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- PDB-2eu1: Crystal structure of the chaperonin GroEL-E461K -

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Basic information

Entry
Database: PDB / ID: 2eu1
TitleCrystal structure of the chaperonin GroEL-E461K
ComponentsGROEL
KeywordsCHAPERONE/PEPTIDE BINDING PROTEIN / chaperonin / GROEL / HSP60 / E461K / CHAPERONE-PEPTIDE BINDING PROTEIN 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
GROEL; domain 2 / TCP-1-like chaperonin intermediate domain / GROEL; domain 1 / GroEL-like equatorial domain / GroEL / GroEL / Chaperonin Cpn60, conserved site / Chaperonins cpn60 signature. / Chaperonin Cpn60/GroEL / GroEL-like equatorial domain superfamily ...GROEL; domain 2 / TCP-1-like chaperonin intermediate domain / GROEL; domain 1 / GroEL-like equatorial domain / GroEL / GroEL / 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 / 3-Layer(bba) Sandwich / 2-Layer Sandwich / Orthogonal Bundle / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.29 Å
AuthorsCabo-Bilbao, A. / Spinelli, S. / Sot, B. / Agirre, J. / Mechaly, A.E. / Muga, A. / Guerin, D.M.A.
Citation
Journal: J.Struct.Biol. / Year: 2006
Title: Crystal structure of the temperature-sensitive and allosteric-defective chaperonin GroEL(E461K).
Authors: Cabo-Bilbao, A. / Spinelli, S. / Sot, B. / Agirre, J. / Mechaly, A.E. / Muga, A. / Guerin, D.M.A.
#1: Journal: J.Mol.Biol. / Year: 2005
Title: Crystal structure of wild-type chaperonin GROEL
Authors: Bartolucci, C. / Lamba, D. / Grazulis, S. / Manakova, E. / Heumann, H.
#2: Journal: Nat Struct Mol Biol / Year: 2004
Title: A mutant chaperonin with rearranged inter-ring electrostatic contacts and temperature-sensitive dissociation.
Authors: B Trevor Sewell / Robert B Best / Shaoxia Chen / Alan M Roseman / George W Farr / Arthur L Horwich / Helen R Saibil /
Abstract: The chaperonin GroEL assists protein folding through ATP-dependent, cooperative movements that alternately create folding chambers in its two rings. The substitution E461K at the interface between ...The chaperonin GroEL assists protein folding through ATP-dependent, cooperative movements that alternately create folding chambers in its two rings. The substitution E461K at the interface between these two rings causes temperature-sensitive, defective protein folding in Escherichia coli. To understand the molecular defect, we have examined the mutant chaperonin by cryo-EM. The normal out-of-register alignment of contacts between subunits of opposing wild-type rings is changed in E461K to an in-register one. This is associated with loss of cooperativity in ATP binding and hydrolysis. Consistent with the loss of negative cooperativity between rings, the cochaperonin GroES binds simultaneously to both E461K rings. These GroES-bound structures were unstable at higher temperature, dissociating into complexes of single E461K rings associated with GroES. Lacking the allosteric signal from the opposite ring, these complexes cannot release their GroES and become trapped, dead-end states.
#3: Journal: Nature / Year: 1995
Title: Conformational variability in the refined structure of the chaperonin GroEL at 2.8 A resolution
Authors: Braig, K. / Adams, P.D. / Brunger, A.T.
#4: Journal: Nature / Year: 1997
Title: The crystal structure of the asymmetric GroEL-GroES-(ADP)7 chaperonin complex.
Authors: Z Xu / A L Horwich / P B Sigler /
Abstract: Chaperonins assist protein folding with the consumption of ATP. They exist as multi-subunit protein assemblies comprising rings of subunits stacked back to back. In Escherichia coli, asymmetric ...Chaperonins assist protein folding with the consumption of ATP. They exist as multi-subunit protein assemblies comprising rings of subunits stacked back to back. In Escherichia coli, asymmetric intermediates of GroEL are formed with the co-chaperonin GroES and nucleotides bound only to one of the seven-subunit rings (the cis ring) and not to the opposing ring (the trans ring). The structure of the GroEL-GroES-(ADP)7 complex reveals how large en bloc movements of the cis ring's intermediate and apical domains enable bound GroES to stabilize a folding chamber with ADP confined to the cis ring. Elevation and twist of the apical domains double the volume of the central cavity and bury hydrophobic peptide-binding residues in the interface with GroES, as well as between GroEL subunits, leaving a hydrophilic cavity lining that is conducive to protein folding. An inward tilt of the cis equatorial domain causes an outward tilt in the trans ring that opposes the binding of a second GroES. When combined with new functional results, this negative allosteric mechanism suggests a model for an ATP-driven folding cycle that requires a double toroid.
#5: Journal: Protein Sci. / Year: 2005
Title: Ionic interactions at both inter-ring contact sites of GroEL are involved in transmission of the allosteric signal: a time-resolved infrared difference study
Authors: Sot, B. / von Germar, F. / Mantele, W. / Valpuesta, J.M. / Taneva, S.G. / Muga, A.
#6: Journal: J.Biol.Chem. / Year: 2002
Title: Salt bridges at the inter-ring interface regulate the thermostat of GroEL
Authors: Sot, B. / Galan, A. / Valpuesta, J.M. / Bertrand, S. / Muga, A.
#7: Journal: J.Biol.Chem. / Year: 2003
Title: GroEL stability and function. Contribution of the ionic interactions at the inter-ring contact sites
Authors: Sot, B. / Banuelos, S. / Valpuesta, J.M. / Muga, A.
History
DepositionOct 28, 2005Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 29, 2006Provider: repository / Type: Initial release
Revision 1.1May 1, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Derived calculations / Version format compliance
Revision 1.3Jan 31, 2018Group: Database references / Category: citation_author / Item: _citation_author.name
Revision 1.4Oct 20, 2021Group: Database references / Category: database_2 / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details
Revision 1.5Aug 23, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: GROEL
B: GROEL
C: GROEL
D: GROEL
E: GROEL
F: GROEL
G: GROEL
H: GROEL
I: GROEL
J: GROEL
K: GROEL
L: GROEL
M: GROEL
N: GROEL


