The resolution revolution in cryoEM requires high-quality sample preparation: a rapid pipeline to a high-resolution map of yeast fatty acid synthase.

Autor:	Joppe M; Institute of Organic Chemistry and Chemical Biology, Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Max-von-Laue-Strasse 15, 60438 Frankfurt am Main, Germany., D'Imprima E; Department of Structural Biology, Max Planck Institute of Biophysics, Max-von-Laue Strasse 3, 60438 Frankfurt am Main, Germany., Salustros N; Department of Structural Biology, Max Planck Institute of Biophysics, Max-von-Laue Strasse 3, 60438 Frankfurt am Main, Germany., Paithankar KS; Institute of Organic Chemistry and Chemical Biology, Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Max-von-Laue-Strasse 15, 60438 Frankfurt am Main, Germany., Vonck J; Department of Structural Biology, Max Planck Institute of Biophysics, Max-von-Laue Strasse 3, 60438 Frankfurt am Main, Germany., Grininger M; Institute of Organic Chemistry and Chemical Biology, Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Max-von-Laue-Strasse 15, 60438 Frankfurt am Main, Germany., Kühlbrandt W; Department of Structural Biology, Max Planck Institute of Biophysics, Max-von-Laue Strasse 3, 60438 Frankfurt am Main, Germany.
Jazyk:	angličtina
Zdroj:	IUCrJ [IUCrJ] 2020 Jan 25; Vol. 7 (Pt 2), pp. 220-227. Date of Electronic Publication: 2020 Jan 25 (Print Publication: 2020).
DOI:	10.1107/S2052252519017366
Abstrakt:	Single-particle electron cryo-microscopy (cryoEM) has undergone a 'resolution revolution' that makes it possible to characterize megadalton (MDa) complexes at atomic resolution without crystals. To fully exploit the new opportunities in molecular microscopy, new procedures for the cloning, expression and purification of macromolecular complexes need to be explored. Macromolecular assemblies are often unstable, and invasive construct design or inadequate purification conditions and sample-preparation methods can result in disassembly or denaturation. The structure of the 2.6 MDa yeast fatty acid synthase (FAS) has been studied by electron microscopy since the 1960s. Here, a new, streamlined protocol for the rapid production of purified yeast FAS for structure determination by high-resolution cryoEM is reported. Together with a companion protocol for preparing cryoEM specimens on a hydrophilized graphene layer, the new protocol yielded a 3.1 Å resolution map of yeast FAS from 15 000 automatically picked particles within a day. The high map quality enabled a complete atomic model of an intact fungal FAS to be built. (© Mirko Joppe et al. 2020.)
Databáze:	MEDLINE
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