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Best practices for high data-rate macromolecular crystallography (HDRMX).

ABSTRACT: In macromolecular crystallography, higher flux, smaller beams, and faster detectors open the door to experiments with very large numbers of very small samples that can reveal polymorphs and dynamics but require re-engineering of approaches to the clustering of images both at synchrotrons and XFELs (X-ray free electron lasers). The need for the management of orders of magnitude more images and limitations of file systems favor a transition from simple one-file-per-image systems such as CBF to image container systems such as HDF5. This further increases the load on computers and networks and requires a re-examination of the presentation of metadata. In this paper, we discuss three important components of this problem-improved approaches to the clustering of images to better support experiments on polymorphs and dynamics, recent and upcoming changes in metadata for Eiger images, and software to rapidly validate images in the revised Eiger format.

SUBMITTER: Bernstein HJ

PROVIDER: S-EPMC6952294 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Best practices for high data-rate macromolecular crystallography (HDRMX).

Bernstein Herbert J HJ Andrews Lawrence C LC Diaz Jorge A JA Jakoncic Jean J Nguyen Thu T Sauter Nicholas K NK Soares Alexei S AS Wei Justin Y JY Wlodek Maciej R MR Xerri Mario A MA

Structural dynamics (Melville, N.Y.) 20200109 1

In macromolecular crystallography, higher flux, smaller beams, and faster detectors open the door to experiments with very large numbers of very small samples that can reveal polymorphs and dynamics but require re-engineering of approaches to the clustering of images both at synchrotrons and XFELs (X-ray free electron lasers). The need for the management of orders of magnitude more images and limitations of file systems favor a transition from simple one-file-per-image systems such as CBF to ima ...[more]

PMID: 31934601

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Project description:This article describes the High-Pressure Freezing Laboratory for Macromolecular Crystallography (HPMX) at the ESRF, and highlights new and complementary research opportunities that can be explored using this facility. The laboratory is dedicated to investigating interactions between macromolecules and gases in crystallo, and finds applications in many fields of research, including fundamental biology, biochemistry, and environmental and medical science. At present, the HPMX laboratory offers the use of different high-pressure cells adapted for helium, argon, krypton, xenon, nitrogen, oxygen, carbon dioxide and methane. Important scientific applications of high pressure to macromolecules at the HPMX include noble-gas derivatization of crystals to detect and map the internal architecture of proteins (pockets, tunnels and channels) that allows the storage and diffusion of ligands or substrates/products, the investigation of the catalytic mechanisms of gas-employing enzymes (using oxygen, carbon dioxide or methane as substrates) to possibly decipher intermediates, and studies of the conformational fluctuations or structure modifications that are necessary for proteins to function. Additionally, cryo-cooling protein crystals under high pressure (helium or argon at 2000 bar) enables the addition of cryo-protectant to be avoided and noble gases can be employed to produce derivatives for structure resolution. The high-pressure systems are designed to process crystals along a well defined pathway in the phase diagram (pressure-temperature) of the gas to cryo-cool the samples according to the three-step `soak-and-freeze method'. Firstly, crystals are soaked in a pressurized pure gas atmosphere (at 294 K) to introduce the gas and facilitate its interactions within the macromolecules. Samples are then flash-cooled (at 100 K) while still under pressure to cryo-trap macromolecule-gas complexation states or pressure-induced protein modifications. Finally, the samples are recovered after depressurization at cryo-temperatures. The final section of this publication presents a selection of different typical high-pressure experiments carried out at the HPMX, showing that this technique has already answered a wide range of scientific questions. It is shown that the use of different gases and pressure conditions can be used to probe various effects, such as mapping the functional internal architectures of enzymes (tunnels in the haloalkane dehalogenase DhaA) and allosteric sites on membrane-protein surfaces, the interaction of non-inert gases with proteins (oxygen in the hydrogenase ReMBH) and pressure-induced structural changes of proteins (tetramer dissociation in urate oxidase). The technique is versatile and the provision of pressure cells and their application at the HPMX is gradually being extended to address new scientific questions.

Dataset Information

Best practices for high data-rate macromolecular crystallography (HDRMX).

Publications

Best practices for high data-rate macromolecular crystallography (HDRMX).

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