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- EMDB-13271: Subtomogram averaging of apoferritin at 2.86 angstrom resolution -

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Basic information

Entry
Database: EMDB / ID: EMD-13271
TitleSubtomogram averaging of apoferritin at 2.86 angstrom resolution
Map data
Sample
  • Complex: apoferritin
Biological speciesEquus caballus (horse)
Methodsubtomogram averaging / cryo EM / Resolution: 2.86 Å
AuthorsNi T / Mendonca L / Frosio T / Sheng Y / Clare D / Himes B / Zhang P
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust 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
DepositionJul 29, 2021-
Header (metadata) releaseFeb 2, 2022-
Map releaseFeb 2, 2022-
UpdateFeb 23, 2022-
Current statusFeb 23, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.5
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 3.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13271.png

Downloads & links

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Map

FileDownload / File: emd_13271.map.gz / Format: CCP4 / Size: 21.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.34 Å
Density
Contour LevelBy AUTHOR: 3.5 / Movie #1: 3.5
Minimum - Maximum-7.857311 - 13.48535
Average (Standard dev.)0.05202668 (±0.75914425)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions177177177
Spacing177177177
CellA=B=C: 237.18001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.341.341.34
M x/y/z177177177
origin x/y/z0.0000.0000.000
length x/y/z237.180237.180237.180
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS177177177
D min/max/mean-7.85713.4850.052

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Supplemental data

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Half map: #2

Fileemd_13271_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_13271_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : apoferritin

EntireName: apoferritin
Components
  • Complex: apoferritin

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Supramolecule #1: apoferritin

SupramoleculeName: apoferritin / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Equus caballus (horse)
Recombinant expressionOrganism: Escherichia (bacteria)

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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: 7.5
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: GRAPHENE / Support film - topology: CONTINUOUS / Support film - Film thickness: 0.8 nm / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Average electron dose: 102.0 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: O (octahedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: emClarity / Number subtomograms used: 4826
ExtractionNumber tomograms: 6 / Number images used: 5668
CTF correctionSoftware - Name: emClarity
Final angle assignmentType: OTHER / Software - Name: emClarity / Details: cross-correlation
FSC plot (resolution estimation)

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