Bacterial Protein Secretion Systems pp 377-413 | Cite as
Structural Analysis of Protein Complexes by Cryo Electron Microscopy
Abstract
Structural studies of biocomplexes using single-particle cryo-electron microscopy (cryo-EM) is now a well-established technique in structural biology and has become competitive with X-ray crystallography. The latest advances in EM enable us to determine structures of protein complexes at 3–5 Å resolution for an extremely broad range of sizes from ~200 kDa up to hundreds of megadaltons (Bartesaghi et al., Science 348(6239):1147–1151, 2051; Bai et al., Nature 525(7568):212–217, 2015; Vinothkumar et al., Nature 515(7525):80–84, 2014; Grigorieff and Harrison, Curr Opin Struct Biol 21(2):265–273, 2011). The majority of biocomplexes comprise a number of different components and are not amenable to crystallisation. Secretion systems are typical examples of such multi-protein complexes, and structural studies of them are extremely challenging. The only feasible approach to revealing their spatial organisation and functional modification is cryo-EM. The development of systems for digital registration of images and algorithms for the fast and efficient processing of recorded images and subsequent analysis facilitated the determination of structures at near-atomic resolution. In this review we will describe sample preparation for cryo-EM, how data are collected by new detectors, and the logistics of image analysis through the basic steps required for reconstructions of both small and large biological complexes and their refinement to nearly atomic resolution. The processing workflow is illustrated using examples of EM analysis of a Type IV Secretion System.
Key words
Cryo-electron microscopy Sample preparation Single particle analysis Image processing Type IV secretion systemNotes
Acknowledgments
The authors thank Dr. H. White for reading the manuscript and useful discussions that led to improvements in the manuscript. This work was funded by MRC Grant MR/K012401/1 to E.V.O. The authors apologise for not covering all methods fully owing to space constraints.
References
- 1.Steitz TA (2008) A structural understanding of the dynamic ribosome machine. Nat Rev Mol Cell Biol 9(3):242–253PubMedCrossRefGoogle Scholar
- 2.Bartesaghi A, Merk A, Banerjee S, Matthies D, Wu X, Milne JL, Subramaniam S (2015) 2.2 A resolution cryo-EM structure of beta-galactosidase in complex with a cell-permeant inhibitor. Science 348(6239):1147–1151PubMedCrossRefGoogle Scholar
- 3.Bai XC, Yan C, Yang G, Lu P, Ma D, Sun L, Zhou R, Scheres SH, Shi Y (2015) An atomic structure of human gamma-secretase. Nature 525(7568):212–217PubMedPubMedCentralCrossRefGoogle Scholar
- 4.Vinothkumar KR, Zhu J, Hirst J (2014) Architecture of mammalian respiratory complex I. Nature 515(7525):80–84PubMedPubMedCentralCrossRefGoogle Scholar
- 5.Grigorieff N, Harrison SC (2011) Near-atomic resolution reconstructions of icosahedral viruses from electron cryo-microscopy. Curr Opin Struct Biol 21(2):265–273PubMedPubMedCentralCrossRefGoogle Scholar
- 6.Grant T, Grigorieff N (2015) Automatic estimation and correction of anisotropic magnification distortion in electron microscopes. J Struct Biol 192(2):204–208PubMedCrossRefGoogle Scholar
- 7.Scheres SH (2010) Classification of structural heterogeneity by maximum-likelihood methods. Methods Enzymol 482:295–320PubMedPubMedCentralCrossRefGoogle Scholar
