A streamlined approach to structure elucidation using in cellulo crystallized recombinant proteins, InCellCryst.
Journal
Nature communications
ISSN: 2041-1723
Titre abrégé: Nat Commun
Pays: England
ID NLM: 101528555
Informations de publication
Date de publication:
24 Feb 2024
24 Feb 2024
Historique:
received:
02
06
2023
accepted:
02
02
2024
medline:
25
2
2024
pubmed:
25
2
2024
entrez:
24
2
2024
Statut:
epublish
Résumé
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.
Identifiants
pubmed: 38402242
doi: 10.1038/s41467-024-45985-7
pii: 10.1038/s41467-024-45985-7
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
1709Subventions
Organisme : Bundesministerium für Bildung und Forschung (Federal Ministry of Education and Research)
ID : 05K18FLA
Organisme : Deutsche Forschungsgemeinschaft (German Research Foundation)
ID : EXC 306
Informations de copyright
© 2024. The Author(s).
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