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- EMDB-13390: subtomogram averaging of a HIV-1 immature Gag at 4.5 angstrom res... -

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Basic information

Entry
Database: EMDB / ID: EMD-13390
Titlesubtomogram averaging of a HIV-1 immature Gag at 4.5 angstrom resolution
Map datafinal map from emClarity reconstruct (cisTEM reconstruct) using 2D images
Sample
  • Complex: HIV-1 immature Gag virus-like particle
Function / homology
Function and homology information


viral process / viral capsid / nucleic acid binding / zinc ion binding
Similarity search - Function
Gag protein p6 / Gag protein p6 / : / gag protein p24 N-terminal domain / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / Retrovirus capsid, C-terminal / Retrovirus capsid, N-terminal / zinc finger / Zinc knuckle ...Gag protein p6 / Gag protein p6 / : / gag protein p24 N-terminal domain / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / Retrovirus capsid, C-terminal / Retrovirus capsid, N-terminal / zinc finger / Zinc knuckle / Zinc finger, CCHC-type superfamily / Zinc finger, CCHC-type / Zinc finger CCHC-type profile.
Similarity search - Domain/homology
Biological speciesHuman immunodeficiency virus 1
Methodsubtomogram averaging / cryo EM / Resolution: 4.5 Å
AuthorsNi T / Mendonca L / Frosio T / Sheng Y / Clare D / Himes B / Zhang P
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Wellcome Trust United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC) United Kingdom
CitationJournal: Nat Protoc / Year: 2022
Title: High-resolution in situ structure determination by cryo-electron tomography and subtomogram averaging using emClarity.
Authors: Tao Ni / Thomas Frosio / Luiza Mendonça / Yuewen Sheng / Daniel Clare / Benjamin A Himes / Peijun Zhang /
Abstract: Cryo-electron tomography and subtomogram averaging (STA) has developed rapidly in recent years. It provides structures of macromolecular complexes in situ and in cellular context at or below ...Cryo-electron tomography and subtomogram averaging (STA) has developed rapidly in recent years. It provides structures of macromolecular complexes in situ and in cellular context at or below subnanometer resolution and has led to unprecedented insights into the inner working of molecular machines in their native environment, as well as their functional relevant conformations and spatial distribution within biological cells or tissues. Given the tremendous potential of cryo-electron tomography STA in in situ structural cell biology, we previously developed emClarity, a graphics processing unit-accelerated image-processing software that offers STA and classification of macromolecular complexes at high resolution. However, the workflow remains challenging, especially for newcomers to the field. In this protocol, we describe a detailed workflow, processing and parameters associated with each step, from initial tomography tilt-series data to the final 3D density map, with several features unique to emClarity. We use four different samples, including human immunodeficiency virus type 1 Gag assemblies, ribosome and apoferritin, to illustrate the procedure and results of STA and classification. Following the processing steps described in this protocol, along with a comprehensive tutorial and guidelines for troubleshooting and parameter optimization, one can obtain density maps up to 2.8 Å resolution from six tilt series by cryo-electron tomography STA.
History
DepositionAug 14, 2021-
Header (metadata) releaseSep 15, 2021-
Map releaseSep 15, 2021-
UpdateMay 4, 2022-
Current statusMay 4, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 1.5
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13390.png

Downloads & links

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Map

FileDownload / File: emd_13390.map.gz / Format: CCP4 / Size: 229.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationfinal map from emClarity reconstruct (cisTEM reconstruct) using 2D images
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.18 Å/pix.
x 392 pix.
= 462.168 Å		1.18 Å/pix.
x 392 pix.
= 462.168 Å		1.18 Å/pix.
x 392 pix.
= 462.168 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.179 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.5 / Movie #1: 1.5
Minimum - Maximum-5.951717 - 8.29212
Average (Standard dev.)0.0067707007 (±0.33156723)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions392392392
Spacing392392392
CellA=B=C: 462.168 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.1791.1791.179
M x/y/z392392392
origin x/y/z0.0000.0000.000
length x/y/z462.168462.168462.168
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS392392392
D min/max/mean-5.9528.2920.007

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Supplemental data

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Additional map: final map from emClarity avg using subtomograms

Fileemd_13390_additional_1.map
Annotationfinal map from emClarity avg using subtomograms
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2 from emClarity reconstruct (cisTEM reconstruct)...

Fileemd_13390_half_map_1.map
Annotationhalf map 2 from emClarity reconstruct (cisTEM reconstruct) using 2D images
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 1 from emClarity reconstruct (cisTEM reconstruct)...

Fileemd_13390_half_map_2.map
Annotationhalf map 1 from emClarity reconstruct (cisTEM reconstruct) using 2D images
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : HIV-1 immature Gag virus-like particle

EntireName: HIV-1 immature Gag virus-like particle
Components
  • Complex: HIV-1 immature Gag virus-like particle

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Supramolecule #1: HIV-1 immature Gag virus-like particle

SupramoleculeName: HIV-1 immature Gag virus-like particle / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Human immunodeficiency virus 1
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Average electron dose: 3.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software: (Name: emClarity, cisTEM)
Details: The final combined map is reconstructed using cisTEM within emClarity package, with a b-factor of -75
Number subtomograms used: 13844
ExtractionNumber tomograms: 5 / Number images used: 20010
CTF correctionSoftware: (Name: emClarity, CTFFIND)
Final angle assignmentType: OTHER / Software - Name: emClarity / Details: cross-correlation

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