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Yorodumi- EMDB-13271: Subtomogram averaging of apoferritin at 2.86 angstrom resolution -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-13271 | |||||||||
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Title | Subtomogram averaging of apoferritin at 2.86 angstrom resolution | |||||||||
Map data | ||||||||||
Sample |
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Biological species | Equus caballus (horse) | |||||||||
Method | subtomogram averaging / cryo EM / Resolution: 2.86 Å | |||||||||
Authors | Ni T / Mendonca L / Frosio T / Sheng Y / Clare D / Himes B / Zhang P | |||||||||
Funding support | United Kingdom, 1 items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_13271.map.gz | 3.8 MB | EMDB map data format | |
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Header (meta data) | emd-13271-v30.xml emd-13271.xml | 13.5 KB 13.5 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_13271_fsc.xml | 34.7 KB | Display | FSC data file |
Images | emd_13271.png | 153.7 KB | ||
Others | emd_13271_half_map_1.map.gz emd_13271_half_map_2.map.gz | 3 MB 2.9 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-13271 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-13271 | HTTPS FTP |
-Validation report
Summary document | emd_13271_validation.pdf.gz | 467.2 KB | Display | EMDB validaton report |
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Full document | emd_13271_full_validation.pdf.gz | 466.8 KB | Display | |
Data in XML | emd_13271_validation.xml.gz | 23.2 KB | Display | |
Data in CIF | emd_13271_validation.cif.gz | 33.3 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-13271 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-13271 | HTTPS FTP |
-Related structure data
Related structure data | C: citing same article (ref.) |
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Similar structure data | |
EM raw data | EMPIAR-10787 (Title: cryo electron tomography of Apoferritin / Data size: 17.3 Data #1: Unaligned raw tilt-series of apoferritin on graphene grid [tilt series]) |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_13271.map.gz / Format: CCP4 / Size: 21.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Voxel size | X=Y=Z: 1.34 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Half map: #2
File | emd_13271_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_13271_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : apoferritin
Entire | Name: apoferritin |
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Components |
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-Supramolecule #1: apoferritin
Supramolecule | Name: apoferritin / type: complex / ID: 1 / Parent: 0 |
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Source (natural) | Organism: Equus caballus (horse) |
Recombinant expression | Organism: Escherichia (bacteria) |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | subtomogram averaging |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.5 |
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Grid | Model: 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 |
Vitrification | Cryogen name: ETHANE |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film 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 beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |