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The resolution revolution in cryoEM requires high-quality sample preparation: a rapid pipeline to a high-resolution map of yeast fatty acid synthase

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D'Imprima,  Edoardo
Department of Structural Biology, Max Planck Institute of Biophysics, Max Planck Society;

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Salustros,  N.
Department of Structural Biology, Max Planck Institute of Biophysics, Max Planck Society;

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Vonck,  Janet
Department of Structural Biology, Max Planck Institute of Biophysics, Max Planck Society;

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Kühlbrandt,  Werner       
Department of Structural Biology, Max Planck Institute of Biophysics, Max Planck Society;

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Citation

Joppe, M., D'Imprima, E., Salustros, N., Paithankar, K. S., Vonck, J., Grininger, M., et al. (2020). The resolution revolution in cryoEM requires high-quality sample preparation: a rapid pipeline to a high-resolution map of yeast fatty acid synthase. IUCrJ, 7(Pt 2), 220-227. doi:10.1107/S2052252519017366.


Cite as: https://hdl.handle.net/21.11116/0000-0005-D980-5
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.