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- EMDB-13487: Horse spleen apoferritin frozen under anaerobic conditions -

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Basic information

Entry
Database: EMDB / ID: EMD-13487
TitleHorse spleen apoferritin frozen under anaerobic conditions
Map data
Sample
  • Complex: Apoferritin
Biological speciesEquus caballus (horse)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsCherrier MV / Vernede X / Fenel D / Martin L / Arragain B / Neumann E / Fontecilla-Camps JC / Schoehn G / Nicolet Y
Funding support France, 3 items
OrganizationGrant numberCountry
French National Research AgencyANR-15-IDEX-02 France
French National Research AgencyANR-10-INSB-05-02 France
Grenoble Alliance for Integrated Structural Cell Biology (GRAL)ANR-17-EURE-0003 France
CitationJournal: Biomolecules / Year: 2022
Title: Oxygen-Sensitive Metalloprotein Structure Determination by Cryo-Electron Microscopy.
Authors: Mickaël V Cherrier / Xavier Vernède / Daphna Fenel / Lydie Martin / Benoit Arragain / Emmanuelle Neumann / Juan C Fontecilla-Camps / Guy Schoehn / Yvain Nicolet /
Abstract: Metalloproteins are involved in key cell processes such as photosynthesis, respiration, and oxygen transport. However, the presence of transition metals (notably iron as a component of [Fe-S] ...Metalloproteins are involved in key cell processes such as photosynthesis, respiration, and oxygen transport. However, the presence of transition metals (notably iron as a component of [Fe-S] clusters) often makes these proteins sensitive to oxygen-induced degradation. Consequently, their study usually requires strict anaerobic conditions. Although X-ray crystallography has been the method of choice for solving macromolecular structures for many years, recently electron microscopy has also become an increasingly powerful structure-solving technique. We have used our previous experience with cryo-crystallography to develop a method to prepare cryo-EM grids in an anaerobic chamber and have applied it to solve the structures of apoferritin and the 3 [FeS]-containing pyruvate ferredoxin oxidoreductase (PFOR) at 2.40 Å and 2.90 Å resolution, respectively. The maps are of similar quality to the ones obtained under air, thereby validating our method as an improvement in the structural investigation of oxygen-sensitive metalloproteins by cryo-EM.
History
DepositionAug 31, 2021-
Header (metadata) releaseMar 23, 2022-
Map releaseMar 23, 2022-
UpdateApr 6, 2022-
Current statusApr 6, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13487.png

Downloads & links

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Map

FileDownload / File: emd_13487.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.9 Å/pix.
x 256 pix.
= 230.144 Å		0.9 Å/pix.
x 256 pix.
= 230.144 Å		0.9 Å/pix.
x 256 pix.
= 230.144 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.899 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0437
Minimum - Maximum-0.09996207 - 0.17547284
Average (Standard dev.)1.7653121e-05 (±0.010965252)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 230.144 Å
α=β=γ: 90.0 °

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Supplemental data

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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) / Organ: spleen
Recombinant expressionOrganism: Escherichia (bacteria)
Molecular weightTheoretical: 440 kDa/nm

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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

Concentration2.05 mg/mL
BufferpH: 7.4 / Details: PBS
GridModel: Quantifoil R2/1 / Material: COPPER/RHODIUM / Mesh: 300 / Support film - Material: CARBON / Pretreatment - Type: GLOW DISCHARGE / Details: 30 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV / Details: Under anaerobic conditions.
DetailsFrom Sigma (A3641)

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 2-60 / Number grids imaged: 1 / Number real images: 961 / Average exposure time: 5.5 sec. / Average electron dose: 1.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 2.5500000000000003 µm / Calibrated defocus min: 0.9 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5500000000000003 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 45000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 133493
CTF correctionSoftware - Name: RELION (ver. 3.1.1)
Final reconstructionApplied symmetry - Point group: O (octahedral) / Resolution.type: BY AUTHOR / Resolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.1) / Number images used: 66655
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.1.1)
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 3.1.1)

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Atomic model buiding 1

RefinementProtocol: FLEXIBLE FIT / Overall B value: 118

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