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- PDB-8bkz: GroEL:GroES-ATP complex under continuous turnover conditions -

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Basic information

Entry
Database: PDB / ID: 8bkz
TitleGroEL:GroES-ATP complex under continuous turnover conditions
Components
  • Chaperonin GroEL
  • Co-chaperonin GroES
KeywordsCHAPERONE / GroEL / GroES
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
ADENOSINE-5'-TRIPHOSPHATE / : / Chaperonin GroEL / Co-chaperonin GroES
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.3 Å
AuthorsDhurandhar, M. / Torino, S. / Efremov, R.
Funding supportEuropean Union, Belgium, 3items
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 9, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 9, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 30, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Sep 13, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
X: Chaperonin GroEL
Y: Co-chaperonin GroES
A: Chaperonin GroEL
B: Co-chaperonin GroES
C: Chaperonin GroEL
D: Co-chaperonin GroES
E: Chaperonin GroEL
F: Co-chaperonin GroES
G: Chaperonin GroEL
H: Co-chaperonin GroES
I: Chaperonin GroEL
J: Co-chaperonin GroES
K: Chaperonin GroEL
L: Co-chaperonin GroES
M: Chaperonin GroEL
N: Co-chaperonin GroES
O: Chaperonin GroEL
P: Co-chaperonin GroES
Q: Chaperonin GroEL
R: Co-chaperonin GroES
S: Chaperonin GroEL
T: Co-chaperonin GroES
V: Chaperonin GroEL
W: Co-chaperonin GroES
Z: Chaperonin GroEL
AA: Co-chaperonin GroES
BA: Chaperonin GroEL
CA: Co-chaperonin GroES
hetero molecules


Theoretical massNumber of molelcules
Total (without water)955,24970
Polymers947,26028
Non-polymers7,98842
Water63,1433505
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area104930 Å2
ΔGint-605 kcal/mol
Surface area317290 Å2
MethodPISA

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Components

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Protein , 2 types, 28 molecules XACEGIKMOQSVZBAYBDFHJLNPRTWAACA

#1: Protein
Chaperonin GroEL / 60 kDa chaperonin / Chaperonin-60 / Cpn60 / GroEL protein


Mass: 57391.711 Da / Num. of mol.: 14
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: groEL, groL, mopA, b4143, JW4103 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A6F5, chaperonin ATPase
#2: Protein
Co-chaperonin GroES / 10 kDa chaperonin / Chaperonin-10 / Cpn10


Mass: 10269.741 Da / Num. of mol.: 14
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: groES, groS, mopB, b4142, JW4102 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A6F9

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Non-polymers , 4 types, 3547 molecules

#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: Mg
#4: Chemical
ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: K
#5: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 3505 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Hetero 14mer assembled from 2 heptameric rings / Type: COMPLEX / Entity ID: #2 / Source: RECOMBINANT
Molecular weightValue: 0.95 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
125 mMTris base1
250 mMKCl1
310 mMMgCl21
40.5 mMTCEP1
52 mMATP1
60.1 %CHAPS1
SpecimenConc.: 3.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3500 nm / Nominal defocus min: 500 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: JEOL CRYOSPECPORTER
Image recordingElectron dose: 63.6 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 4195

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
1crYOLO1.8particle selection
2SerialEM3.8.0image acquisition
10RELION4initial Euler assignment
11RELION4final Euler assignment
12RELIONclassification
13RELION43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D7 (2x7 fold dihedral)
3D reconstructionResolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 136955 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00364862
ELECTRON MICROSCOPYf_angle_d0.587696
ELECTRON MICROSCOPYf_dihedral_angle_d5.679159
ELECTRON MICROSCOPYf_chiral_restr0.04410682
ELECTRON MICROSCOPYf_plane_restr0.00311396

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