分辨率革命——冷冻电子显微学在结构生物学研究中的进展
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摘要
冷冻电子显微学具有悠久的历史,近年来随着硬件设备的革命性技术突破和软件算法方面技术发展,已经成为结构生物学领域最为重要的研究方法,也代表了未来的发展方向。其中,单颗粒三维结构重构法在最近的几年发展迅速,极大地拓展了研究方法的适用范围,并不断刷新所解析结构的高分辨率记录,成为结构生物学及相关学科最为激动人心的研究领域。本文将针对冷冻电子显微学发展的历史,近期革命性技术突破,以及复旦大学在该领域的布局和进展做简要介绍。
Cryo electron microscopy(cryo-EM)is one of the most important methods in structural biology.In the past five years,cryo-EM has milestone breakthrough due to revolutionary advances in hardware and methodology.The resolution has been pushed to as high as 1.8 ,which significantly extended the research scope of this method.Single particle reconstruction method has become one of the most exciting fields of structural biology and related subjects.Here we will briefly introduce the history of cryo-EM,recent revolutionary breakthrough,and the facility at Fudan University.
Cryo electron microscopy(cryo-EM)is one of the most important methods in structural biology.In the past five years,cryo-EM has milestone breakthrough due to revolutionary advances in hardware and methodology.The resolution has been pushed to as high as 1.8 ,which significantly extended the research scope of this method.Single particle reconstruction method has become one of the most exciting fields of structural biology and related subjects.Here we will briefly introduce the history of cryo-EM,recent revolutionary breakthrough,and the facility at Fudan University.
引文
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[30]CHEN S,ZHAO Y,WANG Y,et al.Activation and desensitization mechanism of AMPA receptor-TARP complex by cryo-EM[J].Cell,2017,170(6):1234-1246.
[31]CAMPBELL M,VEESLER D,CHENG A,et al.2.8resolution reconstruction of the Thermoplasma acidophilum 20S proteasome using cryo-electron microscopy[J].eLife,2015,4:e06380.
[32]BARTESAGHI A,MERK A,BANERJEE S,et al.2.2resolution cryo-EM structure ofβ-galactosidase in complex with a cell-permeant inhibitor[J].Science,2015,348(6239):1147-1151.
[33]MERK A,BARTESAGHI A,BANERJEE S,et al.Breaking cryo-EM resolution barriers to facilitate drug discovery[J].Cell,2016,165(7):1698-1707.
[34]KHOSHOUEI M,RADJAINIA M,BAUMEISTER W,et al.Cryo-EM structure of haemoglobin at 3.2determined with the Volta phase plate[J].Nat Commun,2017,30(8):16099.
[35]CROWTHER R,DE ROSIER D,KLUG A.The reconstruction of a three-dimensional structure from projections and its application to electron microscopy[J].Proc R Soc Lond B Biol Sci,1972,182(1066):89-102.
[36]SUBRAMANIAM S,ZHANG P,LEFMAN J,et al.Electron tomography:a powerful tool for 3D cellular microscopy[J].ASM News,2003,69(5):240-245.
[37]ZHANG X,GE P,YU X,et al.Cryo-EM structure of the mature dengue virus at 3.5-resolution[J].Nat Struct Mol Biol,2013,20(1):105-110.
[38]FRANK G,SHUKLA S,RAO P,et al.Cryo-EM analysis of the conformational landscape of human P-glycoprotein(ABCB1)during its catalytic cycle[J].Mol Pharmacol,2016,90(1):35-41.
[39]CHEN ZG,SUN L,ZHANG Z,et al.Cryo-EM structure of the bacteriophage T4 isometric head at 3.3-resolution and its relevance to the assembly of icosahedral viruses[J].Proc Natl Acad Sci,2017,114(39):E8184-E8193.
[40]THOMPSON R,WALKER M,SLEBERT CA,et al.An introduction to sample preparation and imaging by cryoelectron microscopy for structural biology[J].Methods,2016,100:3-15.
[41]JAIN T,SHEEHAN P,CRUM J,et al.Spotiton:A prototype for an integrated inkjet dispense and vitrification system for cryo-TEM[J].J Struct Biol,2012,179(1):68-75.
[42]RIGORT A.Recent developments in FEI’s in situ cryoelectron tomography workflow[J].Nat Methods,2016,13:3-4.
[43]TIEMEIJER PC,BISCHOFF M,FREITAG B,et al.Using a monochromator to improve the resolution in TEM to below 0.5.Part II:application to focal series reconstruction[J].Ultramicroscopy,2012,118:35-43.
