title: Breaking Cryo-EM Resolution Barriers to Facilitate Drug Discovery.
abstract: 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 the cancer target 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 lactate dehydrogenase (145 kDa) and glutamate dehydrogenase (334 kDa), respectively. With these results, two perceived barriers in single-particle cryo-EM are overcome: (1) crossing 2 Å resolution and (2) obtaining structures 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.
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
authors.forename: Alan
authors.initials: A
authors.lastname: Merk
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
authors.forename: Alberto
authors.initials: A
authors.lastname: Bartesaghi
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
authors.forename: Soojay
authors.initials: S
authors.lastname: Banerjee
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
authors.forename: Veronica
authors.initials: V
authors.lastname: Falconieri
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
authors.forename: Prashant
authors.initials: P
authors.lastname: Rao
authors.affiliation: National Center for Advancing Translational Sciences, 9800 Medical Center Drive, Rockville, MD 20850, USA.
authors.forename: Mindy I
authors.initials: MI
authors.lastname: Davis
authors.affiliation: National Center for Advancing Translational Sciences, 9800 Medical Center Drive, Rockville, MD 20850, USA.
authors.forename: Rajan
authors.initials: R
authors.lastname: Pragani
authors.affiliation: National Center for Advancing Translational Sciences, 9800 Medical Center Drive, Rockville, MD 20850, USA.
authors.forename: Matthew B
authors.initials: MB
authors.lastname: Boxer
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
authors.forename: Lesley A
authors.initials: LA
authors.lastname: Earl
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
authors.forename: Jacqueline L S
authors.initials: JLS
authors.lastname: Milne
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA. Electronic address: ss1@nih.gov.
authors.forename: Sriram
authors.initials: S
authors.lastname: Subramaniam
mesh_terms: D000634:Aminoquinolines / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002645:Chickens; D020285:Cryoelectron Microscopy; D018360:Crystallography, X-Ray; D055808:Drug Discovery; D005969:Glutamate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000648:ultrastructure*; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000648:ultrastructure*; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000648:ultrastructure*; D008958:Models, Molecular; D011487:Protein Conformation; D013449:Sulfonamides / Q000737:chemistry / Q000494:pharmacology
chemical_list: D000634:Aminoquinolines; C000612070:GSK2837808A; D013449:Sulfonamides; D007770:L-Lactate Dehydrogenase; D007521:Isocitrate Dehydrogenase; D005969:Glutamate Dehydrogenase