Evolution of standardization and dissemination of cryo-EM structures and data jointly by the community, PDB, and EMDB
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100560
ISSN1083-351X
AutoresWah Chiu, Michael F. Schmid, Grigore Pintilie, Catherine L. Lawson,
Tópico(s)Enzyme Structure and Function
ResumoCryogenic electron microscopy (cryo-EM) methods began to be used in the mid-1970s to study thin and periodic arrays of proteins. Following a half-century of development in cryo-specimen preparation, instrumentation, data collection, data processing, and modeling software, cryo-EM has become a routine method for solving structures from large biological assemblies to small biomolecules at near to true atomic resolution. This review explores the critical roles played by the Protein Data Bank (PDB) and Electron Microscopy Data Bank (EMDB) in partnership with the community to develop the necessary infrastructure to archive cryo-EM maps and associated models. Public access to cryo-EM structure data has in turn facilitated better understanding of structure–function relationships and advancement of image processing and modeling tool development. The partnership between the global cryo-EM community and PDB and EMDB leadership has synergistically shaped the standards for metadata, one-stop deposition of maps and models, and validation metrics to assess the quality of cryo-EM structures. The advent of cryo-electron tomography (cryo-ET) for in situ molecular cell structures at a broad resolution range and their correlations with other imaging data introduce new data archival challenges in terms of data size and complexity in the years to come. Cryogenic electron microscopy (cryo-EM) methods began to be used in the mid-1970s to study thin and periodic arrays of proteins. Following a half-century of development in cryo-specimen preparation, instrumentation, data collection, data processing, and modeling software, cryo-EM has become a routine method for solving structures from large biological assemblies to small biomolecules at near to true atomic resolution. This review explores the critical roles played by the Protein Data Bank (PDB) and Electron Microscopy Data Bank (EMDB) in partnership with the community to develop the necessary infrastructure to archive cryo-EM maps and associated models. Public access to cryo-EM structure data has in turn facilitated better understanding of structure–function relationships and advancement of image processing and modeling tool development. The partnership between the global cryo-EM community and PDB and EMDB leadership has synergistically shaped the standards for metadata, one-stop deposition of maps and models, and validation metrics to assess the quality of cryo-EM structures. The advent of cryo-electron tomography (cryo-ET) for in situ molecular cell structures at a broad resolution range and their correlations with other imaging data introduce new data archival challenges in terms of data size and complexity in the years to come. Cryogenic electron microscopy (cryo-EM) refers to an imaging method using a transmission electron microscope (TEM) operated with an electron energy typically between 100 and 300 kV to collect images of frozen specimens. This corresponds to a wavelength range of 0.02 to 0.037 Å, which is not a limiting factor to resolve atomic details. However, achieving atomic resolution imaging of biological specimens required developing workarounds for many roadblocks, most fundamentally radiation damage and specimen dehydration in the microscope's vacuum, which were circumvented by cryo-EM. A penalty cost of this energy range of electrons is their high probability to cause radiation damage in biological molecules, initially by chemical bond breakage and followed by release of damaged fragments (1Glaeser R.M. Limitations to significant information in biological electron microscopy as a result of radiation damage.J. Ultrastruct. 