The resolution revolution in cryoEM requires new sample preparation procedures: a rapid pipeline to high resolution maps of yeast FAS

  • 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 or 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. We report a new, streamlined protocol for the rapid production of purified yeast FAS for structure determination by high-resolution cryoEM. Together with a companion protocol for preparing cryoEM specimens on a hydrophilized graphene layer, our new protocol has yielded a 3.1 Å map of yeast FAS from 15,000 automatically picked particles within a day. The high map quality enabled us to build a complete atomic model of an intact fungal FAS.

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Author:Mirko JoppeORCiDGND, Edoardo D'ImprimaORCiDGND, Nina SalustrosORCiD, Karthik Shivaji PaithankarORCiDGND, Janet VonckORCiD, Martin GriningerORCiDGND, Werner KühlbrandtORCiDGND
Parent Title (English):bioRxiv
Document Type:Preprint
Date of Publication (online):2018/12/07
Date of first Publication:2018/12/07
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2023/07/01
Page Number:14
Institutes:Biochemie, Chemie und Pharmazie / Biochemie und Chemie
Angeschlossene und kooperierende Institutionen / MPI für Biophysik
Fachübergreifende Einrichtungen / Buchmann Institut für Molekulare Lebenswissenschaften (BMLS)
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 54 Chemie / 540 Chemie und zugeordnete Wissenschaften
5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
Licence (German):License LogoCreative Commons - CC BY-NC-ND - Namensnennung - Nicht kommerziell - Keine Bearbeitungen 4.0 International