- 8.Orlova EV, Saibil HR (2010) Methods for three-dimensional reconstruction of heterogeneous assemblies. Methods Enzymol 482:321–341PubMedCrossRefGoogle Scholar
- 9.Cheng Y, Grigorieff N, Penczek PA, Walz T (2015) A primer to single-particle cryo-electron microscopy. Cell 161(3):438–449PubMedPubMedCentralCrossRefGoogle Scholar
- 10.Costa TR, Felisberto-Rodrigues C, Meir A, Prevost MS, Redzej A, Trokter M, Waksman G (2015) Secretion systems in Gram-negative bacteria: structural and mechanistic insights. Nat Rev Microbiol 13(6):343–359PubMedCrossRefGoogle Scholar
- 11.Ilangovan A, Connery S, Waksman G (2015) Structural biology of the Gram-negative bacterial conjugation systems. Trends Microbiol 23(5):301–310PubMedCrossRefGoogle Scholar
- 12.Fronzes R, Schafer E, Wang L, Saibil HR, Orlova EV, Waksman G (2009) Structure of a type IV secretion system core complex. Science 323(5911):266–268PubMedCrossRefGoogle Scholar
- 13.Rivera-Calzada A, Fronzes R, Savva CG, Chandran V, Lian PW, Laeremans T, Pardon E, Steyaert J, Remaut H, Waksman G, Orlova EV (2013) Structure of a bacterial type IV secretion core complex at subnanometre resolution. EMBO J 32(8):1195–1204PubMedPubMedCentralCrossRefGoogle Scholar
- 14.Low HH, Gubellini F, Rivera-Calzada A, Braun N, Connery S, Dujeancourt A, Lu F, Redzej A, Fronzes R, Orlova EV, Waksman G (2014) Structure of a type IV secretion system. Nature 508(7497):550–553PubMedPubMedCentralCrossRefGoogle Scholar
- 15.Spence JCH (2003) High resolution microscopy, 3rd edn. Oxford University Press, New YorkGoogle Scholar
- 16.Dubochet J, Adrian M, Chang JJ, Homo JC, Lepault J, McDowall AW, Schultz P (1988) Cryo-electron microscopy of vitrified specimens. Q Rev Biophys 21(2):129–228PubMedCrossRefGoogle Scholar
- 17.Jaffe JS, Glaeser RM (1987) Difference Fourier analysis of “surface features” of bacteriorhodopsin using glucose-embedded and frozen-hydrated purple membrane. Ultramicroscopy 23(1):17–28PubMedCrossRefGoogle Scholar
- 18.Dubochet J, Lepault J, Freeman R, Berriman A, Homo JC (1982) Electron microscopy of frozen water and aqueous solutions. J Microsc 124(3):219–237CrossRefGoogle Scholar
- 19.Lepault J, Dubochet J (1986) Electron microscopy of frozen hydrated specimens: preparation and characteristics. Methods Enzymol 127:719–730PubMedCrossRefGoogle Scholar
- 20.Cabra V, Samso M (2015) Do’s and don’ts of cryo-electron microscopy: a primer on sample preparation and high quality data collection for macromolecular 3D reconstruction. J Vis Exp 95:52311Google Scholar
- 21.Grassucci RA, Taylor DJ, Frank J (2007) Preparation of macromolecular complexes for cryo-electron microscopy. Nat Protoc 2(12):3239–3246PubMedPubMedCentralCrossRefGoogle Scholar
- 22.Adrian M, Dubochet J, Lepault J, McDowall AW (1984) Cryo-electron microscopy of viruses. Nature 308(5954):32–36PubMedCrossRefGoogle Scholar
- 23.Baker LA, Rubinstein JL (2010) Radiation damage in electron cryomicroscopy. Methods Enzymol 481:371–388PubMedCrossRefGoogle Scholar
- 24.Tivol WF, Briegel A, Jensen GJ (2008) An improved cryogen for plunge freezing. Microsc Microanal 14(5):375–379PubMedPubMedCentralCrossRefGoogle Scholar
- 25.Vos MR, Bomans PH, Frederik PM, Sommerdijk NA (2008) The development of a glove-box/Vitrobot combination: air-water interface events visualized by cryo-TEM. Ultramicroscopy 108(11):1478–1483PubMedCrossRefGoogle Scholar
