Breaking Cryo-EM Resolution Barriers to Facilitate Drug Discovery

Abstract

SUMMARY Recent advances in single-particle cryoelecton microscopy (cryo-EM) are enabling generation of numerous near-atomic-resolution structures for well-ordered protein complexes with sizes ≥ ~200 kDa. Whether cryo-EM methods are equally useful for high-resolution structural analysis of smaller, dynamic protein complexes such as those involved in cellular metabolism remains an important question. Here, we present 3.8 Å resolution cryo-EM structures of isocitrate dehydrogenase (93 kDa) and identify the nature of conformational changes induced by binding of the allosteric small-molecule inhibitor ML309. We also report 2.8-Å- and 1.8-Å-resolution structures of the cancer targets lactate dehydrogenase (145 kDa) and glutamate dehydrogenase (334 kDa), respectively. With these results, two perceived barriers in single-particle cryo-EM are overcome: our tests demonstrated crossing 2 Å resolution and obtaining maps of proteins with sizes < 100 kDa, demonstrating that cryo-EM can be used to investigate a broad spectrum of drug-target interactions and dynamic conformational states.

Mechanistic insight into eukaryotic 60S ribosomal subunit biogenesis by cryo-electron microscopy

“…(Weis et al 2015;Greber et al 2016;Wu et al 2016) allowed the building of complete atomic models of assembly factors for which no previous high-resolution X-ray or NMR structures were available, providing detailed insight into the molecular interactions and mechanisms that govern preribosome maturation. With the rise of cryo-EM as the method of choice for determination of high-resolution structures of large assemblies (Bai et al 2015a;Nogales and Scheres 2015) and the continued technical advances of this method (Merk et al 2016), including the development of better electron detectors (McMullan et al 2014) and more powerful particle sorting algorithms (Bai et al 2015b), the determination of structures of more dynamic or more transient ribosome biogenesis intermediates at near-atomic resolution will become feasible in the future. Applied to cases where the resolution is not fully sufficient for de novo tracing of protein chains and building of complete atomic models, the combination of cryo-EM and CX-MS (Walzthoeni et al 2013) holds great promise for the detailed analysis of even highly dynamic molecular assemblies, including biogenesis intermediates that are located far upstream in the pathway.…”

Prix Nobel de Chimie 2017 : Jacques Dubochet, Joachim Frank et Richard Henderson

“…2016 : Reconstruction 3D à 1,8 Å de résolution [2] laquelle il est soumis doit être limitée, ce qui génère, de facto, des images qui seront bruitées et quasiment dénuées de toute utilité. Seule la moyenne d'un grand nombre d'images présente un intérêt pour « récupérer » l'information à haute résolution.…”

The potential use of single-particle electron microscopy as a tool for structure-based inhibitor design

“…Currently, proteins smaller than $200 kDa prove a major challenge for EM, although there are exceptions to this (Merk et al, 2016). While developments are under way to improve this, and while there has already been significant progress through the use of direct detectors, this size limitation still hinders the use of EM for several important classes of drug targets, including GPCRs.…”