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- EMDB-27819: 3D Reconstruction of PPG Matrix-Landed 20S Proteasome Core Partic... -

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Basic information

Entry
Database: EMDB / ID: EMD-27819
Title3D Reconstruction of PPG Matrix-Landed 20S Proteasome Core Particle Protein Complexes
Map data
Sample
  • Complex: 20S Proteasome Core Particle
KeywordsMatrix-Landed / Mass Spectrometry / CELL CYCLE
Biological speciesThermoplasma acidophilum (acidophilic)
Methodsingle particle reconstruction / negative staining / Resolution: 15.0 Å
AuthorsSalome AZ / Westphall MS
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM118110 United States
CitationJournal: Anal Chem / Year: 2022
Title: Matrix-Landing Mass Spectrometry for Electron Microscopy Imaging of Native Protein Complexes.
Authors: Austin Z Salome / Kenneth W Lee / Timothy Grant / Michael S Westphall / Joshua J Coon /
Abstract: Recently, we described the use of a chemical matrix for landing and preserving the cations of protein-protein complexes within a mass spectrometer (MS) instrument. By use of a glycerol-landing ...Recently, we described the use of a chemical matrix for landing and preserving the cations of protein-protein complexes within a mass spectrometer (MS) instrument. By use of a glycerol-landing matrix, we used negative stain transmission electron microscopy (TEM) to obtain a three-dimensional (3D) reconstruction of landed GroEL complexes. Here, we investigate the utilities of other chemical matrices for their abilities to land, preserve, and allow for direct imaging of these cationic particles using TEM. We report here that poly(propylene) glycol (PPG) offers superior performance over glycerol for matrix landing. We demonstrated the utility of the PPG matrix landing using three protein-protein complexes─GroEL, the 20S proteasome core particle, and β-galactosidase─and obtained a 3D reconstruction of each complex from matrix-landed particles. These structures have no detectable differences from the structures obtained using conventional preparation methods, suggesting the structures are well preserved at least to the resolution limit of the reconstructions (∼20 Å). We conclude that matrix landing offers a direct approach to couple native MS with TEM for protein structure determination.
History
DepositionAug 9, 2022-
Header (metadata) releaseJan 11, 2023-
Map releaseJan 11, 2023-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27819.png

Downloads & links

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Map

FileDownload / File: emd_27819.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
3.4 Å/pix.
x 128 pix.
= 435.2 Å		3.4 Å/pix.
x 128 pix.
= 435.2 Å		3.4 Å/pix.
x 128 pix.
= 435.2 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 3.4 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 5.89
Minimum - Maximum-3.9417577 - 8.956083
Average (Standard dev.)-0.015330709 (±0.84098464)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 435.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_27819_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_27819_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : 20S Proteasome Core Particle

EntireName: 20S Proteasome Core Particle
Components
  • Complex: 20S Proteasome Core Particle

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Supramolecule #1: 20S Proteasome Core Particle

SupramoleculeName: 20S Proteasome Core Particle / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Thermoplasma acidophilum (acidophilic)

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation statecell

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Sample preparation

BufferpH: 7
StainingType: NEGATIVE / Material: Uranyl Acetate
GridModel: Homemade / Material: COPPER / Mesh: 400
DetailsProtein complex solution was ionized and converted to gas-phase via electrospray ionization. Proteasome gas-phase ions were collected for analysis.

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Electron microscopy

MicroscopeFEI TECNAI 12
Image recordingFilm or detector model: OTHER / Average electron dose: 100.0 e/Å2 / Details: NanoSprint15 MK-II 15 Mpix camera (AMT Imaging)
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm

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Image processing

Startup modelType of model: OTHER
Final reconstructionApplied symmetry - Point group: D7 (2x7 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 15.0 Å / Resolution method: OTHER / Software - Name: cisTEM / Number images used: 15695
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE

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