- 26.Jensen GJ (2010) Cryo-EM. Part A: sample preparation and data collection. Preface. Methods Enzymol 481:xv–xviCrossRefGoogle Scholar
- 27.Boyle WS, Smith GE (1970) Charge coupled semiconductor devices. J Bell Syst Tech 49(4):587–593CrossRefGoogle Scholar
- 28.McMullan G, Cattermole DM, Chen S, Henderson R, Llopart X, Summerfield C, Tlustos L, Faruqi AR (2007) Electron imaging with Medipix2 hybrid pixel detector. Ultramicroscopy 107(4–5):401–413PubMedCrossRefGoogle Scholar
- 29.Faruqi AR, Henderson R (2007) Electronic detectors for electron microscopy. Curr Opin Struct Biol 17(5):549–555PubMedCrossRefGoogle Scholar
- 30.Ramachandra R, Bouwer JC, Mackey MR, Bushong E, Peltier ST, Xuong NH, Ellisman MH (2014) Improving signal to noise in labeled biological specimens using energy-filtered TEM of sections with a drift correction strategy and a direct detection device. Microsc Microanal 20(3):706–714PubMedPubMedCentralCrossRefGoogle Scholar
- 31.Veesler D, Campbell MG, Cheng A, Fu CY, Murez Z, Johnson JE, Potter CS, Carragher B (2013) Maximizing the potential of electron cryomicroscopy data collected using direct detectors. J Struct Biol 184(2):193–202PubMedCrossRefGoogle Scholar
- 32.Cunningham IA (1999) Practical digital imaging and PACS. Advanced Medical Publishing for American Association of Physicists in Medicine, USAGoogle Scholar
- 33.McMullan G, Chen S, Henderson R, Faruqi AR (2009) Detective quantum efficiency of electron area detectors in electron microscopy. Ultramicroscopy 109(9):1126–1143PubMedPubMedCentralCrossRefGoogle Scholar
- 34.McMullan G, Faruqi AR, Clare D, Henderson R (2014) Comparison of optimal performance at 300keV of three direct electron detectors for use in low dose electron microscopy. Ultramicroscopy 147:156–163PubMedPubMedCentralCrossRefGoogle Scholar
- 35.Ruskin RS, Yu Z, Grigorieff N (2013) Quantitative characterization of electron detectors for transmission electron microscopy. J Struct Biol 184(3):385–393PubMedCrossRefGoogle Scholar
- 36.Bammes BE, Rochat RH, Jakana J, Chen DH, Chiu W (2012) Direct electron detection yields cryo-EM reconstructions at resolutions beyond 3/4 Nyquist frequency. J Struct Biol 177(3):589–601PubMedPubMedCentralCrossRefGoogle Scholar
- 37.Milazzo AC, Moldovan G, Lanman J, Jin L, Bouwer JC, Klienfelder S, Peltier ST, Ellisman MH, Kirkland AI, Xuong NH (2010) Characterization of a direct detection device imaging camera for transmission electron microscopy. Ultramicroscopy 110(7):744–747PubMedPubMedCentralCrossRefGoogle Scholar
- 38.Campbell MG, Cheng A, Brilot AF, Moeller A, Lyumkis D, Veesler D, Pan J, Harrison SC, Potter CS, Carragher B, Grigorieff N (2012) Movies of ice-embedded particles enhance resolution in electron cryo-microscopy. Structure 20(11):1823–1828PubMedPubMedCentralCrossRefGoogle Scholar
- 39.Li X, Mooney P, Zheng S, Booth CR, Braunfeld MB, Gubbens S, Agard DA, Cheng Y (2013) Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat Methods 10(6):584–590PubMedPubMedCentralCrossRefGoogle Scholar
- 40.Abrishami V, Vargas J, Li X, Cheng Y, Marabini R, Sorzano CO, Carazo JM (2015) Alignment of direct detection device micrographs using a robust optical flow approach. J Struct Biol 189(3):163–176PubMedCrossRefGoogle Scholar
