkpatrick–Baez pair. With optimized substrates, novel opto-mechanical holders and a next-generation high-voltage power supply, the size of an X-ray beam was rapidly and repeatedly switched in <10 s. Of equal importance, it is also shown that compensation of piezoelectric creep ensures that the X-ray beam size remains stable for more than 1 h after making a major change. The era of high-speed adaptive X-ray optics for synchrotron radiation and XFEL beamlines has begun.

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Abstract: Crystallography chips are fixed-target supports consisting of a film (for example Kapton) or wafer (for example silicon) that is processed using semiconductor-microfabrication techniques to yield an array of wells or through-holes in which single microcrystals can be lodged for raster-scan probing. Although relatively expensive to fabricate, chips offer an efficient means of high-throughput sample presentation for serial diffraction data collection at synchrotron or X-ray free-electron laser (XFEL) sources. Truly efficient loading of a chip (one microcrystal per well and no wastage during loading) is nonetheless challenging. The wells or holes must match the microcrystal size of interest, requiring that a large stock of chips be maintained. Raster scanning requires special mechanical drives to step the chip rapidly and with micrometre precision from well to well. Here, a `chip-less' adaptation is described that essentially eliminates the challenges of loading and precision scanning, albeit with increased, yet still relatively frugal, sample usage. The device consists simply of two sheets of Mylar with the crystal solution sandwiched between them. This sheet-on-sheet (SOS) sandwich structure has been employed for serial femtosecond crystallography data collection with micrometre-sized crystals at an XFEL. The approach is also well suited to time-resolved pump–probe experiments, in particular for long time delays. The SOS sandwich enables measurements under XFEL beam conditions that would damage conventional chips, as documented here. The SOS sheets hermetically seal the sample, avoiding desiccation of the sample provided that the X-ray beam does not puncture the sheets. This is the case with a synchrotron beam but not with an XFEL beam. In the latter case, desiccation, setting radially outwards from each punched hole, sets lower limits on the speed and line spacing of the raster scan. It is shown that these constraints are easily accommodated.

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Abstract: Lens epithelium-derived growth factor (LEDGF)/p75 is the dominant binding partner of HIV-1 integrase in human cells. The crystal structure of the HIV integrase-binding domain (IBD) of LEDGF has been determined in the absence of ligand. IBD was overexpressed in Escherichia coli, purified and crystallized by sitting-drop vapour diffusion. X-ray diffraction data were collected at Diamond Light Source to a resolution of 2.05 Å. The crystals belonged to space group P21, with eight polypeptide chains in the asymmetric unit arranged as an unusual octamer composed of four domain-swapped IBD dimers. IBD exists as a mixture of monomers and dimers in concentrated solutions, but the dimers are unlikely to be biologically relevant.