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Title:Combined EM/X-ray imaging yields a quasi-atomic model of the adenovirus-related bacteriophage PRD1 and shows key capsid and membrane interactions.
Authors:C. San Martin, R. M. Burnett, de Haas, R. Heinkel, T. W. Rutten, S. D. Fuller, S. J. Butcher, D. H. Bamford
Sample:Bacteriophage PRD1 sus1 mutant
Aggregation state:Icosahedral (14 angstroms resolution)
Red flagLatest update:2011-05-26
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Sample
Sample name: Bacteriophage PRD1 sus1 mutant
Oligomeric state: A pseudo T=25 assembly
Theoretical molecular weight of the sample: 70
Components:
ID Type Name Exp. MW (MDa) Oligomeric details Recombinant expression Synthetic Organism GO identifier InterPro identifier Virus identifier Details
1virusPRD1PRD100.068.0.01.0004a T=25 virion
Experiment
Sample preparation:
pHSample conc.DetailsStainingSample support details
7.21 mg/mL20 mM Tris HCl
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
ETHANE60%23 KHOMEMADE PLUNGERBlot for 2 s before plunging into ethane slush msVitrification instrument: EMBL plunger. vitrification carried out at 23 degrees at ambient humidity
Imaging:
MicroscopeVoltageIllumination modeImaging modeCs:Defocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI/PHILIPS CM200FEG/ST200 kVFLOOD BEAMBRIGHT FIELD2 mm1300 nm4100 nm36000FIELD EMISSION GUNSO16344 mm

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
eucentricGATAN LIQUID NITROGEN°° eV105 K K K mrad6 e/Å2the images are taken a underfocused although the entry indicates a positive defocus value
Processing
Software:EMBL-ICOS, MRC
CTF correction:normalized sum of ctf multiplied images
Resolution by author:14 Å
Resolution method:14 by FSC between independent reconstructions
Processing details:final maps were calculated as a normalized sum of image ffts using the svd versions of matbg and then normalized using an overall profile de Haas, F., Paatero, A. O., Mindich, L., Bamford, D. H. and Fuller, S. D. (1999). A symmetry mismatch at the site of RNA packaging by the polymerase complex of dsRNA bacteriophage phi-6. Journal of Molecular Biology 294, 357-372. Mancini, E. J., Clarke, M., Gowen, B., Rutten, T. and Fuller, S. D. (2000). Cryo-electron microscopy reveals the functional organization of an enveloped virus, Semliki Forest virus. Molecular Cell 5, 255-266. San Martin, C., Burnett, R. M., de Haas, F., Heinkel, R., Rutten, T., Fuller, S. D., Butcher, S. J. and Bamford, D. H. (2001). Combined EM/X-ray imaging yields a quasi-atomic model of the adenovirus-related bacteriophage PRD1 and shows key capsid and membrane interactions. Structure (Camb) 9, 917-30. Baker, T. S., Olson, N. H., and Fuller, S. D. (1999). Adding the third dimension to virus life cycles: Three-Dimensional Reconstruction of Icosahedral Viruses from Cryo-Electron Micrographs. Microbiology and Molecular Biology Reviews 63, 862-922. Butcher, S. J., Bamford, D. H., and Fuller, S. D. (1995). DNA packaging orders the membrane of bacteriophage PRD1. Embo J 14, 6078-6086. Ferlenghi, I., Gowen, B., de Haas, F., Mancini, E. J., Garoff, H., Sjoberg, M., and Fuller, S. D. (1998). The first step: activation of the Semliki Forest virus spike protein precursor causes a localized conformational change in the trimeric spike. J Mol Biol 283, 71-81. Fuller, S. D., Berriman, J. A., Butcher, S. J., and Gowen, B. E. (1995). Low pH induces swiveling of the glycoprotein heterodimers in the Semliki Forest virus spike complex. Cell 81, 715-725. Fuller, S. D., Butcher, S. J., Cheng, R. H., and Baker, T. S. (1996). Three-dimensional reconstruction of icosahedral particles--the uncommon line. J Struct Biol 116, 48-55. Mancini, E. J., Clarke, M., Gowen, B., Rutten, T., and Fuller, S. D. (2000). Cryo-electron microscopy reveals the functional organization of an enveloped virus, Semliki Forest virus. Molecular Cell 5, 255-266. Mancini, E. J., de Haas, F., and Fuller, S. D. (1997). High-resolution icosahedral reconstruction: fulfilling the promise of cryo-electron microscopy. Structure 5, 741-750. San Martin, C., Burnett,R. M., de Haas, F., Heinkel, R., Rutten, T., Fuller, S. D., Butcher, S. J., and Bamford, D. H. (2001). Combined EM/X-ray imaging yields a quasi-atomic model of the adenovirus-related bacteriophage PRD1 and shows key capsid and membrane interactions. Structure (Camb) 9, 917-930. San Martin, C., Huiskonen, J. T., Bamford, J. K., Butcher, S. J., Fuller, S. D., Bamford, D. H., and Burnett, R. M. (2002). Minor proteins, mobile arms and membrane-capsid interactions in the bacteriophage PRD1 capsid. Nat Struct Biol 9, 756-763. Sheehan, B., Fuller, S. D., Pique, M. E., and Yeager, M. (1996). AVS software for visualization in molecular microscopy. J Struct Biol 116, 99-106.
Unit cell:
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
297.0 μm/pixel18linkimages were scanned at 7 micron steps size and then averaged to give a final size of 14 micronsZEISS SCAI
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails
1HB7 rigid body refinementminimizing R factor and clashesX-plor and emfit (M. Rossmann Cheng, R., Kuhn, R., Olson, N., Rossmann, M., and Baker, T. (1995). Nucleocapsid and glycoprotein organization in an enveloped virus. Cell 80, 621-630.)400RECIPROCALThe amplitude and distance scales were determined by comparison with an initial quasi atomic model
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