Better, faster, and even cheap

doi:10.1126/science.abd8035

GSTDTAP > 气候变化

DOI	10.1126/science.abd8035
	Better, faster, and even cheap
	Micah Rapp; Bridget Carragher
	2020-10-09
发表期刊	Science
出版年	2020
英文摘要	Cryo–electron microscopy (cryo-EM) enables access to structures of proteins that were previously intractable, including large protein complexes such as the ribosome ([ 1 ][1]), integral membrane proteins ([ 2 ][2], [ 3 ][3]), and highly heterogeneous or conformationally dynamic systems ([ 4 ][4]). Each sample is a vitrified layer of protein suspended over a support film on an EM grid. Despite recent advances in cryo-EM (the so-called “resolution revolution”) ([ 5 ][5], [ 6 ][6]), major barriers persist, including loss of the highest-resolution information through electron beam damage and blurring from sample movement (which is most pronounced initially when the sample is least damaged). Typically, tens of thousands of images must be averaged to compensate for signal loss. On page 223 of this issue, Naydenova et al. ([ 7 ][7]) describe a new specimen support film (see the figure) that not only improves both the quality of images and the efficiency of collection, but also does so at a relatively low price. Like much of society, the strengths and weaknesses of cryo-EM have been highlighted by the coronavirus disease 2019 (COVID-19) pandemic. Structural biology has contributed to our understanding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with more than 330 structures deposited into the Protein Data Bank since early March. At the time of writing, 264 of those structures were produced by x-ray crystallography and 72 by cryo-EM. The astonishing throughput of crystallography was demonstrated in recent work on the main SARS-CoV-2 protease, where ∼1500 datasets were collected in a single day ([ 8 ][8]). In contrast, cryo-EM datasets typically require on the order of 24 hours of data collection, even though the process is largely automated ([ 9 ][9]). High-end microscopes are not only under extraordinary demand, but are also associated with very high capital and annual maintenance costs ([ 10 ][10]). These differences have made x-ray crystallography better suited to the rapid turnaround required by the pharmaceutical drug discovery pipeline ([ 11 ][11]). These timelines also present a barrier for academic research. Increasing throughput will lower costs and enable more researchers to use cryo-EM to solve structures and accelerate scientific discovery. Naydenova et al. propose a new specimen support film, dubbed “HexAuFoil,” that provides many advantages over conventional support films. In cryo-EM, images are typically taken of proteins embedded in a layer of vitrified ice suspended over a support films containing holes ∼1 to 2 µm in diameter. By making these holes smaller (200 to 300 nm in diameter) and packing them more tightly together (a nontrivial feat), they substantially increased data throughput. They obtained 200 images for every microscope stage movement (versus a typical 10 to 30 images) and propose that as many as 800 images could be obtained with an appropriately configured microscope. A second approach to increasing throughput that Naydenova et al. addressed is to increase the data quality by minimizing the information loss from both radiation damage and sample movement. They present a fascinating and exhaustive analysis showing that the primary cause of specimen movement during imaging is a buckling of the vitrified ice layer. On the basis of these insights, they created a support film with an optimal substrate thickness relative to the hole diameter, which reduces the total movement of the sample to <1 Å during the course of an exposure. They could mathematically extrapolate the data to a three-dimensional map before the onset of radiation damage. Standard support films have recently been used to obtain the first truly atomic-level cryo-EM reconstructions ([ 12 ][12], [ 13 ][13]) of apoferritin, a very stable test specimen. The advances developed by Naydenova et al. should bring this goal closer for less well-behaved proteins. Although software is also now available to correct for the effects of beam-induced movement ([ 14 ][14]), this approach does not provide the concomitant benefit of faster data collection. High-resolution cryo-EM is still in a state of active method development, its true potential not yet realized. Thus, it is exciting that Naydenova et al. , with one accessible, inexpensive hardware development, will allow all practitioners to acquire better images much more rapidly as soon as the grids become commercially available. The promise is of a future of high-resolution structures of a wide range of proteins, in an ensemble of conformational or compositional states ([ 15 ][15]), produced with much higher throughput. 1. [↵][16]1. A. Brown, 2. S. Shao , Curr. Opin. Struct. Biol. 52, 1 (2018). [OpenUrl][17][CrossRef][18] 2. [↵][19]1. Y. Cheng , Curr. Opin. Struct. Biol. 52, 58 (2018). [OpenUrl][20][CrossRef][21][PubMed][22] 3. [↵][23]1. M. Dong et al ., Nat. Commun. 11, 4137 (2020). [OpenUrl][24] 4. [↵][25]1. T. Nakane, 2. D. Kimanius, 3. E. Lindahl, 4. S. H. W. Scheres , eLife 7, e36861 (2018). [OpenUrl][26][CrossRef][27][PubMed][28] 5. [↵][29]1. W. Kühlbrandt , Science 343, 1443 (2014). [OpenUrl][30][Abstract/FREE Full Text][31] 6. [↵][32]1. Y. Cheng , Science 361, 876 (2018). [OpenUrl][33][Abstract/FREE Full Text][34] 7. [↵][35]1. K. Naydenova, 2. P. Jia, 3. C. J. Russo , Science 370, 223 (2020). [OpenUrl][36][CrossRef][37] 8. [↵][38]1. A. Douangamath et al ., bioRxiv 118117 (2020). 9. [↵][39]1. D. Lyumkis , J. Biol. Chem. 294, 5181 (2019). [OpenUrl][40][Abstract/FREE Full Text][41] 10. [↵][42]1. K. Naydenova et al ., IUCrJ 6, 1086 (2019). [OpenUrl][43][CrossRef][44][PubMed][45] 11. [↵][46]1. G. Scapin, 2. C. S. Potter, 3. B. Carragher , Cell Chem. Biol. 25, 1318 (2018). [OpenUrl][47] 12. [↵][48]1. T. Nakane et al ., bioRxiv 110189 (2020). 13. [↵][49]1. K. M. Yip, 2. N. Fischer, 3. E. Paknia, 4. A. Chari, 5. H. Stark , bioRxiv 106740 (2020). 14. [↵][50]1. D. Tegunov, 2. L. Xue, 3. C. Dienemann, 4. P. Cramer, 5. J. Mahamid , bioRxiv 136341 (2020). 15. [↵][51]1. A. Dance , Nat. Methods 17, 879 (2020). [OpenUrl][52] Acknowledgments: Supported by NIH grants GM103310 and OD019994 and Simons Foundation grant SF349247. 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领域	气候变化 ; 资源环境
URL	查看原文
引用统计
文献类型	期刊论文
条目标识符	http://119.78.100.173/C666/handle/2XK7JSWQ/298087
专题	气候变化资源环境科学
推荐引用方式 GB/T 7714	Micah Rapp,Bridget Carragher. Better, faster, and even cheap[J]. Science,2020.
APA	Micah Rapp,&Bridget Carragher.(2020).Better, faster, and even cheap.Science.
MLA	Micah Rapp,et al."Better, faster, and even cheap".Science (2020).