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Molecular structure determination by electron microscopy of unstained crystalline specimens.J. Mol. Biol. 1975; 94: 425-440Crossref PubMed Scopus (814) Google Scholar). The adoption of maintaining the specimen at low temperature was the very beginning of cryo-EM, enabling reduction of radiation damage in biological specimens in order to obtain near atomic-resolution electron microscopic data (6Hayward S.B. Stroud R.M. Projected structure of purple membrane determined to 3.7 A resolution by low temperature electron microscopy.J. Mol. Biol. 1981; 151: 491-517Crossref PubMed Scopus (64) Google Scholar, 7Jeng T.W. Chiu W. Low dose electron microscopy of the crotoxin complex thin crystal.J. Mol. Biol. 1983; 164: 329-346Crossref PubMed Scopus (38) Google Scholar). Another constraint of high-energy electrons is the need for biological specimens to be in a high vacuum, which is incompatible with their need to be in a hydrated state to be functional. The pioneering work of Taylor and Glaeser in the mid 1970s demonstrated the feasibility of immersing a thin protein crystal directly in liquid nitrogen and recording electron diffraction data beyond 3 Å resolution with the specimen kept at liquid nitrogen temperature (8Taylor K.A. Glaeser R.M. Electron diffraction of frozen, hydrated protein crystals.Science. 1974; 186: 1036-1037Crossref PubMed Scopus (289) Google Scholar). This initial breakthrough was further extended by the innovative approach of Dubochet and colleagues to freeze biological samples very quickly and effectively in liquid ethane with its high heat transfer coefficient so that the water is frozen into a vitrified rather than a crystalline state (9Dubochet J. McDowall A.W. Vitrification of pure water for electron microscopy.J. Microsc. 1981; 124: 3-4Crossref Scopus (278) Google Scholar, 10Dubochet J. Lepault J. Freeman R. Berriman J.A. Homo J.-C. Electron microscopy of frozen water and aqueous solutions.J. 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The idea of using low temperature to reduce radiation damage and push toward atomic resolution imaging and structure determination was pursued successfully in 1980 to 1990 with thin protein crystals preserved in glucose instead of ice, examined in the electron microscope at low temperatures. The advantage of utilizing thin crystals was the ease of performing image averaging as in crystallography. The detection of 3.5 Å diffraction intensities in the Fourier power spectrum of the image of glucose-embedded crotoxin complex crystal kept at low temperature (12Jeng T.-W. Chiu W. Zemlin F. Zeitler E. Electron imaging of crotoxin complex thin crystal at 3.5 Å.J. Mol. Biol. 1984; 175: 93-97Crossref PubMed Scopus (33) Google Scholar) pointed to the possibility of retrieving the phase information directly from electron images for structure determination. 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Fujiyoshi Y. Structural determinants of water permeation through aquaporin-1.Nature. 2000; 407: 599-605Crossref PubMed Scopus (1394) Google Scholar) (Fig. 1). The data collection for each of these projects was laborious because of low yield of high-resolution images later found to be attributable to electron-beam-induced movement (17Henderson R. Glaeser R.M. Quantitative analysis of image contrast in electron micrographs of beam-sensitive crystals.Ultramicroscopy. 1985; 16: 139-150Crossref Scopus (146) Google Scholar). These experiments led to the first few cryo-EM structures with models deposited to PDB. Paralleling the excitement of these electron crystallographic structure determinations at near atomic resolution, single-particle cryo-EM began its independent path using Dubochet's ice-embedding protocol to study single particles of spherical viruses, membrane channels, and ribosomes at nanometer resolution, as exemplified by some early structures (18Adrian M. Dubochet J. Lepault J. 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Realizing the potential of electron cryo-microscopy.Q. Rev. Biophys. 2004; 37: 3-13Crossref PubMed Scopus (173) Google Scholar). Toward the end of this period, some protein crystallographers began to integrate cryo-EM and X-ray crystallography in two ways. One was to fit the crystal structure of a molecular component into a low-resolution cryo-EM map of a large molecular complex in order to shed light on biological assembly principles. This approach began attracting the modeling community to develop tools that combine both crystallographic and cryo-EM data to generate structural models (31Wriggers W. Milligan R.A. Andrew McCammon J. Situs: A package for docking crystal structures into low-resolution maps from electron microscopy.J. Struct. Biol. 1999; 125: 185-195Crossref PubMed Scopus (447) Google Scholar). Notably, several biologically important protein structures were reported with this approach such as actin-myosin filament (32Rayment I. Holden H.M. Whittaker M. Yohn C.B. 