1. Single-particle cryo-EM at atomic resolution
- Author
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Peter Tiemeijer, Abhay Kotecha, Erwin de Jong, Takanori Nakane, Dimitri Y. Chirgadze, D. Karia, Steven W. Hardwick, Andrija Sente, Maarten Bischoff, Lina Malinauskaite, Jamie McCormack, Garib N. Murshudov, S. Masiulis, A. Radu Aricescu, Patricia M. G. E. Brown, Tomasz Uchański, Jonas Miehling, Sjors H.W. Scheres, Lingbo Yu, Ioana T. Grigoras, Jeroen Keizer, Evgeniya V. Pechnikova, Greg McMullan, and Tomas Malinauskas
- Subjects
0303 health sciences ,Multidisciplinary ,Materials science ,Cryo-electron microscopy ,Drug discovery ,Resolution (electron density) ,Small molecule ,Article ,03 medical and health sciences ,Structural bioinformatics ,0302 clinical medicine ,Protein structure ,Chemical physics ,Particle ,Molecule ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
The three-dimensional positions of atoms in protein molecules define their structure and the roles they perform in biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more mechanistic insights into protein function may be inferred. With breakthroughs in electron detection and image processing technology, electron cryo-microscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years(1,2). However, obtaining cryo-EM reconstructions with sufficient resolution to visualise individual atoms in proteins has thus far been elusive. Here, we show that using a new electron source, energy filter and camera, we obtained a 1.7 Å resolution cryo-EM reconstruction for a human membrane protein, the β3 GABA(A) receptor homopentamer(3). Such maps allow a detailed understanding of small molecule coordination, visualisation of solvent molecules and alternative conformations for multiple amino acids, as well as unambiguous building of ordered acidic side chains and glycans. Applied to mouse apoferritin, our strategy led to a 1.22 Å resolution reconstruction that, for the first time, offers a genuine atomic resolution view of a protein molecule using single particle cryo-EM. Moreover, the scattering potential from many hydrogen atoms can be visualised in difference maps, allowing a direct analysis of hydrogen bonding networks. The technological advances described here, combined with further approaches to accelerate data acquisition and improve sample quality, provide a route towards routine application of cryo-EM in high-throughput screening of small molecule modulators and structure-based drug discovery.
- Published
- 2020