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Fixed target combined with spectral mapping: approaching 100% hit rates for serial crystallography.

ABSTRACT: The advent of ultrafast highly brilliant coherent X-ray free-electron laser sources has driven the development of novel structure-determination approaches for proteins, and promises visualization of protein dynamics on sub-picosecond timescales with full atomic resolution. Significant efforts are being applied to the development of sample-delivery systems that allow these unique sources to be most efficiently exploited for high-throughput serial femtosecond crystallography. Here, the next iteration of a fixed-target crystallography chip designed for rapid and reliable delivery of up to 11?259 protein crystals with high spatial precision is presented. An experimental scheme for predetermining the positions of crystals in the chip by means of in situ spectroscopy using a fiducial system for rapid, precise alignment and registration of the crystal positions is presented. This delivers unprecedented performance in serial crystallography experiments at room temperature under atmospheric pressure, giving a raw hit rate approaching 100% with an effective indexing rate of approximately 50%, increasing the efficiency of beam usage and allowing the method to be applied to systems where the number of crystals is limited.

SUBMITTER: Oghbaey S

PROVIDER: S-EPMC5937680 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Fixed target combined with spectral mapping: approaching 100% hit rates for serial crystallography.

Oghbaey Saeed S Sarracini Antoine A Ginn Helen M HM Pare-Labrosse Olivier O Kuo Anling A Marx Alexander A Epp Sascha W SW Sherrell Darren A DA Eger Bryan T BT Zhong Yinpeng Y Loch Rolf R Mariani Valerio V Alonso-Mori Roberto R Nelson Silke S Lemke Henrik T HT Owen Robin L RL Pearson Arwen R AR Stuart David I DI Ernst Oliver P OP Mueller-Werkmeister Henrike M HM Miller R J Dwayne RJ

Acta crystallographica. Section D, Structural biology 20160727 Pt 8

The advent of ultrafast highly brilliant coherent X-ray free-electron laser sources has driven the development of novel structure-determination approaches for proteins, and promises visualization of protein dynamics on sub-picosecond timescales with full atomic resolution. Significant efforts are being applied to the development of sample-delivery systems that allow these unique sources to be most efficiently exploited for high-throughput serial femtosecond crystallography. Here, the next iterat ...[more]

PMID: 27487825

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Project description: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.

Dataset Information

Fixed target combined with spectral mapping: approaching 100% hit rates for serial crystallography.

Publications

Fixed target combined with spectral mapping: approaching 100% hit rates for serial crystallography.

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