[44]FISCHER N,NEUMANN P,KONEVEGA A,et al.Structure of the E.coli ribosome-EF-Tu complex at<3resolution by Cs-corrected cryo-EM[J].Nature,2015,520(7548):567-570.
[45]DANEV R,BUIJSSE B,KHOSHOUEI M,et al.Volta potential phase plate for in-focus phase contrast transmission electron microscopy[J].Proc Natl Acad Sci U S A,2014,111(44):15635-15640.
[46]KHOSHOUEI M,PFEFFER S,BAUMEISTER W,et al.Subtomogram analysis using the Volta phase plate[J].J Struct Biol,2017,197(2):94-101.
[2]THORNTON JM,TODD AE,MILBURN D,et al.From structure to function:approaches and limitations[J].Nat Struct Biol,2000,7(Suppl):991-994.
[3]SALI A,GLAESER R,EARNEST T,et al.From words to literature in structural proteomics[J].Nature,2003,422(6928):216-225.
[4]SAUNDERS M,WISHNIA A,KIRKWOOD JG.The nuclear magnetic resonance spectrum of ribonuclease[J].J Am Chem Soc,1957,79:3289-3290.
[5]MARION D.An introduction to biological NMR spectroscopy[J].Mol Cell Proteomics,2013,12(11):3006-3025.
[6]KENDREW JC,BODO G,DINTZIS HM,et al.A threedimensional model of the myoglobin molecule obtained by x-ray analysis[J].Nature,1958,181(4610):662-666.
[7]KENDREW JC,DICKERSON RE,STRANDBERGBE,et al.Structure of myoglobin:A three-dimensional Fourier synthesis at 2resolution[J].Nature,1960,185(4711):422-427.
[8]PERUTZ MF,ROSSMANN MG,CULLIS AF,et al.Structure of haemoglobin:a three-dimensional Fourier synthesis at 5.5-resolution,obtained by X-ray analysis[J].Nature,1960,185(4711):416-422.
[9]ROSSMANN MG,BLOW DM.The detection of sub-units within the crystallographic asymmetric unit[J].Acta Cryst,1962,15:24-31.
[10]SHI Y.A glimpse of structural biology through X-ray crystallography[J].Cell,2014,159(5):995-1014.
[11]RHODESG.Crystallography made crystal clear:A guide for users of macromolecular models[M].3rd Edition.Cambridge:Academic Press,2006.
[12]FRANK J.Three-dimensional electron microscopy of macromolecular assemblies:visualization of biological molecules in their native state[M].Oxford:Oxford University Press,2006.
[13]ZHOU ZH.Atomic resolution cryo electron microscopy of macromolecular complexes[J].Adv Protein Chem Struct Biol,2011,82:1-35.
[14]TAYLOR KA,GLAESER RM.Electron diffraction of frozen,hydrated protein crystals[J].Science,1974,186(4168):1036-1037.
[15]ADRIAN M,DUBOCHET J,LEPAULT J,et al.Cryoelectron microscopy of viruses[J].Nature,1984,308(5954):32-36.
[16]HENDERSON R,BALDWIN JM,CESKA TA,et al.Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy[J].J Mol Biol,1990,213(4):899-929.
[17]DE ROSIER D,KLUG A.Reconstruction of three dimensional structures from electron micrographs[J].Nature,1968,217(5124):130-134.
[18]ZHANG X,JIN L,FANG Q,et al.3.3 A cryo-EM structure of a nonenveloped virus reveals a priming mechanism for cell entry[J].Cell,2010,141(3):472-482.
[19]LIAO M,CAO E,JULIUS D,et al.Structure of the TRPV1 ion channel determined by electron cryomicroscopy[J].Nature,2013,504(7478):107-112.
[20]CAO E,LIAO M,CHENG Y,et al.TRPV1 structures in distinct conformations reveal activation mechanisms[J].Nature,2013,504(7478):113-118.
[21]BAMMES B,ROCHAT R,JAKANA J,et al.Direct electron detection yields cryo-EM reconstructions at resolutions beyond 3/4 Nyquist frequency[J].J Struct Biol,2012,177(3):589-601.
[22]LI X,MOONEY P,ZHENG S,et al.Electron counting and beam-induced motion correction enables near atomic resolution single particle cryoEM[J].Nat Methods,2013,10(6):584-590.
[23]BRILOT AF,CHEN JZ,CHENG A,et al.Beam-induced motion of vitrified specimen on holey carbon film[J].J Struct Biol,2012,177(3):630-637.
[24]MCMULLAN G,CHEN S,HENDERSON R,et al.Detective quantum efficiency of electron area detectors in electron microscopy[J].Ultramicroscopy,2009,109(9):1126-1143.
