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- EMDB-4610: CryoEM reconstruction of Cowpea Mosaic Virus (CPMV) bound to an A... -

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Basic information

Entry
Database: EMDB / ID: EMD-4610
TitleCryoEM reconstruction of Cowpea Mosaic Virus (CPMV) bound to an Affimer reagent
Map datapostprocessed map of cryoEM map of Cowpea Mosaic virus bound to an Affimer protein
Sample
  • Complex: Cowpea mosaic virus
    • Complex: Affimer
      • Protein or peptide: Affimer binding protein
    • Virus: Cowpea mosaic virus
      • Protein or peptide: RNA2 polyprotein
      • Protein or peptide: Cowpea mosaic virus large subunit
Function / homology
Function and homology information


transport of virus in host, cell to cell / host cell plasmodesma / T=3 icosahedral viral capsid / host cell nucleus / GTP binding / structural molecule activity / DNA binding / RNA binding
Similarity search - Function
Large coat protein / RNA2 polyprotein / Large coat protein / Small coat protein / Viral coat protein subunit
Similarity search - Domain/homology
Biological speciessynthetic construct (others) / Cowpea mosaic virus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsHesketh EL / Tiede C / Adamson H / Adams TL / Byrne MJ / Meshcheriakova Y / Lomonossoff GP / Kruse I / McPherson MJ / Tomlinson DC / Ranson NA
Funding support United Kingdom, 3 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research CouncilBB/L021250/1 United Kingdom
Wellcome Trust090932/Z/09/Z United Kingdom
Biotechnology and Biological Sciences Research CouncilBB/R00160X/1 United Kingdom
CitationJournal: Nat Commun / Year: 2015
Title: Mechanisms of assembly and genome packaging in an RNA virus revealed by high-resolution cryo-EM.
Authors: Emma L Hesketh / Yulia Meshcheriakova / Kyle C Dent / Pooja Saxena / Rebecca F Thompson / Joseph J Cockburn / George P Lomonossoff / Neil A Ranson /
Abstract: Cowpea mosaic virus is a plant-infecting member of the Picornavirales and is of major interest in the development of biotechnology applications. Despite the availability of >100 crystal structures of ...Cowpea mosaic virus is a plant-infecting member of the Picornavirales and is of major interest in the development of biotechnology applications. Despite the availability of >100 crystal structures of Picornavirales capsids, relatively little is known about the mechanisms of capsid assembly and genome encapsidation. Here we have determined cryo-electron microscopy reconstructions for the wild-type virus and an empty virus-like particle, to 3.4 Å and 3.0 Å resolution, respectively, and built de novo atomic models of their capsids. These new structures reveal the C-terminal region of the small coat protein subunit, which is essential for virus assembly and which was missing from previously determined crystal structures, as well as residues that bind to the viral genome. These observations allow us to develop a new model for genome encapsidation and capsid assembly.
History
DepositionFeb 13, 2019-
Header (metadata) releaseOct 2, 2019-
Map releaseDec 18, 2019-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0481
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0481
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6qoz
  • Surface level: 0.0481
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6qoz
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4610.png

Downloads & links

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Map

FileDownload / File: emd_4610.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationpostprocessed map of cryoEM map of Cowpea Mosaic virus bound to an Affimer protein
Voxel sizeX=Y=Z: 1.4 Å
Density
Contour LevelBy AUTHOR: 0.0481 / Movie #1: 0.0481
Minimum - Maximum-0.109593965 - 0.27533975
Average (Standard dev.)0.0020623095 (±0.013281776)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 537.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.41.41.4
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z537.600537.600537.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.1100.2750.002

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Supplemental data

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Half map: Unfiltered half map of cryoEM map of Cowpea...

Fileemd_4610_half_map_1.map
AnnotationUnfiltered half map of cryoEM map of Cowpea Mosaic virus bound to an Affimer protein
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered half map of cryoEM map of Cowpea...

