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A pipeline for structure determination of in vivo-grown crystals using in cellulo diffraction.

ABSTRACT: While structure determination from micrometre-sized crystals used to represent a challenge, serial X-ray crystallography on microfocus beamlines at synchrotron and free-electron laser facilities greatly facilitates this process today for microcrystals and nanocrystals. In addition to typical microcrystals of purified recombinant protein, these advances have enabled the analysis of microcrystals produced inside living cells. Here, a pipeline where crystals are grown in insect cells, sorted by flow cytometry and directly analysed by X-ray diffraction is presented and applied to in vivo-grown crystals of the recombinant CPV1 polyhedrin. When compared with the analysis of purified crystals, in cellulo diffraction produces data of better quality and a gain of ?0.35 Å in resolution for comparable beamtime usage. Importantly, crystals within cells are readily derivatized with gold and iodine compounds through the cellular membrane. Using the multiple isomorphous replacement method, a near-complete model was autobuilt from 2.7 Å resolution data. Thus, in favourable cases, an in cellulo pipeline can replace the complete workflow of structure determination without compromising the quality of the resulting model. In addition to its efficiency, this approach maintains the protein in a cellular context throughout the analysis, which reduces the risk of disrupting transient or labile interactions in protein-protein or protein-ligand complexes.

SUBMITTER: Boudes M

PROVIDER: S-EPMC4822565 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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A pipeline for structure determination of in vivo-grown crystals using in cellulo diffraction.

Boudes Marion M Garriga Damià D Fryga Andrew A Caradoc-Davies Tom T Coulibaly Fasséli F

Acta crystallographica. Section D, Structural biology 20160330 Pt 4

While structure determination from micrometre-sized crystals used to represent a challenge, serial X-ray crystallography on microfocus beamlines at synchrotron and free-electron laser facilities greatly facilitates this process today for microcrystals and nanocrystals. In addition to typical microcrystals of purified recombinant protein, these advances have enabled the analysis of microcrystals produced inside living cells. Here, a pipeline where crystals are grown in insect cells, sorted by flo ...[more]

PMID: 27050136

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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

A pipeline for structure determination of in vivo-grown crystals using in cellulo diffraction.

Publications

A pipeline for structure determination of in vivo-grown crystals using in cellulo diffraction.

Similar Datasets

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