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Abstract: X-ray diffraction (XRD) of microcrystals is signal-to-noise limited by the inherently weak diffraction. As such, Electron diffraction (ED) is increasingly used to measure diffraction data from submicron crystals, or those deemed too small for XRD due the stronger interaction of electrons with matter. However, many samples which are too thin for XRD are often too thick for ED using the currently available electron beam energies (<300 keV) and hence require thinning by focussed ion beam milling (FIB) which adds additional sample preparation steps. In addition to determining structures from nanocrystals, ED provides Coulomb potential data which are complementary to that obtained with XRD. As such ED data may be necessary to answer particular scientific questions. The macromolecular crystallography beamline, VMXm, at Diamond Light Source, has been optimised for maximising the S:N in XRD experiments with a variable focus high-energy (>20 KeV) X-ray beam, with in-vacuum endstation and the use of low background cryoTEM grids for crystal mounting [1], [2]. This has allowed VMXm to collect high-resolution rotation data from single crystals measuring ∼1.2 μm which were only previously tractable using an X-ray Free Electron Laser [3]. This has pushed the amenable sample envelope at synchrotrons to new dimensions and perhaps near to the practical limit of XRD. Indeed, simulations have predicted the limit to be ∼0.5 μm thick in the case of lysozyme, assuming photoelectron escape [4]. This XRD beamline opens up the possibilities to directly compare XRD and ED datasets and understand the complementarity of these experiments. In this work we present data from cubic human insulin crystals that have been thinned by FIB milling from ∼10 μm to various submicron thicknesses. 200 kV ED data were then collected from these lamellae before XRD data were measured from the same lamellae using VMXm. It was possible to obtain a complete XRD dataset to 2.45 Å using a 1.68 μm3 illuminated volume and a 2.04 Å ED dataset from the same 0.25 μm lamella. We have demonstrated that the data quality is comparable between ED and VMXm from the same crystal, while giving an opportunity to directly compare X-ray and electron derived maps. This includes the comparison of the radiation damage each experiment imparts on the sample [5] as well as the information content [6]. This work indicates that the usable sample envelope for synchrotron X-rays extends to much thinner samples than had been previously thought. It is also the first demonstration of ED and XRD measured from the same crystal volume enabling direct comparison of X-ray and electron derived data. Ultimately, the work will inform the design and use of high energy (MeV) ED instruments such as HeXI and how those can be complemented by XRD derived information from beamlines such as VMXm.

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Abstract: X-ray diffraction (XRD) of microcrystals is signal-to-noise limited by due to the inherently weak diffraction. Therefore, it is key that the beamline instrumentation and the sample itself introduce minimal noise. The VMXm beamline, at Diamond Light Source, has been optimised for maximising the S:N in experiments with a variable focus high-energy (>20 KeV) X-ray beam, with in-vacuum endstation and the use of low background cryoTEM grids for crystal mounting [1], [2]. This has allowed VMXm to collect high-resolution rotation data from single crystals measuring ~1.2 μm which were only previously tractable using an X-ray Free Electron Laser [3]. This has pushed the amenable sample envelope at synchrotrons to new dimensions and perhaps near to the practical limit of XRD. Indeed, simulations have predicted the limit to be ~0.5 μm thick in the case of lysozyme, assuming photoelectron escape [4]. Electron diffraction (ED) is frequently used to measure diffraction data from submicron crystals. Many samples which are too thin for XRD are often too thick for ED using the currently available electron beam energies (<300 keV) and hence require thinning by focussed ion beam milling (FIB). In addition to determining structures from nanocrystals, ED provides Coulomb potential data which are complementary to that obtained with XRD. As such ED data may be necessary to answer particular scientific questions. In this work we present data from cubic human insulin crystals that have been thinned by FIB milling from ~10 μm to various submicron thicknesses. 200 kV ED data were then collected from these lamellae before XRD data were measured from the same lamellae using VMXm. It was possible to obtain a complete XRD dataset to 2.45 Å using a 1.68 μm3 illuminated volume and a 2.04 Å ED dataset from the same 0.25 μm lamella. We have demonstrated that the data quality is comparable between ED and VMXm from the same crystal, while giving an opportunity to directly compare X-ray and electron derived maps. This includes the comparison of the radiation damage each experiment imparts on the sample [5] as well as the information content [6]. This work indicates that the usable sample envelope for synchrotron X-rays extends to much thinner samples than had been previously thought. It is also the first demonstration of ED and XRD measured from the same crystal volume enabling direct comparison of X-ray and electron derived data. Ultimately, the work will inform the design and use of high energy (MeV) ED instruments such as HeXI and how those can be complemented by XRD derived information from beamlines such as VMXm.