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Some of them were indeed deposited to the PDB (Fig. 3). The second use of cryo-EM maps by crystallographers was to build an initial low-resolution model of a large complex and then apply phase extension to solve its high-resolution crystal structure. This approach was successful to solve challenging crystallographic projects at that time such as ribosome subunit and large icosahedral virus structures (36Ban N. Freeborn B. Nissen P. Penczek P. Grassucci R.A. Sweet R. Frank J. Moore P.B. Steitz T.A. A 9 Å resolution X-ray crystallographic map of the large ribosomal subunit.Cell. 1998; 93: 1105-1115Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 37Grimes J.M. Jakana J. Ghosh M. Basak A.K. Roy P. Chiu W. Stuart D.I. Prasad B.V.V. An atomic model of the outer layer of the bluetongue virus core derived from X-ray crystallography and electron cryomicroscopy.Structure. 1997; 5: 885-893Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). With the aforementioned achievements and strong potential for use of cryo-EM as a supplementary source of structural data for large biological complexes, Kim Henrick, former director of the European PDB group at the European Bioinformatics Institute (EBI), recognized the need for a public archive of cryo-EM maps, enabling reuse of a growing body of novel structural results that could not be archived in PDB. In 2002, he and his colleagues launched the EMDB for archiving single-particle reconstructions with any symmetry, subtomogram averages of subcellular tomograms, and electron crystallography maps of thin crystals, along with metadata describing the full experimental workflow (38Henrick K. Newman R. Tagari M. Chagoyen M. EMDep: A web-based system for the deposition and validation of high-resolution electron microscopy macromolecular structural information.J. Struct. Biol. 2003; 144: 228-237Crossref PubMed Scopus (39) Google Scholar). Henrick, in collaboration with Helen Berman, former director of the USA PDB group at the Research Collaboratory for Structural Bioinformatics (RCSB), led efforts to integrate the EMDB data model for cryo-EM maps with PDB's data dictionary, significantly extending it beyond its original context of crystallography. In a 2004 meeting co-organized with Michael Rossmann (Purdue), input was sought from an international group of cryo-EM practitioners (Fig. 4, A and B). In 2006, Henrick and Berman recruited Wah Chiu, former director of National Center for Macromolecular Imaging, an NIH-supported Cryo-EM Resource Center, to form the Unified Data Resource for 3D Electron Microscopy collaborative project (EMDataResource (EMDR); emdataresource.org, previously called EMDataBank). The project was targeted partly to reach out to the broader cryo-EM community and partly to leverage the long-standing experience of the protein crystallography community in developing and maintaining public archives for structure coordinates and experimental metadata (39Berman H.M. Lawson C.L. Vallat B. Gabanyi M.J. Anticipating innovations in structural biology.Q. Rev. Biophys. 2018; 51e8Crossref PubMed Scopus (4) Google Scholar). This partnership has continuously sought community consensus to refine the meta data dictionary, data formats, and validation standards through 19 in-person workshops held mainly at EBI and RCSB (40Lawson C.L. Berman H.M. Chiu W. Evolving data standards for cryo-EM structures.Struct. Dyn. 2020; 7014701Crossref PubMed Scopus (14) Google Scholar). In 2008, the first map plus model joint deposition system ("one stop shop") was developed by the EMDataResource project team in collaboration with the worldwide PDB (wwPDB) (41Lawson C.L. Baker M.L. Best C. Bi C. 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Significantly, the participants overwhelmingly recommended that all published cryo-EM maps should be deposited to EMDB and all models to PDB prior to publication and that journals should develop policies to encourage this practice. Through numerous communications with editors of prominent journals, it has gradually become the rule rather than an exception that a manuscript cannot be published without accession codes for EMDB and PDB if applicable. Owing to advances in software development, cryo-EM structure resolution improved steadily from subnanometer to near atomic resolution by the late 2000s. In 2008, several single-particle structures were reported to reach ∼4 Å resolution where polypeptide backbone tracing was possible (43Ludtke S.J. Baker M.L. Chen D.-H. Song J.-L. Chuang D.T. Chiu W. De novo backbone trace of GroEL from single particle electron cryomicroscopy.Structure. 2008; 16: 441-448Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 44Jiang W. Baker M.L. 