TY - JOUR

T1 - The resolution revolution in cryoEM requires high-quality sample preparation

T2 - a rapid pipeline to a high-resolution map of yeast fatty acid synthase

AU - Joppe, Mirko

AU - D'Imprima, Edoardo

AU - Salustros, Nina

AU - Paithankar, Karthik S.

AU - Vonck, Janet

AU - Grininger, Martin

AU - Kuehlbrandt, Werner

PY - 2020

Y1 - 2020

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 angstrom 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.

AB - 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 angstrom 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

KW - STABILITY OPTIMIZATION

KW - EM STRUCTURE

KW - ADSORPTION

KW - SYSTEM

KW - FAS

KW - SYNTHETASE

KW - MICROSCOPY

KW - PROTEINS

KW - COMPLEX

KW - LIGAND

U2 - 10.1107/S2052252519017366

DO - 10.1107/S2052252519017366

M3 - Journal article

C2 - 32148850

VL - 7

SP - 220

EP - 227

JO - I U Cr J

JF - I U Cr J

SN - 2052-2525

ER -