Single-particle cryo-EM at atomic resolution
Top Cited Papers
- 21 October 2020
- journal article
- research article
- Published by Springer Science and Business Media LLC in Nature
- Vol. 587 (7832), 152-156
- https://doi.org/10.1038/s41586-020-2829-0
Abstract
The three-dimensional positions of atoms in protein molecules define their structure and their roles 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. Electron cryo-microscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years1,2. However, it has proved difficult to obtain cryo-EM reconstructions with sufficient resolution to visualize individual atoms in proteins. Here we use a new electron source, energy filter and camera to obtain a 1.7 Å resolution cryo-EM reconstruction for a human membrane protein, the β3 GABAA receptor homopentamer3. Such maps allow a detailed understanding of small-molecule coordination, visualization of solvent molecules and alternative conformations for multiple amino acids, and unambiguous building of ordered acidic side chains and glycans. Applied to mouse apoferritin, our strategy led to a 1.22 Å resolution reconstruction that 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 visualized in difference maps, allowing a direct analysis of hydrogen-bonding networks. Our technological advances, 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.Keywords
This publication has 46 references indexed in Scilit:
- Quantitative characterization of electron detectors for transmission electron microscopyJournal of Structural Biology, 2013
- Using a monochromator to improve the resolution in TEM to below 0.5Å. Part I: Creating highly coherent monochromated illuminationUltramicroscopy, 2012
- The GIF Quantum, a next generation post-column imaging energy filterUltramicroscopy, 2010
- Current and future aberration correctors for the improvement of resolution in electron microscopyPhilosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences, 2009
- Future trends in aberration-corrected electron microscopyPhilosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences, 2009
- Detective quantum efficiency of electron area detectors in electron microscopyUltramicroscopy, 2009
- Automated molecular microscopy: The new Leginon systemJournal of Structural Biology, 2005
- A new 200 kV Ω‐filter electron microscopeJournal of Microscopy, 1999
- Quantitative energy-filtered electron microscopy of biological molecules in iceUltramicroscopy, 1992
- Phase annealing in SHELX-90: direct methods for larger structuresActa Crystallographica Section A Foundations of Crystallography, 1990