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- PDB-8wuc: Cryo-EM structure of H. thermoluteolus GroEL-GroES2 football complex -

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Basic information

Entry
Database: PDB / ID: 8wuc
TitleCryo-EM structure of H. thermoluteolus GroEL-GroES2 football complex
Components
  • Chaperonin GroEL
  • Co-chaperonin GroES
KeywordsCHAPERONE / Chaperonin / ATPase / protein folding
Function / homology
Function and homology information


chaperonin ATPase / protein folding chaperone / isomerase activity / ATP-dependent protein folding chaperone / unfolded protein binding / protein refolding / ATP binding / cytoplasm
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'-DIPHOSPHATE / Co-chaperonin GroES / Chaperonin GroEL
Similarity search - Component
Biological speciesHydrogenophilus thermoluteolus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsLiao, Z. / Gopalasingam, C.C. / Kameya, M. / Gerle, C. / Shigematsu, H. / Ishii, M. / Arakawa, T. / Fushinobu, S.
Funding support Japan, 2items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)23H00322 Japan
Japan Science and TechnologyJPMJSP2108 Japan
CitationJournal: Structure / Year: 2024
Title: Structural insights into thermophilic chaperonin complexes.
Authors: Zengwei Liao / Chai C Gopalasingam / Masafumi Kameya / Christoph Gerle / Hideki Shigematsu / Masaharu Ishii / Takatoshi Arakawa / Shinya Fushinobu /
Abstract: Group I chaperonins are dual heptamer protein complexes that play significant roles in protein homeostasis. The structure and function of the Escherichia coli chaperonin are well characterized. ...Group I chaperonins are dual heptamer protein complexes that play significant roles in protein homeostasis. The structure and function of the Escherichia coli chaperonin are well characterized. However, the dynamic properties of chaperonins, such as large ATPase-dependent conformational changes by binding of lid-like co-chaperonin GroES, have made structural analyses challenging, and our understanding of these changes during the turnover of chaperonin complex formation is limited. In this study, we used single-particle cryogenic electron microscopy to investigate the structures of GroES-bound chaperonin complexes from the thermophilic hydrogen-oxidizing bacteria Hydrogenophilus thermoluteolus and Hydrogenobacter thermophilus in the presence of ATP and AMP-PNP. We captured the structure of an intermediate state chaperonin complex, designated as an asymmetric football-shaped complex, and performed analyses to decipher the dynamic structural variations. Our structural analyses of inter- and intra-subunit communications revealed a unique mechanism of complex formation through the binding of a second GroES to a bullet-shaped complex.
History
DepositionOct 20, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 27, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Chaperonin GroEL
a: Co-chaperonin GroES
B: Chaperonin GroEL
b: Co-chaperonin GroES
C: Chaperonin GroEL
c: Co-chaperonin GroES
D: Chaperonin GroEL
d: Co-chaperonin GroES
E: Chaperonin GroEL
e: Co-chaperonin GroES
F: Chaperonin GroEL
f: Co-chaperonin GroES
G: Chaperonin GroEL
g: Co-chaperonin GroES
H: Chaperonin GroEL
h: Co-chaperonin GroES
I: Chaperonin GroEL
i: Co-chaperonin GroES
J: Chaperonin GroEL
j: Co-chaperonin GroES
K: Chaperonin GroEL
k: Co-chaperonin GroES
L: Chaperonin GroEL
l: Co-chaperonin GroES
M: Chaperonin GroEL
m: Co-chaperonin GroES
N: Chaperonin GroEL
n: Co-chaperonin GroES
hetero molecules


Theoretical massNumber of molelcules
Total (without water)934,60756
Polymers928,28628
Non-polymers6,32128
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 56081.340 Da / Num. of mol.: 14 / Source method: isolated from a natural source / Source: (natural) Hydrogenophilus thermoluteolus (bacteria) / Strain: TH-1 / References: UniProt: A0A2Z6DW38, chaperonin ATPase
#2: Protein
Co-chaperonin GroES / 10 kDa chaperonin / Chaperonin-10 / Cpn10


Mass: 10224.831 Da / Num. of mol.: 14
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Hydrogenophilus thermoluteolus (bacteria)
Strain: TH-1 / Gene: groES, groS, HPTL_0309 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A2Z6DVV7
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / 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 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1H. thermoluteolus GroEL-GroES2 football complex bound with ADP-Mg2+COMPLEX6.76 mg/mL native GroEL was mixed with 3.7 mg/mL recombinant GroES followed by addition of 2 mM ATP in imaging buffer supplemented by detergents (0.1% 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate (CHAPS), 0.05% n-Octyl-beta-D-glucopyranoside)#1-#20MULTIPLE SOURCES
2H. thermoluteolus GroELCOMPLEX#11NATURAL
3H. thermoluteolus GroESCOMPLEX#21RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
11954 kDa/nmNO
21808 kDa/nmNO
3173 kDa/nmNO
Source (natural)Organism: Hydrogenophilus thermoluteolus (bacteria) / Strain: TH-1
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3) / Plasmid: pET 23a
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2100 mMPotassium chlorideKCl1
35 mMMagnesium chlorideMgCl21
42 mMAdenosine triphosphate1
50.1 %3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate1
60.05 %n-Octyl-beta-D-glucopyranoside1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Grids were freshly treated using Gatan Solarus II / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 281 K / Details: Blot force 0, blot time 8 s

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 120000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 4.95 sec. / Electron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1233

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFIND4.1CTF correction
10RELION4initial Euler assignment
11RELION4final Euler assignment
12RELION4classification
13cryoSPARC4.33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D7 (2x7 fold dihedral)
3D reconstructionResolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 103957 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00465912
ELECTRON MICROSCOPYf_angle_d0.64988984
ELECTRON MICROSCOPYf_dihedral_angle_d7.4719394
ELECTRON MICROSCOPYf_chiral_restr0.04310668
ELECTRON MICROSCOPYf_plane_restr0.00311578

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