The Protein Journal

, Volume 32, Issue 8, pp 635–640 | Cite as

Using cryoEM Reconstruction and Phase Extension to Determine Crystal Structure of Bacteriophage ϕ6 Major Capsid Protein



Bacteriophage ϕ6 is a double-stranded RNA virus that has been extensively studied as a model organism. Here we describe structure determination of ϕ6 major capsid protein P1. The protein crystallized in base centered orthorhombic space group C2221. Matthews’s coefficient indicated that the crystals contain from four to seven P1 subunits in the crystallographic asymmetric unit. The self-rotation function had shown presence of fivefold axes of non-crystallographic symmetry in the crystals. Thus, electron density map corresponding to a P1 pentamer was excised from a previously determined cryoEM reconstruction of the ϕ6 procapsid at 7 Å resolution and used as a model for molecular replacement. The phases for reflections at higher than 7 Å resolution were obtained by phase extension employing the fivefold non-crystallographic symmetry present in the crystal. The averaged 3.6 Å-resolution electron density map was of sufficient quality to allow model building.


Molecular replacement Cryo-electron microscopy Non-crystallographic symmetry Virus capsid protein Phase extension 



Non-crystallographic symmetry


Cryo-electron microscopy



This research was supported by MarieCurie FP7-PEOPLE-2012-CIG, project number 333916, and by Academy of Sciences Czech Republic (RVO: 61388963). Crystallographic data were collected at the Southeast Regional Collaborative Access Team 22-ID beamline at the Advanced Photon Source, Argonne National Laboratory. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract W-31-109-Eng-38.


