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- EMDB-16157: Map of GroEL:GroES-(ADP) complex plunge frozen 200 ms after react... -

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Basic information

Entry
Database: EMDB / ID: EMD-16157
TitleMap of GroEL:GroES-(ADP) complex plunge frozen 200 ms after reaction initiation with ATP
Map data
Sample
  • Complex: Hetero 14mer assembled from 2 heptameric rings
    • Protein or peptide: GroEL
    • Protein or peptide: GroES
KeywordsGroEL / GroES / CHAPERONE
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.3 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 22, 2022-
Header (metadata) releaseAug 30, 2023-
Map releaseAug 30, 2023-
UpdateSep 13, 2023-
Current statusSep 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16157.png

Downloads & links

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Map

FileDownload / File: emd_16157.map.gz / Format: CCP4 / Size: 229.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.96 Å
Density
Contour LevelBy AUTHOR: 0.021
Minimum - Maximum-0.018173344 - 0.08832749
Average (Standard dev.)0.0002744572 (±0.003948514)
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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Mask #1

Fileemd_16157_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_16157_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_16157_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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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
    • Protein or peptide: GroEL
    • Protein or peptide: GroES

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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: all
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 950 KDa

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Macromolecule #1: GroEL

MacromoleculeName: GroEL / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAAKDVKFGN DARVKMLRGV NVLADAVKVT LGPKGRNVVL DKSFGAPTIT KDGVSVAREI ELEDKFENMG AQMVKEVASK ANDAAGDGTT TATVLAQAII TEGLKAVAAG MNPMDLKRGI DKAVTAAVEE LKALSVPCSD SKAIAQVGTI SANSDETVGK LIAEAMDKVG ...String:
MAAKDVKFGN DARVKMLRGV NVLADAVKVT LGPKGRNVVL DKSFGAPTIT KDGVSVAREI ELEDKFENMG AQMVKEVASK ANDAAGDGTT TATVLAQAII TEGLKAVAAG MNPMDLKRGI DKAVTAAVEE LKALSVPCSD SKAIAQVGTI SANSDETVGK LIAEAMDKVG KEGVITVEDG TGLQDELDVV EGMQFDRGYL SPYFINKPET GAVELESPFI LLADKKISNI REMLPVLEAV AKAGKPLLII AEDVEGEALA TLVVNTMRGI VKVAAVKAPG FGDRRKAMLQ DIATLTGGTV ISEEIGMELE KATLEDLGQA KRVVINKDTT TIIDGVGEEA AIQGRVAQIR QQIEEATSDY DREKLQERVA KLAGGVAVIK VGAATEVEMK EKKARVEDAL HATRAAVEEG VVAGGGVALI RVASKLADLR GQNEDQNVGI KVALRAMEAP LRQIVLNCGE EPSVVANTVK GGDGNYGYNA ATEEYGNMID MGILDPTKVT RSALQYAASV AGLMITTECM VTDLPKNDAA DLGAAGGMGG MGGMGGMM

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Macromolecule #2: GroES

MacromoleculeName: GroES / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MANIRPLHDR VIVKRKEVET KSAGGIVLTG SAAAKSTRGE VLAVGNGRIL ENGEVKPLDV KVGDIVIFND GYGVKSEKID NEEVLIMSES DILAIVEA

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

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Image processing

Startup modelType of model: OTHER
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
Final reconstructionApplied symmetry - Point group: C7 (7 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 6.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0) / Number images used: 1433
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL

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