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- PDB-8ba7: CryoEM structure of nucleotide-free GroEL-Rubisco. -

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Basic information

Entry
Database: PDB / ID: 8ba7
TitleCryoEM structure of nucleotide-free GroEL-Rubisco.
ComponentsChaperonin GroEL
KeywordsCHAPERONE / GroEL / Rubisco / complex
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
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.4 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 1.0Oct 25, 2023Provider: repository / Type: Initial release
Revision 1.1Dec 27, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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


Theoretical massNumber of molelcules
Total (without water)801,64714
Polymers801,64714
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "I"
d_2ens_1chain "B"
d_3ens_1chain "C"
d_4ens_1chain "D"
d_5ens_1chain "E"
d_6ens_1chain "F"
d_7ens_1chain "G"
d_8ens_1chain "H"
d_9ens_1chain "M"
d_10ens_1chain "J"
d_11ens_1chain "K"
d_12ens_1chain "A"
d_13ens_1chain "L"
d_14ens_1chain "N"

NCS domain segments:

Component-ID: 1 / Ens-ID: ens_1 / Beg auth comp-ID: ALA / Beg label comp-ID: ALA / End auth comp-ID: PRO / End label comp-ID: PRO / Auth seq-ID: 2 - 525 / Label seq-ID: 1 - 524

Dom-IDAuth asym-IDLabel asym-ID
d_1II
d_2BB
d_3CC
d_4DD
d_5EE
d_6FF
d_7GG
d_8HH
d_9MM
d_10JJ
d_11KK
d_12AA
d_13LL
d_14NN

NCS oper:
IDCodeMatrixVector
1given(-0.888053733044, -0.459524584792, 0.0140614080916), (-0.459725030762, 0.88736588006, -0.035138170076), (0.0036692392485, -0.0376689643758, -0.999283536243)182.709509692, 47.6705927114, 166.095049162
2given(-0.192736190505, -0.981113246845, 0.0164182135063), (-0.981205083643, 0.192539092529, -0.012856192334), (0.00945223267345, -0.0185874880916, -0.999782556651)170.311258615, 142.273882767, 164.453389868
3given(0.645743294444, -0.763537990442, -0.00503337196158), (-0.763538893937, -0.645758252864, 0.00215320792138), (-0.00489439753321, 0.00245275568354, -0.999985014319)89.3974579652, 191.399359114, 163.729042145
4given(0.999278413901, 0.0266718925539, -0.0270418501262), (0.0265613702151, -0.999637332481, -0.0044381440092), (-0.0271504166257, 0.00371667291367, -0.999624450091)0.194457220404, 158.893096855, 165.107494254
5given(0.601888207126, 0.798089099632, -0.0280066986904), (0.797650262558, -0.602509507817, -0.0271357998147), (-0.0385310882855, -0.00600683266438, -0.999239347302)-30.9603453874, 68.9243117208, 166.409169056
6given(-0.243405504403, 0.969595731044, -0.0252562619499), (0.969425185487, 0.242362155244, -0.0384108767043), (-0.0311218599986, -0.0338334752443, -0.99894280406)21.1590948047, -11.3077363011, 168.277943295
7given(0.62610194965, -0.779418578668, 0.0224282828439), (0.779615470551, 0.626258504901, -5.58366603302E-5), (-0.0140023827508, 0.0175203957248, 0.999748452867)89.7677355798, -30.6384641362, -0.338967012842
8given(-0.899675583466, -0.435665506076, 0.0279179392142), (0.436505399309, -0.898726595773, 0.0418753199864), (0.00684696199705, 0.0498605341462, 0.998732720124)180.293481293, 115.392467668, -4.91889324147
9given(0.623631593676, 0.78164133189, -0.0109755933746), (-0.781501054536, 0.623727812393, 0.0148228811448), (0.0184319594057, -0.000666579194766, 0.999829894804)-32.8314549302, 89.7772643283, -1.57058270964
10given(-0.217618976883, 0.975967142045, -0.011406951792), (-0.97534197372, -0.217009917378, 0.0401837038975), (0.0367425529837, 0.0198704154045, 0.999127194801)17.2928414117, 170.294811398, -4.55928590458
11given(-0.907222722264, 0.420642863493, -0.00255217552277), (0.420209821945, 0.905977718669, -0.0512647911907), (-0.0192519544051, -0.0475810326423, -0.998681835013)114.457085499, -20.2949098173, 168.418557857
12given(-0.900508182594, 0.434690709337, 0.0113578297707), (-0.433703566176, -0.899736619545, 0.0487363533344), (0.0314042953647, 0.0389615536919, 0.998747098903)111.969659884, 182.402182965, -5.7210236337
13given(-0.223398741374, -0.974123592448, 0.0342961949646), (0.974709428754, -0.22304423181, 0.0138852495046), (-0.0058763806696, 0.0365307718654, 0.999315250988)170.432385699, 21.4171202491, -2.41074955891

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Components

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


Mass: 57260.504 Da / Num. of mol.: 14
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K12 / Gene: groEL, groL, mopA, b4143, JW4103 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A6F5, chaperonin ATPase

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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 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.802 MDa / Experimental value: YES
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12
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 K2 BASE (4k x 4k) / Num. of grids imaged: 2

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategory
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
CTF correctionType: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 65453 / Num. of class averages: 2 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 162.5 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002454376
ELECTRON MICROSCOPYf_angle_d0.500673430
ELECTRON MICROSCOPYf_chiral_restr0.05868932
ELECTRON MICROSCOPYf_plane_restr0.00279632
ELECTRON MICROSCOPYf_dihedral_angle_d4.04037742
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2IELECTRON MICROSCOPYNCS constraints0.000700194906772
ens_1d_3IELECTRON MICROSCOPYNCS constraints0.000703587554365
ens_1d_4IELECTRON MICROSCOPYNCS constraints0.000706698213168
ens_1d_5IELECTRON MICROSCOPYNCS constraints0.000705543819535
ens_1d_6IELECTRON MICROSCOPYNCS constraints0.000709774422471
ens_1d_7IELECTRON MICROSCOPYNCS constraints0.000704875700804
ens_1d_8IELECTRON MICROSCOPYNCS constraints0.000705027562488
ens_1d_9IELECTRON MICROSCOPYNCS constraints0.00070310473895
ens_1d_10IELECTRON MICROSCOPYNCS constraints0.000704843290692
ens_1d_11IELECTRON MICROSCOPYNCS constraints0.000709825915334
ens_1d_12IELECTRON MICROSCOPYNCS constraints0.000704585639912
ens_1d_13IELECTRON MICROSCOPYNCS constraints0.000707064074394
ens_1d_14IELECTRON MICROSCOPYNCS constraints0.000710620706057

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