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- EMDB-16125: Structure of GroEL:GroES-ATP complex plunge frozen 200 ms after r... -

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Basic information

Entry
Database: EMDB / ID: EMD-16125
TitleStructure of GroEL:GroES-ATP complex plunge frozen 200 ms after reaction initiation
Map data
Sample
  • Complex: GroEL and GroES
    • Protein or peptide: Chaperonin GroEL
    • Protein or peptide: Co-chaperonin GroES
  • Ligand: MAGNESIUM ION
  • Ligand: POTASSIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: water
KeywordsGroEL / GroES / CHAPERONE
Function / homology
Function and homology information


GroEL-GroES complex / chaperonin ATPase / virion assembly / chaperone cofactor-dependent protein refolding / protein folding chaperone / isomerase activity / ATP-dependent protein folding chaperone / response to radiation / unfolded protein binding / protein folding ...GroEL-GroES complex / chaperonin ATPase / virion assembly / chaperone cofactor-dependent protein refolding / protein folding chaperone / isomerase activity / ATP-dependent protein folding chaperone / response to radiation / unfolded protein binding / protein folding / protein-folding chaperone binding / response to heat / protein refolding / magnesium ion binding / ATP hydrolysis activity / ATP binding / identical protein binding / membrane / metal ion binding / cytosol
Similarity search - Function
Chaperonin GroES, conserved site / Chaperonins cpn10 signature. / Chaperonin 10 Kd subunit / GroES chaperonin family / GroES chaperonin superfamily / Chaperonin 10 Kd subunit / Chaperonin Cpn60, conserved site / Chaperonins cpn60 signature. / Chaperonin Cpn60/GroEL / GroEL-like equatorial domain superfamily ...Chaperonin GroES, conserved site / Chaperonins cpn10 signature. / Chaperonin 10 Kd subunit / GroES chaperonin family / GroES chaperonin superfamily / Chaperonin 10 Kd subunit / Chaperonin Cpn60, conserved site / Chaperonins cpn60 signature. / Chaperonin Cpn60/GroEL / GroEL-like equatorial domain superfamily / TCP-1-like chaperonin intermediate domain superfamily / GroEL-like apical domain superfamily / TCP-1/cpn60 chaperonin family / Chaperonin Cpn60/GroEL/TCP-1 family / GroES-like superfamily
Similarity search - Domain/homology
Chaperonin GroEL / Co-chaperonin GroES
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsDhurandhar M / Efremov R / Torino S
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 10, 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_16125.png

Downloads & links

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Map

FileDownload / File: emd_16125.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: 0.025
Minimum - Maximum-0.097782485 - 0.28029191
Average (Standard dev.)0.00018379878 (±0.005963762)
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_16125_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_16125_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_16125_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 : GroEL and GroES

EntireName: GroEL and GroES
Components
  • Complex: GroEL and GroES
    • Protein or peptide: Chaperonin GroEL
    • Protein or peptide: Co-chaperonin GroES
  • Ligand: MAGNESIUM ION
  • Ligand: POTASSIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: water

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Supramolecule #1: GroEL and GroES

SupramoleculeName: GroEL and GroES / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: Chaperonin GroEL

MacromoleculeName: Chaperonin GroEL / type: protein_or_peptide / ID: 1 / Number of copies: 14 / Enantiomer: LEVO / EC number: chaperonin ATPase
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12
Molecular weightTheoretical: 57.391711 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAAKDVKFGN DARVKMLRGV NVLADAVKVT LGPKGRNVVL DKSFGAPTIT KDGVSVAREI ELEDKFENMG AQMVKEVASK ANDAAGDGT TTATVLAQAI ITEGLKAVAA GMNPMDLKRG IDKAVTAAVE ELKALSVPCS DSKAIAQVGT ISANSDETVG K LIAEAMDK ...String:
MAAKDVKFGN DARVKMLRGV NVLADAVKVT LGPKGRNVVL DKSFGAPTIT KDGVSVAREI ELEDKFENMG AQMVKEVASK ANDAAGDGT TTATVLAQAI ITEGLKAVAA GMNPMDLKRG IDKAVTAAVE ELKALSVPCS DSKAIAQVGT ISANSDETVG K LIAEAMDK VGKEGVITVE DGTGLQDELD VVEGMQFDRG YLSPYFINKP ETGAVELESP FILLADKKIS NIREMLPVLE AV AKAGKPL LIIAEDVEGE ALATLVVNTM RGIVKVAAVK APGFGDRRKA MLQDIATLTG GTVISEEIGM ELEKATLEDL GQA KRVVIN KDTTTIIDGV GEEAAIQGRV AQIRQQIEEA TSDYDREKLQ ERVAKLAGGV AVIKVGAATE VEMKEKKARV EDAL HATRA AVEEGVVAGG GVALIRVASK LADLRGQNED QNVGIKVALR AMEAPLRQIV LNCGEEPSVV ANTVKGGDGN YGYNA ATEE YGNMIDMGIL DPTKVTRSAL QYAASVAGLM ITTECMVTDL PKNDAADLGA AGGMGGMGGM GGMM

UniProtKB: Chaperonin GroEL

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Macromolecule #2: Co-chaperonin GroES

MacromoleculeName: Co-chaperonin GroES / type: protein_or_peptide / ID: 2 / Number of copies: 14 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12
Molecular weightTheoretical: 10.472016 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MANIRPLHDR VIVKRKEVET KSAGGIVLTG SAAAKSTRGE VLAVGNGRIL ENGEVKPLDV KVGDIVIFND GYGVKSEKID NEEVLIMSE SDILAIVEA

UniProtKB: Co-chaperonin GroES

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 14 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #4: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 4 / Number of copies: 14 / Formula: K
Molecular weightTheoretical: 39.098 Da

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Macromolecule #5: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 14 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 1339 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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
GridModel: Quantifoil R2/1 / Material: COPPER / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 3 / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / 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: D7 (2x7 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0) / Number images used: 27784
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8bmt:
Structure of GroEL:GroES-ATP complex plunge frozen 200 ms after reaction initiation

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