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Abstract: VMXm joins the suite of operational macromolecular crystallography beamlines at Diamond Light Source. It has been designed to optimize rotation data collections from protein crystals less than 10 µm and down to below 1 µm in size. The beamline has a fully focused beam of 0.3 × 2.3 µm (vertical × horizontal) with a tuneable energy range (6–28 keV) and high flux (1.6 × 1012 photons s−1 at 12.5 keV). The crystals are housed within a vacuum chamber to minimize background scatter from air. Crystals are plunge-cooled on cryo-electron microscopy grids, allowing much of the liquid surrounding the crystals to be removed. These factors improve the signal-to-noise during data collection and the lifetime of the microcrystals can be prolonged by exploiting photoelectron escape. A novel in vacuo sample environment has been designed which also houses a scanning electron microscope to aid with sample visualization. This combination of features at VMXm allows measurements at the physical limits of X-ray crystallography on biomacromolecules to be explored and exploited.

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Abstract: This chapter describes additions to the DIALS software package for processing serial still-shot crystallographic data, and the implementation of a pipeline, xia2.ssx, for processing and merging serial crystallography data using DIALS programs. To integrate partial still-shot diffraction data, a 3D gaussian profile model was developed that can describe anisotropic spot shapes. This model is optimised by maximum likelihood methods using the pixel-intensity distributions of strong diffraction spots, enabling simultaneous refinement of the profile model and Ewald-sphere offsets. We demonstrate the processing of an example SSX dataset where the improved partiality estimates lead to better model statistics compared with post-refined isotropic models. We also demonstrate some of the workflows available for merging SSX data, including processing time/dose resolved data series, where data can be separated at the point of merging after scaling and discuss the program outputs used to investigate the data throughout the pipeline.

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Abstract: rocessing of single-crystal X-ray diffraction data from area detectors can be separated into two steps. First, raw intensities are obtained by integration of the diffraction images, and then data correction and reduction are performed to determine structure-factor amplitudes and their uncertainties. The second step considers the diffraction geometry, sample illumination, decay, absorption and other effects. While absorption is only a minor effect in standard macromolecular crystallography (MX), it can become the largest source of uncertainty for experiments performed at long wavelengths. Current software packages for MX typically employ empirical models to correct for the effects of absorption, with the corrections determined through the procedure of minimizing the differences in intensities between symmetry-equivalent reflections; these models are well suited to capturing smoothly varying experimental effects. However, for very long wavelengths, empirical methods become an unreliable approach to model strong absorption effects with high fidelity. This problem is particularly acute when data multiplicity is low. This paper presents an analytical absorption correction strategy (implemented in new software AnACor) based on a volumetric model of the sample derived from X-ray tomography. Individual path lengths through the different sample materials for all reflections are determined by a ray-tracing method. Several approaches for absorption corrections (spherical harmonics correction, analytical absorption correction and a combination of the two) are compared for two samples, the membrane protein OmpK36 GD, measured at a wavelength of λ = 3.54 Å, and chlorite dismutase, measured at λ = 4.13 Å. Data set statistics, the peak heights in the anomalous difference Fourier maps and the success of experimental phasing are used to compare the results from the different absorption correction approaches. The strategies using the new analytical absorption correction are shown to be superior to the standard spherical harmonics corrections. While the improvements are modest in the 3.54 Å data, the analytical absorption correction outperforms spherical harmonics in the longer-wavelength data (λ = 4.13 Å), which is also reflected in the reduced amount of data being required for successful experimental phasing.