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- EMDB-15162: Structure of the human mitochondrial HSPD1 single ring from grids... -

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Basic information

Entry
Database: EMDB / ID: EMD-15162
TitleStructure of the human mitochondrial HSPD1 single ring from grids produced in 14ms
Map dataFinal postprocessed map
Sample
  • Organelle or cellular component: human mitochondrial heat shock protein family member D1 (HSPD1)
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.8 Å
AuthorsKlebl DP / Wang Y / Thompson RF / Muench SP
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Front Mol Biosci / Year: 2022
Title: It started with a Cys: Spontaneous cysteine modification during cryo-EM grid preparation.
Authors: David P Klebl / Yiheng Wang / Frank Sobott / Rebecca F Thompson / Stephen P Muench /
Abstract: Advances in single particle cryo-EM data collection and processing have seen a significant rise in its use. However, the influences of the environment generated through grid preparation, by for ...Advances in single particle cryo-EM data collection and processing have seen a significant rise in its use. However, the influences of the environment generated through grid preparation, by for example interactions of proteins with the air-water interface are poorly understood and can be a major hurdle in structure determination by cryo-EM. Initial interactions of proteins with the air-water interface occur quickly and proteins can adopt preferred orientation or partially unfold within hundreds of milliseconds. It has also been shown previously that thin-film layers create hydroxyl radicals. To investigate the potential this might have in cryo-EM sample preparation, we studied two proteins, HSPD1, and beta-galactosidase, and show that cysteine residues are modified in a time-dependent manner. In the case of both HSPD1 and beta-galactosidase, this putative oxidation is linked to partial protein unfolding, as well as more subtle structural changes. We show these modifications can be alleviated through increasing the speed of grid preparation, the addition of DTT, or by sequestering away from the AWI using continuous support films. We speculate that the modification is oxidation by reactive oxygen species which are formed and act at the air-water interface. Finally, we show grid preparation on a millisecond timescale outruns cysteine modification, showing that the reaction timescale is in the range of 100s to 1,000s milliseconds and offering an alternative approach to prevent spontaneous cysteine modification and its consequences during cryo-EM grid preparation.
History
DepositionJun 14, 2022-
Header (metadata) releaseAug 31, 2022-
Map releaseAug 31, 2022-
UpdateAug 31, 2022-
Current statusAug 31, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15162.png

Downloads & links

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Map

FileDownload / File: emd_15162.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFinal postprocessed map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.23 Å/pix.
x 200 pix.
= 246. Å		1.23 Å/pix.
x 200 pix.
= 246. Å		1.23 Å/pix.
x 200 pix.
= 246. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.23 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0172
Minimum - Maximum-0.01064137 - 0.04398941
Average (Standard dev.)0.000685095 (±0.0030901693)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 246.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15162_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: second Half map for fast HSPD1

Fileemd_15162_half_map_1.map
Annotationsecond Half map for fast HSPD1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map for fast HSPD1

Fileemd_15162_half_map_2.map
AnnotationHalf map for fast HSPD1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : human mitochondrial heat shock protein family member D1 (HSPD1)

EntireName: human mitochondrial heat shock protein family member D1 (HSPD1)
Components
  • Organelle or cellular component: human mitochondrial heat shock protein family member D1 (HSPD1)

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Supramolecule #1: human mitochondrial heat shock protein family member D1 (HSPD1)

SupramoleculeName: human mitochondrial heat shock protein family member D1 (HSPD1)
type: organelle_or_cellular_component / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)
Molecular weightExperimental: 60 kDa/nm
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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

Concentration2.3 mg/mL
BufferpH: 7.7 / Details: 25 mM Tris, 150 mM NaCl, 5 mM MgCl2 pH 7.7
GridModel: Quantifoil / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: HOMEMADE PLUNGER / Details: Home-made high speed plunging device.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 56.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.3000000000000003 µm / Nominal defocus min: 1.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 166797
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 5218
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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