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- EMDB-2487: Determination of protein structure at 8.5 Angstrom resolution usi... -

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Basic information

Entry
Database: EMDB / ID: EMD-2487
TitleDetermination of protein structure at 8.5 Angstrom resolution using cryo-electron microscopy and subtomogram averaging
Map dataReconstruction of the immature retroviral CANC hexamer
Sample
  • Sample: M-MPV CANC Gag lattice
  • Protein or peptide: M-MPV dPro CANC protein
KeywordsCryo-electron Tomography / Sub-tomogram Averaging / Retrovirus / Capsid
Biological speciesMason-Pfizer monkey virus
Methodsubtomogram averaging / cryo EM / Resolution: 8.5 Å
AuthorsSchur FKM / Hagen W / de Marco A / Briggs JAG
CitationJournal: J Struct Biol / Year: 2013
Title: Determination of protein structure at 8.5Å resolution using cryo-electron tomography and sub-tomogram averaging.
Authors: Florian K M Schur / Wim J H Hagen / Alex de Marco / John A G Briggs /
Abstract: Cryo-electron tomography combined with image processing by sub-tomogram averaging is unique in its power to resolve the structures of proteins and macromolecular complexes in situ. Limitations of the ...Cryo-electron tomography combined with image processing by sub-tomogram averaging is unique in its power to resolve the structures of proteins and macromolecular complexes in situ. Limitations of the method, including the low signal to noise ratio within individual images from cryo-tomographic datasets and difficulties in determining the defocus at which the data was collected, mean that to date the very best structures obtained by sub-tomogram averaging are limited to a resolution of approximately 15 Å. Here, by optimizing data collection and defocus determination steps, we have determined the structure of assembled Mason-Pfizer monkey virus Gag protein using sub-tomogram averaging to a resolution of 8.5 Å. At this resolution alpha-helices can be directly and clearly visualized. These data demonstrate for the first time that high-resolution structural information can be obtained from cryo-electron tomograms using sub-tomogram averaging. Sub-tomogram averaging has the potential to allow detailed studies of unsolved and biologically relevant structures under biologically relevant conditions.
History
DepositionOct 18, 2013-
Header (metadata) releaseNov 13, 2013-
Map releaseNov 13, 2013-
UpdateDec 11, 2013-
Current statusDec 11, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.209
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.209
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2487.tif

Downloads & links

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Map

FileDownload / File: emd_2487.map.gz / Format: CCP4 / Size: 6.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the immature retroviral CANC hexamer
Voxel sizeX=Y=Z: 2.025 Å
Density
Contour LevelBy AUTHOR: 0.209 / Movie #1: 0.209
Minimum - Maximum-0.63227189 - 0.7348187
Average (Standard dev.)-0.00071977 (±0.08508004)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions120120120
Spacing120120120
CellA=B=C: 243.00002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.0252.0252.025
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z243.000243.000243.000
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS120120120
D min/max/mean-0.6320.735-0.001

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Supplemental data

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Sample components

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Entire : M-MPV CANC Gag lattice

EntireName: M-MPV CANC Gag lattice
Components
  • Sample: M-MPV CANC Gag lattice
  • Protein or peptide: M-MPV dPro CANC protein

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Supramolecule #1000: M-MPV CANC Gag lattice

SupramoleculeName: M-MPV CANC Gag lattice / type: sample / ID: 1000 / Oligomeric state: helical / Number unique components: 1

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Macromolecule #1: M-MPV dPro CANC protein

MacromoleculeName: M-MPV dPro CANC protein / type: protein_or_peptide / ID: 1 / Oligomeric state: helical / Recombinant expression: Yes
Source (natural)Organism: Mason-Pfizer monkey virus
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statehelical array

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Sample preparation

BufferpH: 7.7 / Details: 50mM Tris, 100mM NaCl,1uM Zn
GridDetails: 300mesh copper, glow discharged
VitrificationCryogen name: ETHANE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.3 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 42000
Specialist opticsEnergy filter - Name: GIF2002 postcolumn energy filter
Sample stageSpecimen holder: Liquid nitrogen cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt series - Axis1 - Min angle: -45 ° / Tilt series - Axis1 - Max angle: 60 °
DateMay 21, 2013
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (2k x 2k) / Average electron dose: 40 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Phase flipping of individual tilts
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 8.5 Å / Resolution method: OTHER / Software - Name: TOM, AV3
Details: Reconstruction carried out using sub-tomogram averaging
Number subtomograms used: 121346
DetailsSubtomogram averaging was performed using scripts derived from the TOM (Nickell et al, 2005) and AV3 (Foerster et al, 2005) packages.

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