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- Title
A streamlined approach to structure elucidation using in cellulo crystallized recombinant proteins, InCellCryst.
- Authors
Schönherr, Robert; Boger, Juliane; Lahey-Rudolph, J. Mia; Harms, Mareike; Kaiser, Jacqueline; Nachtschatt, Sophie; Wobbe, Marla; Duden, Rainer; König, Peter; Bourenkov, Gleb; Schneider, Thomas R.; Redecke, Lars
- 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. SX experiments require thousands of high-quality microcrystals. Here, authors present a pipeline for protein crystallization in insect cells, including compartment screening and identification of physiological ligands from data collection in cellulo.
- Subjects
RECOMBINANT proteins; MOLECULAR cloning; X-ray crystallography; FREE electron lasers; RECOMBINANT viruses; X-ray diffraction; SYNCHROTRONS
- Publication
Nature Communications, 2024, Vol 15, Issue 1, p1
- ISSN
2041-1723
- Publication type
Article
- DOI
10.1038/s41467-024-45985-7