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- EMDB-15178: Structure of the human mitochondrial HSPD1 single ring (blotted b... -

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Basic information

Entry
Database: EMDB / ID: EMD-15178
TitleStructure of the human mitochondrial HSPD1 single ring (blotted but no DTT)
Map dataFinal post-processed 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: 3.4 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 15, 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_15178.png

Downloads & links

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Map

FileDownload / File: emd_15178.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFinal post-processed map
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 300 pix.
= 246.6 Å		0.82 Å/pix.
x 300 pix.
= 246.6 Å		0.82 Å/pix.
x 300 pix.
= 246.6 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.822 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.316
Minimum - Maximum-0.986842 - 1.7632399
Average (Standard dev.)0.0053036553 (±0.050603494)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 246.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15178_msk_1.map
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AxesZYX

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Additional map: Final map without post-processing

Fileemd_15178_additional_1.map
AnnotationFinal map without post-processing
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Half map: Half map 1

Fileemd_15178_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

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Half map: Half map 2

Fileemd_15178_half_map_2.map
AnnotationHalf map 2
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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 R1.2/1.3 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 39.9 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 45334
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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