Journal Article

PUBDB-2024-01564

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png A streamlined approach to structure elucidation using in cellulo crystallized recombinant proteins, InCellCryst

Schönherr, R. ; Boger, J. ; Lahey-Rudolph, J. M. ; Harms, M. ; Kaiser, J. ; Nachtschatt, S. ; Wobbe, M. ; Duden, R. ; König, P. ; Bourenkov, G.XFEL.EU*EMBL* ; Schneider, T. R.EMBL* ; Redecke, L. (Corresponding author)DESY*

2024
Nature Publishing Group UK [London]

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Please use a persistent id in citations: doi:10.1038/s41467-024-45985-7  doi:10.3204/PUBDB-2024-01564

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.

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Contributing Institute(s):

1. EMBL-User (EMBL-User)
2. EMBL (EMBL)
3. Universität zu Lübeck (U Lübeck)

Research Program(s):

1. 633 - Life Sciences – Building Blocks of Life: Structure and Function (POF4-633) (POF4-633)
2. 6G3 - PETRA III (DESY) (POF4-6G3) (POF4-6G3)

Experiment(s):

1. PETRA Beamline P14 (PETRA III)
2. PETRA Beamline P12 (PETRA III)

Appears in the scientific report 2024

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