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- PDB-8ba8: CryoEM structure of GroEL-ADP.BeF3-Rubisco. -

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Basic information

Entry
Database: PDB / ID: 8ba8
TitleCryoEM structure of GroEL-ADP.BeF3-Rubisco.
ComponentsChaperonin GroEL
KeywordsCHAPERONE / GroEL / nucleotide / Rubisco / Chameleon
Function / homology
Function and homology information


GroEL-GroES complex / chaperonin ATPase / virion assembly / chaperone cofactor-dependent protein refolding / isomerase activity / ATP-dependent protein folding chaperone / response to radiation / unfolded protein binding / protein folding / response to heat ...GroEL-GroES complex / chaperonin ATPase / virion assembly / chaperone cofactor-dependent protein refolding / isomerase activity / ATP-dependent protein folding chaperone / response to radiation / unfolded protein binding / protein folding / response to heat / protein refolding / magnesium ion binding / ATP hydrolysis activity / ATP binding / membrane / identical protein binding / cytosol
Similarity search - Function
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
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / BERYLLIUM TRIFLUORIDE ION / : / Chaperonin GroEL
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsGardner, S. / Saibil, H.R.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/M009513/1 United Kingdom
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Structural basis of substrate progression through the bacterial chaperonin cycle.
Authors: Scott Gardner / Michele C Darrow / Natalya Lukoyanova / Konstantinos Thalassinos / Helen R Saibil /
Abstract: The bacterial chaperonin GroEL-GroES promotes protein folding through ATP-regulated cycles of substrate protein binding, encapsulation, and release. Here, we have used cryoEM to determine structures ...The bacterial chaperonin GroEL-GroES promotes protein folding through ATP-regulated cycles of substrate protein binding, encapsulation, and release. Here, we have used cryoEM to determine structures of GroEL, GroEL-ADP·BeF, and GroEL-ADP·AlF-GroES all complexed with the model substrate Rubisco. Our structures provide a series of snapshots that show how the conformation and interactions of non-native Rubisco change as it proceeds through the GroEL-GroES reaction cycle. We observe specific charged and hydrophobic GroEL residues forming strong initial contacts with non-native Rubisco. Binding of ATP or ADP·BeF to GroEL-Rubisco results in the formation of an intermediate GroEL complex displaying striking asymmetry in the ATP/ADP·BeF-bound ring. In this ring, four GroEL subunits bind Rubisco and the other three are in the GroES-accepting conformation, suggesting how GroEL can recruit GroES without releasing bound substrate. Our cryoEM structures of stalled GroEL-ADP·AlF-Rubisco-GroES complexes show Rubisco folding intermediates interacting with GroEL-GroES via different sets of residues.
History
DepositionOct 11, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 27, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Chaperonin GroEL
B: Chaperonin GroEL
C: Chaperonin GroEL
D: Chaperonin GroEL
E: Chaperonin GroEL
F: Chaperonin GroEL
G: Chaperonin GroEL
H: Chaperonin GroEL
I: Chaperonin GroEL
J: Chaperonin GroEL
K: Chaperonin GroEL
L: Chaperonin GroEL
M: Chaperonin GroEL
N: Chaperonin GroEL
hetero molecules


Theoretical massNumber of molelcules
Total (without water)810,81463
Polymers803,48414
Non-polymers7,33149
Water1267
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 1 types, 14 molecules ABCDEFGHIJKLMN

#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 K-12 (bacteria) / Strain: K-12 / Gene: groEL, groL, mopA, b4143, JW4103 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A6F5, chaperonin ATPase

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Non-polymers , 5 types, 56 molecules

#2: Chemical
ChemComp-BEF / BERYLLIUM TRIFLUORIDE ION


Mass: 66.007 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: BeF3
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


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


Mass: 39.098 Da / Num. of mol.: 14 / Source method: isolated from a natural source / Formula: K
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 7 / 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: GroEL-ADP.BeFx-Rubisco / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.802 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pTrcESL
Buffer solutionpH: 7.5
SpecimenConc.: 2.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: SPOTITON / Cryogen name: ETHANE / Humidity: 80 % / Chamber temperature: 293 K
Details: The grid was prepared using a chameleon (SPT Labtech).

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3000 nm / Nominal defocus min: 1400 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Image recordingElectron dose: 40.2 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2

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Processing

EM software
IDNameVersionCategory
10RELION3.1initial Euler assignment
11cryoSPARC3.3.2final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 7712622
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 202582 / Num. of class averages: 2 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 173.15 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003954796
ELECTRON MICROSCOPYf_angle_d0.747174046
ELECTRON MICROSCOPYf_chiral_restr0.08448988
ELECTRON MICROSCOPYf_plane_restr0.0089625
ELECTRON MICROSCOPYf_dihedral_angle_d10.87397882

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