Quantifying the heterogeneity of macromolecular machines by mass photometry.
Journal
Nature communications
ISSN: 2041-1723
Titre abrégé: Nat Commun
Pays: England
ID NLM: 101528555
Informations de publication
Date de publication:
14 04 2020
14 04 2020
Historique:
received:
20
01
2020
accepted:
12
03
2020
entrez:
15
4
2020
pubmed:
15
4
2020
medline:
25
7
2020
Statut:
epublish
Résumé
Sample purity is central to in vitro studies of protein function and regulation, and to the efficiency and success of structural studies using techniques such as x-ray crystallography and cryo-electron microscopy (cryo-EM). Here, we show that mass photometry (MP) can accurately characterize the heterogeneity of a sample using minimal material with high resolution within a matter of minutes. To benchmark our approach, we use negative stain electron microscopy (nsEM), a popular method for EM sample screening. We include typical workflows developed for structure determination that involve multi-step purification of a multi-subunit ubiquitin ligase and chemical cross-linking steps. When assessing the integrity and stability of large molecular complexes such as the proteasome, we detect and quantify assemblies invisible to nsEM. Our results illustrate the unique advantages of MP over current methods for rapid sample characterization, prioritization and workflow optimization.
Identifiants
pubmed: 32286308
doi: 10.1038/s41467-020-15642-w
pii: 10.1038/s41467-020-15642-w
pmc: PMC7156492
doi:
Substances chimiques
Anaphase-Promoting Complex-Cyclosome
EC 2.3.2.27
Proteasome Endopeptidase Complex
EC 3.4.25.1
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
1772Subventions
Organisme : NIGMS NIH HHS
ID : R01 GM077537
Pays : United States
Organisme : NIGMS NIH HHS
ID : T32 GM008570
Pays : United States
Organisme : NCI NIH HHS
ID : P30 CA021765
Pays : United States
Organisme : Howard Hughes Medical Institute
Pays : United States
Organisme : NIGMS NIH HHS
ID : R37 GM069530
Pays : United States
Organisme : NIGMS NIH HHS
ID : R35 GM128855
Pays : United States
Organisme : NIAID NIH HHS
ID : P30 AI050410
Pays : United States
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