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A further technological breakthrough was made in the mid-2010s in cryo-EM data recording devices different from photographic films or charge-coupled devices. The direct electron detector enabled cryo-EM scientists to overcome two major barriers, resulting in more effective recording of high-resolution images (47McMullan G. Faruqi A.R. Clare D. Henderson R. Comparison of optimal performance at 300keV of three direct electron detectors for use in low dose electron microscopy.Ultramicroscopy. 2014; 147: 156-163Crossref PubMed Scopus (213) Google Scholar). Image blurring attributable to the beam-induced specimen motion was reduced by digitally realigning and averaging multiple frames of the same specimen area, and quantum detective efficiency was also significantly improved (48Campbell M.G. Cheng A. Brilot A.F. Moeller A. Lyumkis D. Veesler D. Pan J. Harrison S.C. Potter C.S. Carragher B. Grigorieff N. 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In parallel, the emergence of new image processing software made structure studies possible even for conformationally heterogeneous particles (52Scheres S.H.W. Classification of structural heterogeneity by maximum-likelihood methods.Methods Enzymol. 2010; 482: 295-320Crossref PubMed Scopus (65) Google Scholar). The commercially available TEM with automated data collection software makes large data set collection more tractable in round-the-clock data collection style. Many near atomic-resolution structures began to emerge unexpectedly and rapidly (Fig. 6). Even though the electron optics of all the high-end instruments should be good enough for atomic-resolution imaging, many projects were still hindered by specimen quality: either from denaturation during cryo-specimen preparation or from the presence of inherently flexible domains. Various experimental and computational approaches have been introduced to resolve some but not all of these difficulties (53Glaeser R.M. 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Methods. 2016; 13: 387-388Crossref PubMed Scopus (173) Google Scholar), which enables cryo-EM scientists to archive and share raw images and intermediate data files associated with their maps deposited to EMDB. Making raw image data broadly available has multiple benefits, including accelerating development of reconstruction software and enriching resources for cryo-EM scientists in training. In this rapid growth phase of cryo-EM, it is necessary to establish rigorous methods to validate the maps and associated models. EMDR has taken an initiative to develop validation tools and simultaneously promote the public awareness of this necessary step in the structure determination as is done in crystallography (Table 1).Table 1EMDataResource challengesActivityParticipants' goalsOutcomes2010 Model Challenge (62Ludtke S.J. Lawson C.L. Kleywegt G.J. Berman H. Chiu W. The 2010 cryo-EM modeling challenge.Biopolymers. 2012; 97: 651-654Crossref PubMed Scopus (20) Google Scholar)Produce best models against 13 selected maps (2.5–24 Å)•130 models submitted by participant groups•Established modeling community for cryo-EM•Identified critical standardization issues•Identified issues to explore in future challenges•Nine articles/special issue of Biopolymers (Sept 2012)2016/17 Map Challenge (63Lawson C.L. Chiu W. Comparing cryo-EM structures.J. Struct. Biol. 2018; 204: 523-526Crossref PubMed Scopus (20) Google Scholar)Produce best maps from seven raw image datasets (2.5–5 Å), compare reconstruction practices•66 maps submitted by 27 participant groups•Map quality depended on level of experience•Identified need for map resolution determination standardization•11 articles/virtual special issue J Struct Biol (2018)2016/17 Model Challenge (63Lawson C.L. Chiu W. Comparing cryo-EM structures.J. Struct. Biol. 2018; 204: 523-526Crossref PubMed Scopus (20) Google Scholar)Produce best models from 12 selected maps (2.5–5 Å), compare modeling practices•63 models submitted by 16 participant groups•Innovative methods introduced for model fit-to-map assessment•Identified need for further review of global fit-to-map metrics•seven articles/virtual special issue J Struct Biol (2018)2019 Model Challenge (64Lawson C.L. Kryshtafovych A. Adams P.D. Afonine P.V. Baker M.L. Barad B.A. Bond P. Burnley T. Cao R. Cheng J. Chojnowski G. Cowtan K. Dill K. DiMaio F. Farrell D.P. et al.Outcomes of the 2019 EMDataResource model challenge: Validation of cryo-EM models at near-atomic resolution.Nat. Methods. 2021; 18: 156-164Crossref PubMed Scopus (29) Google Scholar)Produce best models from fou
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