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- EMDB-26222: 3D Structure Determination of GroEL Protein Complexes using Matri... -

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Basic information

Entry
Database: EMDB / ID: EMD-26222
Title3D Structure Determination of GroEL Protein Complexes using Matrix-Landing Mass Spectrometry
Map dataGroEL ESI mass analysis and soft landing. Charge states 62 through 71 collected for 600 sec. onto a glycerol treated carbon grid. Upon removal from vacuum, sample stained with uranyl acetate.
Sample
  • Complex: GroEL
KeywordsAssembly / Complex / Folding / CHAPERONE
Biological speciesEscherichia (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 15.0 Å
AuthorsWestphall MS / Coon JJ / Grant T
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM118110 United States
CitationJournal: Nat Commun / Year: 2022
Title: Three-dimensional structure determination of protein complexes using matrix-landing mass spectrometry.
Authors: Michael S Westphall / Kenneth W Lee / Austin Z Salome / Jean M Lodge / Timothy Grant / Joshua J Coon /
Abstract: Native mass spectrometry (MS) is increasingly used to provide complementary data to electron microscopy (EM) for protein structure characterization. Beyond the ability to provide mass measurements of ...Native mass spectrometry (MS) is increasingly used to provide complementary data to electron microscopy (EM) for protein structure characterization. Beyond the ability to provide mass measurements of gas-phase biomolecular ions, MS instruments offer the ability to purify, select, and precisely control the spatial location of these ions. Here we present a modified Orbitrap MS system capable of depositing a native MS ion beam onto EM grids. We further describe the use of a chemical landing matrix that preserves the structural integrity of the deposited particles. With this system we obtain a three-dimensional reconstruction of the 800 kDa protein complex GroEL from gas-phase deposited GroEL ions. These data provide direct evidence that non-covalent protein complexes can indeed retain their condensed-phase structures following ionization and vaporization. Finally, we describe how further developments of this technology could pave the way to an integrated MS-EM technology with promise to provide improved cryo-EM sample preparation over conventional plunge-freezing techniques.
History
DepositionFeb 17, 2022-
Header (metadata) releaseApr 6, 2022-
Map releaseApr 6, 2022-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26222.png

Downloads & links

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Map

FileDownload / File: emd_26222.map.gz / Format: CCP4 / Size: 16.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationGroEL ESI mass analysis and soft landing. Charge states 62 through 71 collected for 600 sec. onto a glycerol treated carbon grid. Upon removal from vacuum, sample stained with uranyl acetate.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.4 Å/pix.
x 162 pix.
= 550.8 Å		3.4 Å/pix.
x 162 pix.
= 550.8 Å		3.4 Å/pix.
x 162 pix.
= 550.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.4 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.154
Minimum - Maximum-0.2897674 - 0.57353425
Average (Standard dev.)-0.08522537 (±0.03306577)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-81-81-81
Dimensions162162162
Spacing162162162
CellA=B=C: 550.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Same sample as the soft-landed GroEl but pipetted...

Fileemd_26222_additional_1.map
AnnotationSame sample as the soft-landed GroEl but pipetted onto the grid followed by negative staining.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : GroEL

EntireName: GroEL
Components
  • Complex: GroEL

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Supramolecule #1: GroEL

SupramoleculeName: GroEL / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia (bacteria)
Molecular weightTheoretical: 802.5 KDa

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 7
StainingType: POSITIVE / Material: Uranyl Acetate
GridModel: Homemade / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 5 / Pretreatment - Type: GLOW DISCHARGE
DetailsProtein complex solution was ionized and converted to gas-phase via electrospray ionization. GroEL 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
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm

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Image processing

DetailsNanoSprint15 MK-II 15 Mpix camera (AMT Imaging)
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 15.0 Å / Resolution method: OTHER / Software - Name: cisTEM / Number images used: 3000
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE

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