A streamlined approach to structure elucidation using in cellulo crystallized recombinant proteins, InCellCryst

Robert Schönherr, Juliane Boger, J. Mia Lahey-Rudolph, Mareike Harms, Jacqueline Kaiser, Sophie Nachtschatt, Marla Wobbe, Rainer Duden, Peter König, Gleb Bourenkov, Thomas R. Schneider and Lars Redecke ()
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Robert Schönherr: University of Lübeck
Juliane Boger: University of Lübeck
J. Mia Lahey-Rudolph: University of Lübeck
Mareike Harms: University of Lübeck
Jacqueline Kaiser: University of Lübeck
Sophie Nachtschatt: University of Lübeck
Marla Wobbe: University of Lübeck
Rainer Duden: University of Lübeck
Peter König: University of Lübeck
Gleb Bourenkov: European Molecular Biology Laboratory, Hamburg Unit c/o Deutsches Elektronen-Synchrotron DESY
Thomas R. Schneider: European Molecular Biology Laboratory, Hamburg Unit c/o Deutsches Elektronen-Synchrotron DESY
Lars Redecke: University of Lübeck

Nature Communications, 2024, vol. 15, issue 1, 1-17

Abstract: Abstract With the advent of serial X-ray crystallography on microfocus beamlines at free-electron laser and synchrotron facilities, the demand for protein microcrystals has significantly risen in recent years. However, by in vitro crystallization extensive efforts are usually required to purify proteins and produce sufficiently homogeneous microcrystals. Here, we present InCellCryst, an advanced pipeline for producing homogeneous microcrystals directly within living insect cells. Our baculovirus-based cloning system enables the production of crystals from completely native proteins as well as the screening of different cellular compartments to maximize chances for protein crystallization. By optimizing cloning procedures, recombinant virus production, crystallization and crystal detection, X-ray diffraction data can be collected 24 days after the start of target gene cloning. Furthermore, improved strategies for serial synchrotron diffraction data collection directly from crystals within living cells abolish the need to purify the recombinant protein or the associated microcrystals.

Date: 2024
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DOI: 10.1038/s41467-024-45985-7

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