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- EMDB-16154: Map of the GroEL-ES-ATP complex plunge-frozen 50 ms after mixing ... -

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Basic information

Entry
Database: EMDB / ID: EMD-16154
TitleMap of the GroEL-ES-ATP complex plunge-frozen 50 ms after mixing with ATP
Map data
Sample
  • Complex: Hetero 14mer assembled from 2 heptameric rings
KeywordsGroEL / GroES / CHAPERONE
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.1 Å
AuthorsDhurandhar M / Torino S / Efremov R
Funding supportEuropean Union, Belgium, 3 items
OrganizationGrant numberCountry
European Research Council (ERC)726436European Union
Research Foundation - Flanders (FWO)G0H5916N Belgium
Research Foundation - Flanders (FWO)G054617N Belgium
CitationJournal: Nat Methods / Year: 2023
Title: Time-resolved cryo-EM using a combination of droplet microfluidics with on-demand jetting.
Authors: Stefania Torino / Mugdha Dhurandhar / Annelore Stroobants / Raf Claessens / Rouslan G Efremov /
Abstract: Single-particle cryogenic electron microscopy (cryo-EM) allows reconstruction of high-resolution structures of proteins in different conformations. Protein function often involves transient ...Single-particle cryogenic electron microscopy (cryo-EM) allows reconstruction of high-resolution structures of proteins in different conformations. Protein function often involves transient functional conformations, which can be resolved using time-resolved cryo-EM (trEM). In trEM, reactions are arrested after a defined delay time by rapid vitrification of protein solution on the EM grid. Despite the increasing interest in trEM among the cryo-EM community, making trEM samples with a time resolution below 100 ms remains challenging. Here we report the design and the realization of a time-resolved cryo-plunger that combines a droplet-based microfluidic mixer with a laser-induced generator of microjets that allows rapid reaction initiation and plunge-freezing of cryo-EM grids. Using this approach, a time resolution of 5 ms was achieved and the protein density map was reconstructed to a resolution of 2.1 Å. trEM experiments on GroEL:GroES chaperonin complex resolved the kinetics of the complex formation and visualized putative short-lived conformations of GroEL-ATP complex.
History
DepositionNov 17, 2022-
Header (metadata) releaseAug 9, 2023-
Map releaseAug 9, 2023-
UpdateSep 13, 2023-
Current statusSep 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16154.png

Downloads & links

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Map

FileDownload / File: emd_16154.map.gz / Format: CCP4 / Size: 229.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.96 Å/pix.
x 392 pix.
= 376.32 Å		0.96 Å/pix.
x 392 pix.
= 376.32 Å		0.96 Å/pix.
x 392 pix.
= 376.32 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.96 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0132
Minimum - Maximum-0.028941294 - 0.07191182
Average (Standard dev.)0.00016064393 (±0.003082478)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions392392392
Spacing392392392
CellA=B=C: 376.31998 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_16154_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_16154_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Hetero 14mer assembled from 2 heptameric rings

EntireName: Hetero 14mer assembled from 2 heptameric rings
Components
  • Complex: Hetero 14mer assembled from 2 heptameric rings

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Supramolecule #1: Hetero 14mer assembled from 2 heptameric rings

SupramoleculeName: Hetero 14mer assembled from 2 heptameric rings / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 950 KDa

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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

Concentration8 mg/mL
BufferpH: 7.5
Component:
ConcentrationName
25.0 mMTris base
50.0 mMKCl
10.0 mMMgCl2
0.5 mMTCEP
2.0 mMATP
0.1 %CHAPS
GridModel: Quantifoil R2/1 / Material: COPPER / Support film - Material: CARBON / Support film - topology: CONTINUOUS
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 2 / Number real images: 4195 / Average electron dose: 63.6 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.5 µm
Sample stageSpecimen holder model: JEOL CRYOSPECPORTER / Cooling holder cryogen: NITROGEN

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Image processing

Startup modelType of model: OTHER
Final reconstructionApplied symmetry - Point group: C7 (7 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 7.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0) / Number images used: 2170
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
Final 3D classificationSoftware - Name: RELION
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL

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