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TitleThe resolution revolution in cryoEM requires high-quality sample preparation: a rapid pipeline to a high-resolution map of yeast fatty acid synthase.
Journal, issue, pagesIUCrJ, Vol. 7, Issue Pt 2, Page 220-227, Year 2020
Publish dateMar 1, 2020
AuthorsMirko Joppe / Edoardo D'Imprima / Nina Salustros / Karthik S Paithankar / Janet Vonck / Martin Grininger / Werner Kühlbrandt /
PubMed AbstractSingle-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 ...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.
External linksIUCrJ / PubMed:32148850 / PubMed Central
MethodsEM (single particle)
Resolution3.1 Å
Structure data

10420

6ta1

EMDB-10420, PDB-6ta1:
Fatty acid synthase of S. cerevisiae
Method: EM (single particle) / Resolution: 3.1 Å

Chemicals

ChemComp-NDP:
NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / Nicotinamide adenine dinucleotide phosphate

ChemComp-FMN:
FLAVIN MONONUCLEOTIDE / Flavin mononucleotide

Source
  • saccharomyces cerevisiae (strain atcc 204508 / s288c) (yeast)
KeywordsBIOSYNTHETIC PROTEIN / megasynthase / complex

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