[25]MCMULLAN G,FARUQI AR,HENDERSON R,et al.Experimental observation of the improvement in MTF from backthinning a CMOS direct electron detector[J].Ultramicroscopy,2009,109(9):1144-1147.
[26]MCMULLAN G,CLARK AT,TURCHETTA R,et al.Enhanced imaging in low dose electron microscopy using electron counting[J].Ultramicroscopy,2009,109(12):1411-1416.
[27]KIMANIUS D,FORSBERG B,SCHERES S,et al.Accelerated cryo-EM structure determination with parallelisation using GPUs in relion-2[J].eLife,2016,5:e18722.
[28]LYUMKIS D,BRILOT AF,THEOBALD DL,et al.Likelihood-based classification of cryo-EM images using FREALIGN[J].J Struct Biol,2013,183(3):377-388.
[29]PARK E,CAMPBELL E,MACKINNON R.Structure of a CLC chloride ion channel by cryo-electron microscopy[J].Nature,2017,541(7638):500-505.
[30]CHEN S,ZHAO Y,WANG Y,et al.Activation and desensitization mechanism of AMPA receptor-TARP complex by cryo-EM[J].Cell,2017,170(6):1234-1246.
[31]CAMPBELL M,VEESLER D,CHENG A,et al.2.8resolution reconstruction of the Thermoplasma acidophilum 20S proteasome using cryo-electron microscopy[J].eLife,2015,4:e06380.
[32]BARTESAGHI A,MERK A,BANERJEE S,et al.2.2resolution cryo-EM structure ofβ-galactosidase in complex with a cell-permeant inhibitor[J].Science,2015,348(6239):1147-1151.
[33]MERK A,BARTESAGHI A,BANERJEE S,et al.Breaking cryo-EM resolution barriers to facilitate drug discovery[J].Cell,2016,165(7):1698-1707.
[34]KHOSHOUEI M,RADJAINIA M,BAUMEISTER W,et al.Cryo-EM structure of haemoglobin at 3.2determined with the Volta phase plate[J].Nat Commun,2017,30(8):16099.
[35]CROWTHER R,DE ROSIER D,KLUG A.The reconstruction of a three-dimensional structure from projections and its application to electron microscopy[J].Proc R Soc Lond B Biol Sci,1972,182(1066):89-102.
[36]SUBRAMANIAM S,ZHANG P,LEFMAN J,et al.Electron tomography:a powerful tool for 3D cellular microscopy[J].ASM News,2003,69(5):240-245.
[37]ZHANG X,GE P,YU X,et al.Cryo-EM structure of the mature dengue virus at 3.5-resolution[J].Nat Struct Mol Biol,2013,20(1):105-110.
[38]FRANK G,SHUKLA S,RAO P,et al.Cryo-EM analysis of the conformational landscape of human P-glycoprotein(ABCB1)during its catalytic cycle[J].Mol Pharmacol,2016,90(1):35-41.
[39]CHEN ZG,SUN L,ZHANG Z,et al.Cryo-EM structure of the bacteriophage T4 isometric head at 3.3-resolution and its relevance to the assembly of icosahedral viruses[J].Proc Natl Acad Sci,2017,114(39):E8184-E8193.
[40]THOMPSON R,WALKER M,SLEBERT CA,et al.An introduction to sample preparation and imaging by cryoelectron microscopy for structural biology[J].Methods,2016,100:3-15.
[41]JAIN T,SHEEHAN P,CRUM J,et al.Spotiton:A prototype for an integrated inkjet dispense and vitrification system for cryo-TEM[J].J Struct Biol,2012,179(1):68-75.
[42]RIGORT A.Recent developments in FEI’s in situ cryoelectron tomography workflow[J].Nat Methods,2016,13:3-4.
[43]TIEMEIJER PC,BISCHOFF M,FREITAG B,et al.Using a monochromator to improve the resolution in TEM to below 0.5.Part II:application to focal series reconstruction[J].Ultramicroscopy,2012,118:35-43.
[44]FISCHER N,NEUMANN P,KONEVEGA A,et al.Structure of the E.coli ribosome-EF-Tu complex at<3resolution by Cs-corrected cryo-EM[J].Nature,2015,520(7548):567-570.
[45]DANEV R,BUIJSSE B,KHOSHOUEI M,et al.Volta potential phase plate for in-focus phase contrast transmission electron microscopy[J].Proc Natl Acad Sci U S A,2014,111(44):15635-15640.
[46]KHOSHOUEI M,PFEFFER S,BAUMEISTER W,et al.Subtomogram analysis using the Volta phase plate[J].J Struct Biol,2017,197(2):94-101.