Theoretical massNumber of molelcules
Total (without water)803,48514
Polymers803,48514
Non-polymers00
Water0
1
A: GROEL
B: GROEL
C: GROEL
D: GROEL
E: GROEL
F: GROEL
G: GROEL

A: GROEL
B: GROEL
C: GROEL
D: GROEL
E: GROEL
F: GROEL
G: GROEL


Theoretical massNumber of molelcules
Total (without water)803,48514
Polymers803,48514
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation4_566x,-y+1,-z+11
2
H: GROEL
I: GROEL
J: GROEL
K: GROEL
L: GROEL
M: GROEL
N: GROEL


Theoretical massNumber of molelcules
Total (without water)401,7427
Polymers401,7427
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPQS
3
H: GROEL
I: GROEL
J: GROEL
K: GROEL
L: GROEL
M: GROEL
N: GROEL


Theoretical massNumber of molelcules
Total (without water)401,7427
Polymers401,7427
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area21710 Å2
ΔGint-99 kcal/mol
Surface area150750 Å2
MethodPISA
4
A: GROEL
B: GROEL
C: GROEL
D: GROEL
E: GROEL
F: GROEL
G: GROEL


Theoretical massNumber of molelcules
Total (without water)401,7427
Polymers401,7427
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area22150 Å2
ΔGint-95 kcal/mol
Surface area150340 Å2
MethodPISA, PQS
Unit cell
Length a, b, c (Å)267.695, 290.641, 247.006
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number20
Space group name H-MC2221
DetailsThe biological GroELE461K molecule is formed in the crystal by the association of two rings from neighbouring asymmetric units.

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Components

#1: Protein
GROEL / / Protein Cpn60 / 60 kDa chaperonin


Mass: 57391.773 Da / Num. of mol.: 14 / Mutation: E461K
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli) / References: UniProt: P0A6F5

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.11 Å3/Da / Density % sol: 60.41 %
Crystal growTemperature: 277 K / pH: 8
Details: Reservoir solution: 43% (v/v) MPD, 100mM Imidazole, 170mM MgCl2.6H2O, dissolved in the ratio 1:1.5 with 22.5mg/ml protein, 50mM TRIS-HCL, 10mM MgCl2, pH 8.0, VAPOR DIFFUSION, SITTING DROP, temperature 277K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID29 / Wavelength: 0.97563
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97563 Å / Relative weight: 1
ReflectionResolution: 3.29→39.75 Å / Num. obs: 123350 / % possible obs: 85 %

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Processing

Software
NameVersionClassification
SCALAdata scaling
AMoREphasing
CNS1.1refinement
CCP4(SCALA)data scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 1OEL

1oel


Resolution: 3.29→15 Å / σ(F): 0 / Stereochemistry target values: ENGH & HUBER / Details: MAXIMUM LIKELIHOOD TARGET USING AMPLITUDES
RfactorNum. reflection% reflectionSelection details
Rfree0.296 6133 4.2 %RANDOM
Rwork0.276 ---
obs0.276 121338 --
all-122137 --
Refinement stepCycle: LAST / Resolution: 3.29→15 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms53970 0 0 0 53970
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_bond_d0.011
X-RAY DIFFRACTIONc_bond_d_na
X-RAY DIFFRACTIONc_bond_d_prot
X-RAY DIFFRACTIONc_angle_d
X-RAY DIFFRACTIONc_angle_d_na
X-RAY DIFFRACTIONc_angle_d_prot
X-RAY DIFFRACTIONc_angle_deg1.329
X-RAY DIFFRACTIONc_angle_deg_na
X-RAY DIFFRACTIONc_angle_deg_prot
X-RAY DIFFRACTIONc_dihedral_angle_d
X-RAY DIFFRACTIONc_dihedral_angle_d_na
X-RAY DIFFRACTIONc_dihedral_angle_d_prot
X-RAY DIFFRACTIONc_improper_angle_d
X-RAY DIFFRACTIONc_improper_angle_d_na
X-RAY DIFFRACTIONc_improper_angle_d_prot
X-RAY DIFFRACTIONc_mcbond_it
X-RAY DIFFRACTIONc_mcangle_it
X-RAY DIFFRACTIONc_scbond_it
X-RAY DIFFRACTIONc_scangle_it
Xplor fileSerial no: 1 / Param file: PROTEIN_REP.PARAM

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