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- EMDB-2435: Protein Interactions in the Murine Cytomegalovirus Capsid Reveale... -

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Basic information

Entry
Database: EMDB / ID: EMD-2435
TitleProtein Interactions in the Murine Cytomegalovirus Capsid Revealed by CryoEMProtein
Map dataReconstruction of Murine Cytomegalovirus
Sample
  • Sample: Murine cytomegalovirus capsid
  • Virus: Murid herpesvirus 1 (Murine cytomegalovirus)
Keywordscytomegalovirus / herpes simplex virus type 1 / electron cryo microscopy / three-dimensional / major capsid protein.
Biological speciesMurid herpesvirus 1 (Murine cytomegalovirus)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 9.1 Å
AuthorsHui W / Tang Q / Liu H / Atanasov I / Zhu H / Zhou ZH
CitationJournal: Protein Cell / Year: 2013
Title: Protein interactions in the murine cytomegalovirus capsid revealed by cryoEM.
Authors: Wong H Hui / Qiyi Tang / Hongrong Liu / Ivo Atanasov / Fenyong Liu / Hua Zhu / Z Hong Zhou /
Abstract: Cytomegalovirus (CMV) is distinct among members of the Herpesviridae family for having the largest dsDNA genome (230 kb). Packaging of large dsDNA genome is known to give rise to a highly pressurized ...Cytomegalovirus (CMV) is distinct among members of the Herpesviridae family for having the largest dsDNA genome (230 kb). Packaging of large dsDNA genome is known to give rise to a highly pressurized viral capsid, but molecular interactions conducive to the formation of CMV capsid resistant to pressurization have not been described. Here, we report a cryo electron microscopy (cryoEM) structure of the murine cytomegalovirus (MCMV) capsid at a 9.1 Å resolution and describe the molecular interactions among the ∼3000 protein molecules in the MCMV capsid at the secondary structure level. Secondary structural elements are resolved to provide landmarks for correlating with results from sequence-based prediction and for structure-based homology modeling. The major capsid protein (MCP) upper domain (MCPud) contains α-helices and β-sheets conserved with those in MCPud of herpes simplex virus type 1 (HSV-1), with the largest differences identified as a "saddle loop" region, located at the tip of MCPud and involved in interaction with the smallest capsid protein (SCP). Interactions among the bacteriophage HK97-like floor domain of MCP, the middle domain of MCP, the hook and clamp domains of the triplex proteins (hoop and clamp domains of TRI-1 and clamp domain of TRI-2) contribute to the formation of a mature capsid. These results offer a framework for understanding how cytomegalovirus uses various secondary structural elements of its capsid proteins to build a robust capsid for packaging its large dsDNA genome inside and for attaching unique functional tegument proteins outside.
History
DepositionAug 8, 2013-
Header (metadata) releaseSep 4, 2013-
Map releaseSep 18, 2013-
UpdateNov 20, 2013-
Current statusNov 20, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 15
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 15
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2435.png

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Map

FileDownload / File: emd_2435.map.gz / Format: CCP4 / Size: 817.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of Murine Cytomegalovirus
Voxel sizeX=Y=Z: 1.8 Å
Density
Contour LevelBy AUTHOR: 15.0 / Movie #1: 15
Minimum - Maximum-21.674497599999999 - 45.755878449999997
Average (Standard dev.)1.05608952 (±5.15527439)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin100100530
Dimensions760760380
Spacing760760380
CellA: 1368.0 Å / B: 1368.0 Å / C: 684.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.81.81.8
M x/y/z760760380
origin x/y/z0.0000.0000.000
length x/y/z1368.0001368.000684.000
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS100100530
NC/NR/NS760760380
D min/max/mean-21.67445.7561.056

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Supplemental data

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Sample components

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Entire : Murine cytomegalovirus capsid