Fileemd_4610_half_map_2.map
AnnotationUnfiltered half map of cryoEM map of Cowpea Mosaic virus bound to an Affimer protein
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cowpea mosaic virus

EntireName: Cowpea mosaic virus
Components
  • Complex: Cowpea mosaic virus
    • Complex: Affimer
      • Protein or peptide: Affimer binding protein
    • Virus: Cowpea mosaic virus
      • Protein or peptide: RNA2 polyprotein
      • Protein or peptide: Cowpea mosaic virus large subunit

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Supramolecule #1: Cowpea mosaic virus

SupramoleculeName: Cowpea mosaic virus / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #3: Affimer

SupramoleculeName: Affimer / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #2: Cowpea mosaic virus

SupramoleculeName: Cowpea mosaic virus / type: virus / ID: 2 / Parent: 1 / Macromolecule list: #1-#2 / NCBI-ID: 12264 / Sci species name: Cowpea mosaic virus / Virus type: VIRION / Virus isolate: SPECIES / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Cowpea mosaic virus
Host systemOrganism: Nicotiana benthamiana (plant)

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Macromolecule #1: RNA2 polyprotein

MacromoleculeName: RNA2 polyprotein / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Cowpea mosaic virus / Strain: SB
Molecular weightTheoretical: 20.961564 KDa
Recombinant expressionOrganism: Nicotiana benthamiana (plant)
SequenceString:
GPVCAEASDV YSPCMIASTP PAPFSDVTAV TFDLINGKIT PVGDDNWNTH IYNPPIMNVL RTAAWKSGTI HVQLNVRGAG VKRADWDGQ VFVYLRQSMN PESYDARTFV ISQPGSAMLN FSFDIIGPNS GFEFAESPWA NQTTWYLECV ATNPRQIQQF E VNMRFDPN FRVAGNILMP PFPLSTETPP L

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Macromolecule #2: Cowpea mosaic virus large subunit

MacromoleculeName: Cowpea mosaic virus large subunit / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Cowpea mosaic virus / Strain: SB
Molecular weightTheoretical: 40.858434 KDa
Recombinant expressionOrganism: Nicotiana benthamiana (plant)
SequenceString: MEQNLFALSL DDTSSVRGSL LDTKFAQTRV LLSKAMAGGD VLLDEYLYDV VNGQDFRATV AFLRTHVITG KIKVTATTNI SDNSGCCLM LAINSGVRGK YSTDVYTICS QDSMTWNPGC KKNFSFTFNP NPCGDSWSAE MISRSRVRMT VICVSGWTLS P TTDVIAKL ...String:
MEQNLFALSL DDTSSVRGSL LDTKFAQTRV LLSKAMAGGD VLLDEYLYDV VNGQDFRATV AFLRTHVITG KIKVTATTNI SDNSGCCLM LAINSGVRGK YSTDVYTICS QDSMTWNPGC KKNFSFTFNP NPCGDSWSAE MISRSRVRMT VICVSGWTLS P TTDVIAKL DWSIVNEKCE PTIYHLADCQ NWLPLNRWMG KLTFPQGVTS EVRRMPLSIG GGAGATQAFL ANMPNSWISM WR YFRGELH FEVTKMSSPY IKATVTFLIA FGNLSDAFGF YESFPHRIVQ FAEVEEKCTL VFSQQEFVTA WSTQVNPRTT LEA DGCPYL YAIIHDSTTG TISGDFNLGV KLVGIKDFCG IGSNPGIDGS RLL

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Macromolecule #3: Affimer binding protein

MacromoleculeName: Affimer binding protein / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 9.286674 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
ENSLEIEELA RFAVDEHNKK ENALLEFVRV VKAKEQVVAG TMYYLTLEAK DGGKKKLYEA KVWVKPWENF KELQEFKPV

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7
GridMaterial: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 281 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 1 / Number real images: 2980 / Average exposure time: 2.0 sec. / Average electron dose: 45.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 144258
CTF correctionSoftware - Name: Gctf
Startup modelType of model: EMDB MAP
EMDB ID:

3013

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationNumber classes: 2 / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 15441
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:

4n6u

,

5a32

)
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-6qoz:
CryoEM reconstruction of Cowpea Mosaic Virus (CPMV) bound to an Affimer reagent

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