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Nat Commun. 2019 Jun 19;10(1):2697. doi: 10.1038/s41467-019-10490-9.

Integrated NMR and cryo-EM atomic-resolution structure determination of a half-megadalton enzyme complex.

Author information

1
Institut de Biologie Structurale (IBS), CEA, CNRS, Université Grenoble Alpes, 71, Avenue des Martyrs, F-38044, Grenoble, France.
2
Institut de Biologie Structurale (IBS), CEA, CNRS, Université Grenoble Alpes, 71, Avenue des Martyrs, F-38044, Grenoble, France. leandro.estrozi@ibs.fr.
3
Laboratory of Imaging Sciences, Center for Information Technology, National Institutes of Health, 12 South Drive, MSC 5624, Bethesda, MD, 20892, USA.
4
NMR-Bio, 5 Place Robert Schuman, F-38025, Grenoble, France.
5
Institut de Génomique Fonctionnelle, CNRS UMR-5203, INSERM U1191, University of Montpellier, F-34000, Montpellier, France.
6
Institute of Biophysical Chemistry, Goethe University Frankfurt am Main, 60438 Frankfurt am Main, Germany.
7
Biozentrum University of Basel, Klingelbergstrasse 70, 4056, Basel, Switzerland.
8
Laboratory of Physical Chemistry, ETH Zürich, 8093 Zürich, Switzerland.
9
Graduate School of Science, Tokyo Metropolitan University, Tokyo 192-0397, Japan.
10
Institut de Biologie Structurale (IBS), CEA, CNRS, Université Grenoble Alpes, 71, Avenue des Martyrs, F-38044, Grenoble, France. adrien.favier@ibs.fr.
11
Institut de Biologie Structurale (IBS), CEA, CNRS, Université Grenoble Alpes, 71, Avenue des Martyrs, F-38044, Grenoble, France. paul.schanda@ibs.fr.

Abstract

Atomic-resolution structure determination is crucial for understanding protein function. Cryo-EM and NMR spectroscopy both provide structural information, but currently cryo-EM does not routinely give access to atomic-level structural data, and, generally, NMR structure determination is restricted to small (<30 kDa) proteins. We introduce an integrated structure determination approach that simultaneously uses NMR and EM data to overcome the limits of each of these methods. The approach enables structure determination of the 468 kDa large dodecameric aminopeptidase TET2 to a precision and accuracy below 1 Å by combining secondary-structure information obtained from near-complete magic-angle-spinning NMR assignments of the 39 kDa-large subunits, distance restraints from backbone amides and ILV methyl groups, and a 4.1 Å resolution EM map. The resulting structure exceeds current standards of NMR and EM structure determination in terms of molecular weight and precision. Importantly, the approach is successful even in cases where only medium-resolution cryo-EM data are available.

PMID:
31217444
PMCID:
PMC6584647
DOI:
10.1038/s41467-019-10490-9
[Indexed for MEDLINE]
Free PMC Article

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