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

Marion Boudes, Damia Garriga, Andrew Fryga, Tom Caradoc-Davies, Fasseli Coulibaly

Research output: Contribution to journalArticleResearchpeer-review

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 nearcomplete 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.

Original languageEnglish
Pages (from-to)576-585
Number of pages10
JournalActa Crystallographica Section D: Structural Biology
Volume72
Issue number4
DOIs
Publication statusPublished - 2016

Keywords

  • In cellulo diffraction
  • In vivo crystallization
  • Microcrystallography
  • Polyhedrin

Cite this

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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 {\AA} 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 nearcomplete model was autobuilt from 2.7 {\AA} 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.",
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A pipeline for structure determination of in vivo-grown crystals using in cellulo diffraction. / Boudes, Marion; Garriga, Damia; Fryga, Andrew; Caradoc-Davies, Tom; Coulibaly, Fasseli.

In: Acta Crystallographica Section D: Structural Biology, Vol. 72, No. 4, 2016, p. 576-585.

Research output: Contribution to journalArticleResearchpeer-review

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