EntireName: Murine cytomegalovirus capsid
Components
  • Sample: Murine cytomegalovirus capsid
  • Virus: Murid herpesvirus 1 (Murine cytomegalovirus)

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Supramolecule #1000: Murine cytomegalovirus capsid

SupramoleculeName: Murine cytomegalovirus capsid / type: sample / ID: 1000 / Details: The sample is monodisperse in PBS buffer / Oligomeric state: icosahedral virus capsid / Number unique components: 4
Molecular weightTheoretical: 186.4 MDa

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Supramolecule #1: Murid herpesvirus 1

SupramoleculeName: Murid herpesvirus 1 / type: virus / ID: 1 / Name.synonym: Murine cytomegalovirus
Details: The virus is enveloped but the structure presented here is for the capsid only.
NCBI-ID: 10366 / Sci species name: Murid herpesvirus 1 / Sci species strain: strain Smith / Virus type: OTHER / Virus isolate: STRAIN / Virus enveloped: Yes / Virus empty: Yes / Syn species name: Murine cytomegalovirus
Host (natural)Organism: Murine / Strain: strain Smith / synonym: INVERTEBRATES
Molecular weightTheoretical: 186.4 MDa
Virus shellShell ID: 1 / Name: Capsid / Diameter: 1310 Å / T number (triangulation number): 16

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 7 / Details: PBS
StainingType: NEGATIVE / Details: vitreous ice, no staining
GridDetails: across holes in Quantifoil grids
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 90 K / Instrument: FEI VITROBOT MARK I / Details: Vitrification instrument: FEI Vitrobot / Method: Blot for 7-9 seconds before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 47000
Specialist opticsEnergy filter - Name: FEI
Sample stageSpecimen holder: Autoloader of Titan Krios at liquid nitrogen temperature
Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
TemperatureMin: 80 K / Max: 100 K / Average: 90 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 250,000 times magnification
Legacy - Electron beam tilt params: 0
DateOct 15, 2009
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 6.35 µm / Number real images: 1299 / Average electron dose: 20 e/Å2 / Bits/pixel: 16
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: CTFFIND
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.1 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMIRS / Number images used: 3467
DetailsIndividual particle images were automatically boxed out from micrographs by the autoBox program in the IMIRS package, followed by manual screening to select good capsid particle images that appear perfectly intact and without contamination and signs of specimen charging. Defocus value and astigmatism parameters of each micrographs were determined with CTFFIND. Subsequent data processing includes the determination of particle orientation/center parameters, 3D reconstruction and iterative, projection-based refinement by a distributed computing approach using modular programs in the IMIRS package with recent enhancements. The distributed computing was performed entirely through on six Microsoft Windows personal computers and four Windows servers within a custom-designed MPI network. Astigmatism was taken into consideration during the correction of contrast transfer function (CTF) both in the orientation/center refinement step and the 3D reconstruction step. Using this procedure, we first obtained a 3D map from 6402 particle images from the Polara micrographs. This map has a resolution of about 11 angstrom and was used as the starting model to assist processing the higher resolution particle images of the Titan micrographs. To process the Titan micrographs, we discarded micrographs with specimen charging by evaluating the Fourier transform of the micrographs and selected 669 micrographs for in-depth data processing. From the 669 good Titan micrographs selected out through this process, we boxed 5383 particle images and determined their orientation/center parameters by using the 11 angstrom map as the starting model. These orientation/center parameters were iteratively refined against the latest 3D map by gradually including Fourier data at regions of higher spatial frequency. The iterative process was terminated when the reconstruction converges to a stable solution and no further improvement in the resolution of the map was observed. Our reconstruction converges when the high spatial frequency cut-off of the included image data reached 1/6.9 angstrom-1. The final map was obtained from 3467 particles images, all from the Titan micrographs.

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Atomic model buiding 1

Initial modelPDB ID:

1no7


Chain - Chain ID: A
SoftwareName: Chimera
DetailsManual fitting in Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Chimera fit-to-map

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