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 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.
Highlights
Recent developments in single-particle cryoEM make it possible to determine the structures of large macromolecular complexes that are not available in sufficiently large amounts or that resist crystallization
This is documented by a sharp increase in the number of maps released by the Electron Microscopy Data Bank (EMDB)
We have revisited the process of resolving the structure of yeast fatty acid synthase (FAS), a major milestone in early cryoEM and crystallographic studies, and have derived a rapid protocol for determining its complete structure at high resolution
Summary
Recent developments in single-particle cryoEM make it possible to determine the structures of large macromolecular complexes that are not available in sufficiently large amounts or that resist crystallization. New image-processing algorithms can deal with sample heterogeneity, and analyzing this heterogeneity often provides direct insights into molecular mechanisms (Zivanov et al, 2018; Punjani et al, 2017; Grant et al, 2018; Murphy et al, 2019) It is no longer uncommon for cryoEM to achieve resolutions of 3 Aor better. Yeast FAS is a prime example for revealing cotranslational subunit association as a mechanism in the assembly of eukaryotic proteins (Shiber et al, 2018; Fischer et al, 2020) It is critical for the production of fatty acids in microbes as a platform for chemical synthesis (Gajewski, Pavlovic et al, 2017; Zhu et al, 2017). Requirements for high-quality protein preparations for structural studies are becoming more stringent To meet these requirements, we developed a new protocol for the rapid preparation of recombinantly expressed yeast FAS. The same approach can be used for other macromolecular assemblies
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