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

Abstract

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.

The evolution of SPHIRE-crYOLO particle picking and its application in automated cryo-EM processing workflows

“…crYOLO [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22] . For example, Pang et al 23 used crYOLO to selectively pick particles, which were attached to liposomes; Rogala et al 14 highlighted in their study about mTORC1 that crYOLO was especially useful to exclude particles on carbon; Joppe et al 24 made use of crYOLO in a streamlined pipeline for rapid structure determination of yeast fatty acid synthase; and the new filament mode was recently used by Pospich et al to examine the structural effects of toxins on actin filaments 16 .…”