- 41.Afanasyev P, Ravelli RB, Matadeen R, De Carlo S, van Duinen G, Alewijnse B, Peters PJ, Abrahams JP, Portugal RV, Schatz M, van Heel M (2015) A posteriori correction of camera characteristics from large image data sets. Sci Rep 5:10317PubMedPubMedCentralCrossRefGoogle Scholar
- 42.Glaeser RM (1971) Limitations to significant information in biological electron microscopy as a result of radiation damage. J Ultrastruct Res 36(3):466–482PubMedCrossRefGoogle Scholar
- 43.Chiu W, Jeng TW (1982) Electron radiation sensitivity of protein crystals. Ultramicroscopy 10(1–2):63–69PubMedCrossRefGoogle Scholar
- 44.Frank J (2006) Three dimensional electron microscopy of macromolecular assemblies: visualization of biological molecules in their native state, 2nd edn. Oxford University Press, New YorkCrossRefGoogle Scholar
- 45.Egerton RF, Li P, Malac M (2004) Radiation damage in the TEM and SEM. Micron 35(6):399–409PubMedCrossRefGoogle Scholar
- 46.Burmeister WP (2000) Structural changes in a cryo-cooled protein crystal owing to radiation damage. Acta Crystallogr D Biol Crystallogr 56(Pt 3):328–341PubMedCrossRefGoogle Scholar
- 47.Taylor KA, Glaeser RM (1976) Electron microscopy of frozen hydrated biological specimens. J Ultrastruct Res 55(3):448–456PubMedCrossRefGoogle Scholar
- 48.Chiu W (1986) Electron microscopy of frozen, hydrated biological specimens. Annu Rev Biophys Biophys Chem 15:237–257PubMedCrossRefGoogle Scholar
- 49.Knapek E, Dubochet J (1980) Beam damage to organic material is considerably reduced in cryo-electron microscopy. J Mol Biol 141(2):147–161PubMedCrossRefGoogle Scholar
- 50.Bartesaghi A, Matthies D, Banerjee S, Merk A, Subramaniam S (2014) Structure of beta-galactosidase at 3.2-a resolution obtained by cryo-electron microscopy. Proc Natl Acad Sci U S A 111(32):11709–11714PubMedPubMedCentralCrossRefGoogle Scholar
- 51.Carlson DB, Evans JE (2012) Low-dose imaging techniques for transmission electron microscopy. The transmission electron microscope. InTech, ChinaGoogle Scholar
- 52.Erickson HP, Klug A (1971) Measurement and compensation of defocusing and aberrations by Fourier processing of electron micrographs. Philos Trans R Soc B 261(837):105–118CrossRefGoogle Scholar
- 53.Wade RH (1992) A brief look at imaging and contrast transfer. Ultramicroscopy 46:145–156CrossRefGoogle Scholar
- 54.Thon F (1966) Zur Defokussierungsabh ä ngigkeit des Phasenkontrastes bei der elektronenmikroskopischen Abbildung. Naturforschg 21a:476–478Google Scholar
- 55.Tang G, Peng L, Baldwin PR, Mann DS, Jiang W, Rees I, Ludtke SJ (2007) EMAN2: an extensible image processing suite for electron microscopy. J Struct Biol 157(1):38–46PubMedCrossRefGoogle Scholar
- 56.Rohou A, Grigorieff N (2015) CTFFIND4: fast and accurate defocus estimation from electron micrographs. J Struct Biol 192(2):216–221PubMedCrossRefGoogle Scholar
- 57.van Heel M, Harauz G, Orlova EV, Schmidt R, Schatz M (1996) A new generation of the IMAGIC image processing system. J Struct Biol 116(1):17–24PubMedCrossRefGoogle Scholar
- 58.Wiener N (1964) Extrapolation, interpolation, and smoothing of stationary time series. Wiley, New YorkGoogle Scholar
- 59.de la Rosa-Trevin JM, Oton J, Marabini R, Zaldivar A, Vargas J, Carazo JM, Sorzano CO (2013) Xmipp 3.0: an improved software suite for image processing in electron microscopy. J Struct Biol 184(2):321–328PubMedCrossRefGoogle Scholar
