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.
Highlights
Large structural genomics consortia and platforms have pushed the development of high-throughput pipelines for the structure determination of biological macromolecules
Data from structural biology consortia show that the production of diffraction-quality crystals remains the main bottleneck in X-ray crystallography
Using in vivo crystals of the Bombyx mori CPV1 (CPV1) polyhedrin as a model system, we show that de novo structure determination can be carried out at a resolution of 1.5 Ain $8 days from expression to refinement using data measured on a standard microfocus beamline
Summary
Large structural genomics consortia and platforms have pushed the development of high-throughput pipelines for the structure determination of biological macromolecules. Miniaturization and automation of crystallogenesis has been adopted by most medium-to-large research centres, greatly accelerating the typical structural biology workflow (Abola et al, 2000) Despite these advances, data from structural biology consortia show that the production of diffraction-quality crystals remains the main bottleneck in X-ray crystallography. D72, 576–585 research papers molecular framework to investigate how these proteins spontaneously crystallize in the complex environment of the cells in which they are expressed (Chiu et al, 2012) Such naturally occurring crystals are relatively common and are found in organisms spanning all kingdoms of life (Doye & Poon, 2006). Using in vivo crystals of the Bombyx mori CPV1 (CPV1) polyhedrin as a model system, we show that de novo structure determination can be carried out at a resolution of 1.5 Ain $8 days from expression to refinement using data measured on a standard microfocus beamline
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