  1. 1.
    Mindich L (1999) Precise packaging of the three genomic segments of the double-stranded-RNA bacteriophage phi6. Microbiol Mol Biol Rev MMBR 63:149–160Google Scholar
  2. 2.
    Mindich L (2004) Packaging, replication and recombination of the segmented genome of bacteriophage Phi6 and its relatives. Virus Res 101:83–92CrossRefGoogle Scholar
  3. 3.
    Poranen MM, Tuma R (2004) Self-assembly of double-stranded RNA bacteriophages. Virus Res 101:93–100CrossRefGoogle Scholar
  4. 4.
    Mindich L (2012) Packaging in dsRNA viruses. Adv Exp Med Biol 726:601–608CrossRefGoogle Scholar
  5. 5.
    Poranen MM, Bamford DH (2012) Assembly of large icosahedral double-stranded RNA viruses. Adv Exp Med Biol 726:379–402CrossRefGoogle Scholar
  6. 6.
    Huiskonen JT, de Haas F, Bubeck D, Bamford DH, Fuller SD, Butcher SJ (2006) Structure of the bacteriophage phi6 nucleocapsid suggests a mechanism for sequential RNA packaging. Structure 14:1039–1048CrossRefGoogle Scholar
  7. 7.
    Qiao X, Qiao J, Mindich L (1997) Stoichiometric packaging of the three genomic segments of double-stranded RNA bacteriophage phi6. Proc Natl Acad Sci USA 94:4074–4079CrossRefGoogle Scholar
  8. 8.
    Nemecek D, Cheng N, Qiao J, Mindich L, Steven AC, Heymann JB (2011) Stepwise expansion of the bacteriophage varphi6 procapsid: possible packaging intermediates. J Mol Biol 414:260–271CrossRefGoogle Scholar
  9. 9.
    Kainov DE, Pirttimaa M, Tuma R, Butcher SJ, Thomas GJ Jr, Bamford DH, Makeyev EV (2003) RNA packaging device of double-stranded RNA bacteriophages, possibly as simple as hexamer of P4 protein. J Biol Chem 278:48084–48091CrossRefGoogle Scholar
  10. 10.
    Nemecek D, Boura E, Wu W, Cheng N, Plevka P, Qiao J, Mindich L, Heymann JB, Hurley JH, Steven AC (2013) Subunit folds and maturation pathway of a dsRNA virus capsid. Structure 21:1374–1383Google Scholar
  11. 11.
    Zhou ZH (2008) Towards atomic resolution structural determination by single-particle cryo-electron microscopy. Curr Opin Struct Biol 18:218–228CrossRefGoogle Scholar
  12. 12.
    Grigorieff N, Harrison SC (2011) Near-atomic resolution reconstructions of icosahedral viruses from electron cryo-microscopy. Curr Opin Struct Biol 21:265–273CrossRefGoogle Scholar
  13. 13.
    Bai XC, Fernandez IS, McMullan G, Scheres SH (2013) Ribosome structures to near-atomic resolution from thirty thousand cryo-EM particles. eLife. 2:e00461CrossRefGoogle Scholar
  14. 14.
    Minor W, Cymborowski M, Otwinowski Z, Chruszcz M (2006) HKL-3000: the integration of data reduction and structure solution–from diffraction images to an initial model in minutes. Acta Crystallogr D Biol Crystallogr 62:859–866CrossRefGoogle Scholar
  15. 15.
    Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan RM, Krissinel EB, Leslie AG, McCoy A, McNicholas SJ, Murshudov GN, Pannu NS, Potterton EA, Powell HR, Read RJ, Vagin A, Wilson KS (2011) Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr 67:235–242CrossRefGoogle Scholar
  16. 16.
    Tong L, Rossmann MG (1997) Rotation function calculations with GLRF program. Methods Enzymol 276:594–611CrossRefGoogle Scholar
  17. 17.
    Nemecek D, Qiao J, Mindich L, Steven AC, Heymann JB (2012) Packaging accessory protein P7 and polymerase P2 have mutually occluding binding sites inside the bacteriophage 6 procapsid. J Virol 86:11616–11624CrossRefGoogle Scholar
  18. 18.
    Yang Z, Lasker K, Schneidman-Duhovny D, Webb B, Huang CC, Pettersen EF, Goddard TD, Meng EC, Sali A, Ferrin TE (2012) UCSF Chimera MODELLER, and IMP: an integrated modeling system. J Struct Biol 179:269–278CrossRefGoogle Scholar
  19. 19.
    Pintilie GD, Zhang J, Goddard TD, Chiu W, Gossard DC (2010) Quantitative analysis of cryo-EM density map segmentation by watershed and scale-space filtering, and fitting of structures by alignment to regions. J Struct Biol 170:427–438CrossRefGoogle Scholar
  20. 20.
    McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ (2007) Phaser crystallographic software. J Appl Crystallogr 40:658–674CrossRefGoogle Scholar
  21. 21.
    Kleywegt GJ, Jones TA (1999) Software for handling macromolecular envelopes. Acta Crystallogr D Biol Crystallogr 55:941–944CrossRefGoogle Scholar
  22. 22.
    Kleywegt GJ, Read RJ (1997) Not your average density. Structure 5:1557–1569CrossRefGoogle Scholar
  23. 23.
    El Omari K, Sutton G, Ravantti JJ, Zhang H, Walter TS, Grimes JM, Bamford DH, Stuart DI, Mancini EJ (2013) Plate tectonics of virus shell assembly and reorganization in phage ϕ8, a distant relative of mammalian reoviruses. Structure 21:1384–1395Google Scholar
  24. 24.
    Veesler D, Johnson JE (2013) Cystovirus maturation at atomic resolution. Structure 21:1266–1268CrossRefGoogle Scholar
  25. 25.
    Cowtan K (2008) Fitting molecular fragments into electron density. Acta Crystallogr D Biol Crystallogr 64:83–89CrossRefGoogle Scholar
  26. 26.
    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 66:213–221CrossRefGoogle Scholar
  27. 27.
    Grimes JM, Burroughs JN, Gouet P, Diprose JM, Malby R, Zientara S, Mertens PP, Stuart DI (1998) The atomic structure of the bluetongue virus core. Nature 395:470–478CrossRefGoogle Scholar
  28. 28.
    Pan J, Dong L, Lin L, Ochoa WF, Sinkovits RS, Havens WM, Nibert ML, Baker TS, Ghabrial SA, Tao YJ (2009) Atomic structure reveals the unique capsid organization of a dsRNA virus. Proc Natl Acad Sci USA 106:4225–4230CrossRefGoogle Scholar
  29. 29.
    Duquerroy S, Da Costa B, Henry C, Vigouroux A, Libersou S, Lepault J, Navaza J, Delmas B, Rey FA (2009) The picobirnavirus crystal structure provides functional insights into virion assembly and cell entry. EMBO J 28:1655–1665CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.Central European Institute of TechnologyMasaryk UniversityBrnoCzech Republic
  2. 2.Institute of Organic Chemistry and Biochemistry AS CRPrague 6Czech Republic

Personalised recommendations