TY  - INPR
A1  - Joppe, Mirko
A1  - D'Imprima, Edoardo
A1  - Salustros, Nina
A1  - Paithankar, Karthik Shivaji
A1  - Vonck, Janet
A1  - Grininger, Martin
A1  - Kühlbrandt, Werner
T1  - The resolution revolution in cryoEM requires new sample preparation procedures: a rapid pipeline to high resolution maps of yeast FAS
T2  - bioRxiv
N2  - 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.
Y1  - 2018
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/72653
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-726539
IS  - 829176
ER  -