Revival Update Scientific Developments Supporting Revival Technologies Reported by R. Michael Perry, Ph.D.
Breaking the Next Cryo-EM Resolution Barrier – Atomic Resolution Determination of Proteins! Ka Man Yip, Niels Fischer, Elham Paknia, Ashwin Chari, Holger Stark bioRxiv, 21 May 2020, https://www.biorxiv.org/content/10.1 101/2020.05.21.106740v1, accessed 25 Aug. 2020. (This article is a preprint and has not been certified by peer review.) Summary Single particle cryo-EM is a powerful method to solve the three-dimensional structures of biological macromolecules. The technological development of electron microscopes, detectors, automated procedures in combination with user friendly image processing software and ever-increasing computational power have made cryo-EM a successful and largely expanding technology over the last decade. At resolutions better than 4 Å, atomic model building starts becoming possible but the direct visualization of true atomic positions in protein structure determination requires significantly higher (< 1.5 Å) resolution, which so far could not be attained by cryo-EM. The direct visualization of atom positions is essential for understanding protein-catalyzed chemical reaction mechanisms and to study drug-binding and -interference with protein function. Here we report a 1.25 Å resolution structure of apoferritin obtained by cryo-EM with a newly developed electron microscope providing unprecedented structural details. Our apoferritin structure has almost twice the 3D information content of the current world record reconstruction (at 1.54 Å resolution). For the first time in cryo-EM we can visualize individual atoms in a protein, see density for hydrogen atoms and single atom chemical modifications. Beyond the nominal improvement in resolution we can also show a significant improvement in quality of the cryo-EM density map which is highly relevant for using cryoEM in structure-based drug design.
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Single-Particle Cryo-EM at Atomic Resolution Takanori Nakane, Abhay Kotecha, Andrija Sente, Greg McMullan, Simonas Masiulis, Patricia M.G.E. Brown, IoanaT. Grigoras, Lina Malinauskaite, Tomas Malinauskas, Jonas Miehling, Lingbo Yu, Dimple Karia, Evgeniya V. Pechnikova, Erwin de Jong, Jeroen Keizer, Maarten Bischoff, Jamie McCormack, Peter Tiemeijer, Steven W. Hardwick, Dimitri Y. Chirgadze, Garib Murshudov, A. Radu Aricescu, Sjors H.W. Scheres bioRxiv, 22 May 2020, https://www.biorxiv.org/content/10.1 101/2020.05.22.110189v1 , accessed 25 Aug. 2020. (This article is a preprint and has not been certified by peer review.) Abstract The three-dimensional positions of atoms in protein molecules define their structure and provide mechanistic insights into the roles they perform in complex biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more knowledge about 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. 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 prototypical human membrane protein, the β3 GABAA receptor homopentamer3. 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 1.2 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. Combination of the technological
Cryonics / 4th Quarter 2020
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