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

View ORCID ProfileDiego F. Gauto, View ORCID ProfileLeandro F. Estrozi, View ORCID ProfileCharles D. Schwieters, Gregory Effantin, View ORCID ProfilePavel Macek, View ORCID ProfileRemy Sounier, Astrid C. Sivertsen, Elena Schmidt, Rime Kerfah, Guillaume Mas, Jacques-Philippe Colletier, View ORCID ProfilePeter Güntert, Adrien Favier, View ORCID ProfileGuy Schoehn, View ORCID ProfileJerome Boisbouvier, View ORCID ProfilePaul Schanda
doi: https://doi.org/10.1101/498287
Diego F. Gauto
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale (IBS), 71, avenue des martyrs, F-38044 Grenoble, France.
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  • ORCID record for Diego F. Gauto
Leandro F. Estrozi
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale (IBS), 71, avenue des martyrs, F-38044 Grenoble, France.
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Charles D. Schwieters
2Center for Information Technology, National Institutes of Health, Bethesda, USA.
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Gregory Effantin
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale (IBS), 71, avenue des martyrs, F-38044 Grenoble, France.
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Pavel Macek
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale (IBS), 71, avenue des martyrs, F-38044 Grenoble, France.
6NMR-Bio, 5 place Robert Schumann, F-38025 Grenoble, France.
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Remy Sounier
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale (IBS), 71, avenue des martyrs, F-38044 Grenoble, France.
7Institut de Génomique Fonctionnelle, CNRS UMR-5203 INSERM U1191, University of Montpellier, F-34000 Montpellier, France
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Astrid C. Sivertsen
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale (IBS), 71, avenue des martyrs, F-38044 Grenoble, France.
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Elena Schmidt
3Institute of Biophysical Chemistry, Goethe University Frankfurt am Main, Frankfurt am Main, Germany.
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Rime Kerfah
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale (IBS), 71, avenue des martyrs, F-38044 Grenoble, France.
6NMR-Bio, 5 place Robert Schumann, F-38025 Grenoble, France.
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Guillaume Mas
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale (IBS), 71, avenue des martyrs, F-38044 Grenoble, France.
8Biozentrum University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland.
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Jacques-Philippe Colletier
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale (IBS), 71, avenue des martyrs, F-38044 Grenoble, France.
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Peter Güntert
3Institute of Biophysical Chemistry, Goethe University Frankfurt am Main, Frankfurt am Main, Germany.
4Laboratory of Physical Chemistry, ETH Zürich, Zürich, Switzerland.
5Graduate School of Science, Tokyo Metropolitan University, Tokyo, Japan.
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Adrien Favier
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale (IBS), 71, avenue des martyrs, F-38044 Grenoble, France.
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  • For correspondence: adrien.favier@ibs.fr jerome.boisbouvier@ibs.fr paul.schanda@ibs.fr
Guy Schoehn
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale (IBS), 71, avenue des martyrs, F-38044 Grenoble, France.
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Jerome Boisbouvier
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale (IBS), 71, avenue des martyrs, F-38044 Grenoble, France.
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  • For correspondence: adrien.favier@ibs.fr jerome.boisbouvier@ibs.fr paul.schanda@ibs.fr
Paul Schanda
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale (IBS), 71, avenue des martyrs, F-38044 Grenoble, France.
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  • For correspondence: adrien.favier@ibs.fr jerome.boisbouvier@ibs.fr paul.schanda@ibs.fr
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Abstract

Atomic-resolution structure determination is the key requirement 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 enabled determination of the high-resolution structure 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 specifically labelled 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 (up to 8 Å) cryo-EM data are available, thus paving avenues for the structure determination of challenging biological assemblies.

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Posted December 16, 2018.
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Integrated NMR and cryo-EM atomic-resolution structure determination of a half-megadalton enzyme complex
Diego F. Gauto, Leandro F. Estrozi, Charles D. Schwieters, Gregory Effantin, Pavel Macek, Remy Sounier, Astrid C. Sivertsen, Elena Schmidt, Rime Kerfah, Guillaume Mas, Jacques-Philippe Colletier, Peter Güntert, Adrien Favier, Guy Schoehn, Jerome Boisbouvier, Paul Schanda
bioRxiv 498287; doi: https://doi.org/10.1101/498287
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Integrated NMR and cryo-EM atomic-resolution structure determination of a half-megadalton enzyme complex
Diego F. Gauto, Leandro F. Estrozi, Charles D. Schwieters, Gregory Effantin, Pavel Macek, Remy Sounier, Astrid C. Sivertsen, Elena Schmidt, Rime Kerfah, Guillaume Mas, Jacques-Philippe Colletier, Peter Güntert, Adrien Favier, Guy Schoehn, Jerome Boisbouvier, Paul Schanda
bioRxiv 498287; doi: https://doi.org/10.1101/498287

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