- 60.Smith JM (1999) Ximdisp–a visualization tool to aid structure determination from electron microscope images. J Struct Biol 125(2–3):223–228PubMedCrossRefGoogle Scholar
- 61.Scheres SH (2015) Semi-automated selection of cryo-EM particles in RELION-1.3. J Struct Biol 189(2):114–122PubMedPubMedCentralCrossRefGoogle Scholar
- 62.Langlois R, Pallesen J, Ash JT, Nam Ho D, Rubinstein JL, Frank J (2014) Automated particle picking for low-contrast macromolecules in cryo-electron microscopy. J Struct Biol 186(1):1–7PubMedPubMedCentralCrossRefGoogle Scholar
- 63.Heymann JB, Belnap DM (2007) Bsoft: image processing and molecular modeling for electron microscopy. J Struct Biol 157(1):3–18PubMedCrossRefGoogle Scholar
- 64.Roseman AM (2004) FindEM--a fast, efficient program for automatic selection of particles from electron micrographs. J Struct Biol 145(1–2):91–99PubMedCrossRefGoogle Scholar
- 65.Van Heel M, Portugal RV, Schatz M (2009) Multivariate statistical analysis in single particle (Cryo) electron microscopy. In: Verkley A, Orlova E (eds) An electronic textbook: electron microscopy in life science. 3D-EM Network of Excellence, LondonGoogle Scholar
- 66.Grigorieff N (2007) FREALIGN: high-resolution refinement of single particle structures. J Struct Biol 157(1):117–125PubMedCrossRefGoogle Scholar
- 67.Frank J, Radermacher M, Penczek P, Zhu J, Li Y, Ladjadj M, Leith A (1996) SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J Struct Biol 116(1):190–199PubMedCrossRefGoogle Scholar
- 68.Bartholomew DJ, Steele F, Galbraith J, Moustaki I (2008) Analysis of multivariate social science data. Statistics in the social and behavioral sciences series, 2nd edn. Taylor & Francis, USAGoogle Scholar
- 69.Myung IJ (2003) Tutorial on maximum likelihood estimation. J Math Psyc 47(1):90–100CrossRefGoogle Scholar
- 70.Sigworth FJ (1998) A maximum-likelihood approach to single-particle image refinement. J Struct Biol 122(3):328–339PubMedCrossRefGoogle Scholar
- 71.Scheres SH (2012) A Bayesian view on cryo-EM structure determination. J Mol Biol 415(2):406–418PubMedPubMedCentralCrossRefGoogle Scholar
- 72.Macqueen J (1967) Some methods for classification and analysis of multivariate observations. Proc Fifth Berkeley Symp Math Stat Prob 1:281–297Google Scholar
- 73.Ward JHJ (1963) Hierarchical grouping to optimize an objective function. J Am Stat Assoc 58(301):236–244CrossRefGoogle Scholar
- 74.Guan W, Lockwood A, Inkson BJ, Mobus G (2011) A piezoelectric goniometer inside a transmission electron microscope goniometer. Microsc Microanal 17(5):827–833PubMedCrossRefGoogle Scholar
- 75.Van Heel M (1987) Angular reconstitution: a posteriori assignment of projection directions for 3D reconstruction. Ultramicroscopy 21(2):111–123PubMedCrossRefGoogle Scholar
- 76.van Heel M, Orlova EV, Harauz G, Stark H, Dube P, Zemlin F, Schatz M (1997) Angular reconstitution in three-dimentional electron microscopy: historical and theoretical aspects. Scanning Microsc 11:195–210Google Scholar
- 77.Crowther RA (1971) Procedures for three-dimensional reconstruction of spherical viruses by Fourier synthesis from electron micrographs. Philos Trans R Soc Lond Ser B Biol Sci 261(837):221–230CrossRefGoogle Scholar
- 78.Fuller SD (1987) The T=4 envelope of Sindbis virus is organized by interactions with a complementary T=3 capsid. Cell 48(6):923–934PubMedCrossRefGoogle Scholar
