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- EMDB-3403: Asymmetric cryo-EM reconstruction of phage MS2 reveals genome str... -

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Basic information

Entry
Database: EMDB / ID: EMD-3403
TitleAsymmetric cryo-EM reconstruction of phage MS2 reveals genome structure in situ
Map dataAsymmetric reconstruction of MS2 virion
Sample
  • Sample: Bacteriophage MS2
  • Virus: Enterobacterio phage MS2 (virus)
KeywordsMS2 / RNA genome / structure / cryo-EM
Biological speciesEnterobacterio phage MS2 (virus)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 10.5 Å
AuthorsKoning RI / Gomez-Blanco J / Akopjana I / Vargas J / Kazaks A / Tars K / Carazo JM / Koster AJ
CitationJournal: Nat Commun / Year: 2016
Title: Asymmetric cryo-EM reconstruction of phage MS2 reveals genome structure in situ.
Authors: Roman I Koning / Josue Gomez-Blanco / Inara Akopjana / Javier Vargas / Andris Kazaks / Kaspars Tars / José María Carazo / Abraham J Koster /
Abstract: In single-stranded ribonucleic acid (RNA) viruses, virus capsid assembly and genome packaging are intertwined processes. Using cryo-electron microscopy and single particle analysis we determined the ...In single-stranded ribonucleic acid (RNA) viruses, virus capsid assembly and genome packaging are intertwined processes. Using cryo-electron microscopy and single particle analysis we determined the asymmetric virion structure of bacteriophage MS2, which includes 178 copies of the coat protein, a single copy of the A-protein and the RNA genome. This reveals that in situ, the viral RNA genome can adopt a defined conformation. The RNA forms a branched network of stem-loops that almost all allocate near the capsid inner surface, while predominantly binding to coat protein dimers that are located in one-half of the capsid. This suggests that genomic RNA is highly involved in genome packaging and virion assembly.
History
DepositionMar 30, 2016-
Header (metadata) releaseApr 6, 2016-
Map releaseSep 21, 2016-
UpdateOct 12, 2016-
Current statusOct 12, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-3403.png

Downloads & links

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Map

FileDownload / File: emd_3403.map.gz / Format: CCP4 / Size: 111 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAsymmetric reconstruction of MS2 virion
Voxel sizeX=Y=Z: 1.14 Å
Density
Contour LevelBy AUTHOR: 0.01 / Movie #1: 0.01
Minimum - Maximum-0.02237265 - 0.03816912
Average (Standard dev.)0.00023186 (±0.00549987)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions310310310
Spacing310310310
CellA=B=C: 353.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.141.141.14
M x/y/z310310310
origin x/y/z0.0000.0000.000
length x/y/z353.400353.400353.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-147-147-146
NX/NY/NZ294294294
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS310310310
D min/max/mean-0.0220.0380.000

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Supplemental data

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Sample components

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Entire : Bacteriophage MS2

EntireName: Bacteriophage MS2 (virus)
Components
  • Sample: Bacteriophage MS2
  • Virus: Enterobacterio phage MS2 (virus)

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Supramolecule #1000: Bacteriophage MS2

SupramoleculeName: Bacteriophage MS2 / type: sample / ID: 1000 / Details: Purified by gel filtration
Oligomeric state: One copy of genomic RNA inside a shell of 178 capsid proteins and one A-protein
Number unique components: 1
Molecular weightTheoretical: 3.6 MDa

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Supramolecule #1: Enterobacterio phage MS2

SupramoleculeName: Enterobacterio phage MS2 / type: virus / ID: 1 / Name.synonym: bacteriophage MS2 / NCBI-ID: 12022 / Sci species name: Enterobacterio phage MS2 / Virus type: VIRION / Virus isolate: SUBSPECIES / Virus enveloped: No / Virus empty: No / Syn species name: bacteriophage MS2 / Sci species subspecies: Enterobacterio phage MS2
Host (natural)Organism: Escherichia coli (E. coli) / Strain: XL-1 blue / synonym: BACTERIA(EUBACTERIA)
Molecular weightTheoretical: 3.6 MDa
Virus shellShell ID: 1 / Name: MS2 / Diameter: 275 Å / T number (triangulation number): 3

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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: 7.4 / Details: PBS
StainingType: NEGATIVE / Details: Vitrification
GridDetails: Cu 300 Mesh with quantifoil R2/2 support film, glow discharged in air at 0.2 mbar for 1 minute at 30 mA.
VitrificationCryogen name: ETHANE-PROPANE MIXTURE / Chamber humidity: 70 % / Chamber temperature: 100 K / Instrument: LEICA EM GP
Method: Blotted using filter paper for 1 to 2 seconds before blotting

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 61403 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.02 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 59000
Sample stageSpecimen holder: Nitrogen cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
TemperatureMin: 77 K / Max: 97 K / Average: 87 K
Alignment procedureLegacy - Astigmatism: Cs corrector / Legacy - Electron beam tilt params: 15
DateMay 19, 2014
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Digitization - Sampling interval: 14 µm / Number real images: 751 / Average electron dose: 35 e/Å2
Details: Images are average of 7 frames recorded in movie mode on direct electron detector
Bits/pixel: 16
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Per image
Final two d classificationNumber classes: 5201
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 10.5 Å / Resolution method: OTHER / Software - Name: Scipion, Xmipp, CTFFIND3, Relion / Number images used: 18977
DetailsMovies were aligned using Optical Flow, CTFs were estimated using CTFFIND3, and were used to select the best quality micrographs. A total of 22,441 particles were picked automatically using Xmipp. Particles were classified using 2D reference-free Relion approach. Relion was used for 3D refinement using icosahedral symmetry and gold-standard approach.

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