TY  - JOUR
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 high-quality sample preparation: a rapid pipeline to a high-resolution map of yeast fatty acid synthase
T2  - IUCrJ
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 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.
KW  - purification of protein complexes
KW  - 3D reconstruction and image processing
KW  - single-particle cryoEM
KW  - cryo-electron microscopy
KW  - macromolecular machines
KW  - protein structure
KW  - yeast fatty acid synthase
Y1  - 2020
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/73412
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-734128
SN  - 2052-2525
VL  - 7
IS  - 2
SP  - 220
EP  - 227
PB  - International Union of Crystallography
CY  - Chester
ER  -