- 79.Herman GT (1980) Image reconstruction from projections: the fundamentals of computerized tomography. Academic, New YorkGoogle Scholar
- 80.Penczek PA (2010) Fundamentals of three-dimensional reconstruction from projections, vol 482. Methods in enzymology: Cryo-EM, part B, 3-D reconstruction. Academic, Elsevier, San Diego, CAGoogle Scholar
- 81.Orlova EV, Saibil HR (2011) Structural analysis of macromolecular assemblies by electron microscopy. Chem Rev 111(12):7710–7748PubMedPubMedCentralCrossRefGoogle Scholar
- 82.Harauz G, van Heel M (1986a) Exact filters for general geometry three-dimensional reconstruction. Optik 73:146–156Google Scholar
- 83.Orlov SS (1976) Theory of three dimensional reconstruction—conditions of a complete set of projections. Sov Phys Crystallogr 20:312–314Google Scholar
- 84.Radermacher M (1988) Three-dimensional reconstruction of single particles from random and nonrandom tilt series. J Electron Microsc Tech 9(4):359–394PubMedCrossRefGoogle Scholar
- 85.De Rosier DJ, Klug A (1968) Reconstruction of three dimensional structures from electron micrographs. Nature 217(5124):130–134PubMedCrossRefGoogle Scholar
- 86.Crowther RA, DeRosier DJ, Klug A (1970) The reconstruction of a three-dimensional structure from projections and its application to electron microscopy. Proc R Soc A 317 (1530)Google Scholar
- 87.DeRosier DJ, Moore PB (1970) Reconstruction of three-dimensional images from electron micrographs of structures with helical symmetry. J Mol Biol 52(2):355–369PubMedCrossRefGoogle Scholar
- 88.Penczek PA (2008) Single particle reconstruction. In: Shmueli U (ed) International tables for crystallography. Springer, New York, pp 375–388Google Scholar
- 89.Glaeser RM, Downing KH, DeRosier DJ, Chiu W, Frank J (2007) Electron crystallography of biological macromolecules. Oxford University Press, New YorkGoogle Scholar
- 90.van Heel M, Schatz M (2005) Fourier shell correlation threshold criteria. J Struct Biol 151(3):250–262PubMedCrossRefGoogle Scholar
- 91.Rosenthal PB, Henderson R (2003) Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J Mol Biol 333(4):721–745PubMedCrossRefGoogle Scholar
- 92.Scheres SH, Chen S (2012) Prevention of overfitting in cryo-EM structure determination. Nat Methods 9(9):853–854PubMedPubMedCentralCrossRefGoogle Scholar
- 93.Chen S, McMullan G, Faruqi AR, Murshudov GN, Short JM, Scheres SH, Henderson R (2013) High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy 135:24–35PubMedPubMedCentralCrossRefGoogle Scholar
- 94.Unser M, Trus BL, Steven AC (1987) A new resolution criterion based on spectral signal-to-noise ratios. Ultramicroscopy 23(1):39–51PubMedCrossRefGoogle Scholar
- 95.Unser M, Trus BL, Frank J, Steven AC (1989) The spectral signal-to-noise ratio resolution criterion: computational efficiency and statistical precision. Ultramicroscopy 30(3):429–433PubMedCrossRefGoogle Scholar
- 96.Unser M, Sorzano CO, Thevenaz P, Jonic S, El-Bez C, De Carlo S, Conway JF, Trus BL (2005) Spectral signal-to-noise ratio and resolution assessment of 3D reconstructions. J Struct Biol 149(3):243–255PubMedCrossRefGoogle Scholar
- 97.Penczek PA (2002) Three-dimensional spectral signal-to-noise ratio for a class of reconstruction algorithms. J Struct Biol 138(1–2):34–46PubMedCrossRefGoogle Scholar
- 98.Kessel M, Radermacher M, Frank J (1985) The structure of the stalk surface layer of a brine pond microorganism: correlation averaging applied to a double layered lattice structure. J Microsc 139(Pt 1):63–74PubMedCrossRefGoogle Scholar
- 99.van Heel M, Hollenberg J (1980) The stretching of distorted images of two-dimensional crystals. Electron microscopy at molecular dimensions. Springer, BerlinGoogle Scholar
- 100.Sousa D, Grigorieff N (2007) Ab initio resolution measurement for single particle structures. J Struct Biol 157(1):201–210PubMedCrossRefGoogle Scholar
- 101.Kucukelbir A, Sigworth FJ, Tagare HD (2014) Quantifying the local resolution of cryo-EM density maps. Nat Methods 11(1):63–65PubMedCrossRefGoogle Scholar
- 102.Zhang R, Alushin GM, Brown A, Nogales E (2015) Mechanistic origin of microtubule dynamic instability and its modulation by EB proteins. Cell 162(4):849–859PubMedPubMedCentralCrossRefGoogle Scholar
- 103.Clare DK, Orlova EV (2010) 4.6A cryo-EM reconstruction of tobacco mosaic virus from images recorded at 300 keV on a 4k x 4k CCD camera. J Struct Biol 171(3):303–308PubMedPubMedCentralCrossRefGoogle Scholar
- 104.Brown A, Long F, Nicholls RA, Toots J, Emsley P, Murshudov G (2015) Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions. Acta Crystallogr D Biol Crystallogr 71(Pt 1):136–153PubMedPubMedCentralCrossRefGoogle Scholar
- 105.Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJ (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10(6):845–858PubMedPubMedCentralCrossRefGoogle Scholar
- 106.Yang J, Yan R, Roy A, Xu D, Poisson J, Zhang Y (2015) The I-TASSER suite: protein structure and function prediction. Nat Methods 12(1):7–8PubMedPubMedCentralCrossRefGoogle Scholar
- 107.Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF chimera—a visualization system for exploratory research and analysis. J Comput Chem 25(13):1605–1612PubMedCrossRefGoogle Scholar
- 108.Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60(Pt 12 Pt 1):2126–2132PubMedCrossRefGoogle Scholar
- 109.Topf M, Lasker K, Webb B, Wolfson H, Chiu W, Sali A (2008) Protein structure fitting and refinement guided by cryo-EM density. Structure 16(2):295–307PubMedPubMedCentralCrossRefGoogle Scholar
- 110.Lopez-Blanco JR, Chacon P (2013) iMODFIT: efficient and robust flexible fitting based on vibrational analysis in internal coordinates. J Struct Biol 184(2):261–270PubMedCrossRefGoogle Scholar
- 111.Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW, McCoy AJ, Moriarty NW, Oeffner R, Read RJ, Richardson DC, Richardson JS, Terwilliger TC, Zwart PH (2010) PHENIX: a comprehensive python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66(Pt 2):213–221PubMedPubMedCentralCrossRefGoogle Scholar
- 112.Zhu J, Cheng L, Fang Q, Zhou ZH, Honig B (2010) Building and refining protein models within cryo-electron microscopy density maps based on homology modeling and multiscale structure refinement. J Mol Biol 397(3):835–851PubMedPubMedCentralCrossRefGoogle Scholar
- 113.Lindert S, Alexander N, Wotzel N, Karakas M, Stewart PL, Meiler J (2012) EM-fold: de novo atomic-detail protein structure determination from medium-resolution density maps. Structure 20(3):464–478PubMedPubMedCentralCrossRefGoogle Scholar
- 114.Ramachandran GN, Ramakrishnan C, Sasisekharan V (1963) Stereochemistry of polypeptide chain configurations. J Mol Biol 7:95–99PubMedCrossRefGoogle Scholar