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- PDB-2klr: Solid-state NMR structure of the alpha-crystallin domain in alpha... -

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Basic information

Entry
Database: PDB / ID: 2klr
TitleSolid-state NMR structure of the alpha-crystallin domain in alphaB-crystallin oligomers
ComponentsAlpha-crystallin B chainCRYAB
KeywordsCHAPERONE / Protein / dimer / oligomer / heterogeneity / intermolecular interactions / sHSP / human / small heat-shock protein / Cataract / Desmin-related myopathy / Disease mutation / Eye lens protein / Glycoprotein / Methylation / Oxidation / Phosphoprotein
Function / homology
Function and homology information


microtubule polymerization or depolymerization / negative regulation of intracellular transport / apoptotic process involved in morphogenesis / cardiac myofibril / regulation of programmed cell death / tubulin complex assembly / structural constituent of eye lens / negative regulation of amyloid fibril formation / M band / lens development in camera-type eye ...microtubule polymerization or depolymerization / negative regulation of intracellular transport / apoptotic process involved in morphogenesis / cardiac myofibril / regulation of programmed cell death / tubulin complex assembly / structural constituent of eye lens / negative regulation of amyloid fibril formation / M band / lens development in camera-type eye / muscle organ development / actin filament bundle / HSF1-dependent transactivation / negative regulation of reactive oxygen species metabolic process / negative regulation of protein-containing complex assembly / stress-activated MAPK cascade / synaptic membrane / muscle contraction / cellular response to gamma radiation / response to hydrogen peroxide / negative regulation of cell growth / Z disc / unfolded protein binding / protein folding / response to estradiol / amyloid-beta binding / response to heat / perikaryon / protein refolding / microtubule binding / dendritic spine / lysosome / response to hypoxia / protein stabilization / axon / negative regulation of gene expression / negative regulation of DNA-templated transcription / protein-containing complex binding / negative regulation of apoptotic process / structural molecule activity / cell surface / protein homodimerization activity / protein-containing complex / mitochondrion / extracellular exosome / nucleoplasm / identical protein binding / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Alpha-crystallin B chain, ACD domain / Alpha-crystallin, N-terminal / Alpha crystallin A chain, N terminal / Alpha crystallin/Small heat shock protein, animal type / Immunoglobulin-like - #790 / Hsp20/alpha crystallin family / Small heat shock protein (sHSP) domain profile. / Alpha crystallin/Hsp20 domain / HSP20-like chaperone / Immunoglobulin-like ...Alpha-crystallin B chain, ACD domain / Alpha-crystallin, N-terminal / Alpha crystallin A chain, N terminal / Alpha crystallin/Small heat shock protein, animal type / Immunoglobulin-like - #790 / Hsp20/alpha crystallin family / Small heat shock protein (sHSP) domain profile. / Alpha crystallin/Hsp20 domain / HSP20-like chaperone / Immunoglobulin-like / Sandwich / Mainly Beta
Similarity search - Domain/homology
Alpha-crystallin B chain
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodSOLID-STATE NMR / simulated annealing, molecular dynamics, torsion angle dynamics
Model detailslowest energy, model 1
AuthorsJehle, S. / Rajagopal, P. / Markovic, S. / Bardiaux, B. / Kuehne, R. / Higman, V.A. / Klevit, R.E. / van Rossum, B. / Oschkinat, H.
Citation
Journal: Nat Struct Mol Biol / Year: 2010
Title: Solid-state NMR and SAXS studies provide a structural basis for the activation of alphaB-crystallin oligomers.
Authors: Stefan Jehle / Ponni Rajagopal / Benjamin Bardiaux / Stefan Markovic / Ronald Kühne / Joseph R Stout / Victoria A Higman / Rachel E Klevit / Barth-Jan van Rossum / Hartmut Oschkinat /
Abstract: The small heat shock protein alphaB-crystallin (alphaB) contributes to cellular protection against stress. For decades, high-resolution structural studies on oligomeric alphaB have been confounded by ...The small heat shock protein alphaB-crystallin (alphaB) contributes to cellular protection against stress. For decades, high-resolution structural studies on oligomeric alphaB have been confounded by its polydisperse nature. Here, we present a structural basis of oligomer assembly and activation of the chaperone using solid-state NMR and small-angle X-ray scattering (SAXS). The basic building block is a curved dimer, with an angle of approximately 121 degrees between the planes of the beta-sandwich formed by alpha-crystallin domains. The highly conserved IXI motif covers a substrate binding site at pH 7.5. We observe a pH-dependent modulation of the interaction of the IXI motif with beta4 and beta8, consistent with a pH-dependent regulation of the chaperone function. N-terminal region residues Ser59-Trp60-Phe61 are involved in intermolecular interaction with beta3. Intermolecular restraints from NMR and volumetric restraints from SAXS were combined to calculate a model of a 24-subunit alphaB oligomer with tetrahedral symmetry.
#1: Journal: J.Mol.Biol. / Year: 2009
Title: AlphaB-crystallin: a hybrid solid-state/solution-state NMR investigation reveals structural aspects of the heterogeneous oligomer.
Authors: Jehle, S. / van Rossum, B. / Stout, J.R. / Noguchi, S.M. / Falber, K. / Rehbein, K. / Oschkinat, H. / Klevit, R.E. / Rajagopal, P.
History
DepositionJul 8, 2009Deposition site: BMRB / Processing site: RCSB
Revision 1.0Jul 7, 2010Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Alpha-crystallin B chain
B: Alpha-crystallin B chain


Theoretical massNumber of molelcules
Total (without water)40,3842
Polymers40,3842
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)10 / 200structures with the lowest energy
RepresentativeModel #1lowest energy
Noncrystallographic symmetry (NCS)NCS oper:
IDCodeMatrixVector
1given(1), (1), (1)
2generate(-0.352219, 0.835492, -0.421776), (-0.808833, -0.498466, -0.311962), (-0.470883, 0.231268, 0.851343)-19.63822, 37.0625, -5.49449
3generate(-0.355727, -0.809154, -0.467684), (0.83248, -0.501776, 0.234942), (-0.424777, -0.305763, 0.8521)20.45375, 36.11392, 7.93886
DetailsThe authors state that the NCS Matrix for the hexamer should be applied to model 7 in the ensemble.

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Components

#1: Protein Alpha-crystallin B chain / CRYAB / Alpha(B)-crystallin / Rosenthal fiber component / Heat shock protein beta-5 / HspB5 / Renal ...Alpha(B)-crystallin / Rosenthal fiber component / Heat shock protein beta-5 / HspB5 / Renal carcinoma antigen NY-REN-27


Mass: 20191.930 Da / Num. of mol.: 2
Fragment: Alpha-crystallin domain homodimer in oligomers of alphaB-crystallin
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CRYAB, CRYA2 / Production host: Escherichia coli (E. coli) / References: UniProt: P02511
Sequence detailsTHE HOMOGENEOUS PART OF THE ALPHA-CRYSTALLIN DOMAIN DIMER IN OLIGOMERS OF HUMAN ALPHAB CRYSTALLIN ...THE HOMOGENEOUS PART OF THE ALPHA-CRYSTALLIN DOMAIN DIMER IN OLIGOMERS OF HUMAN ALPHAB CRYSTALLIN IS SHOWN IN THIS STRUCTURE. RESIDUES THAT SHOW A SINGLE NMR SIGNAL SET FOR C', CA AND CB WERE INCLUDED IN THE STRUCTURE CALCULATION. SUBJECT OF THE STUDY WAS THE FULL LENGTH PROTEIN (UNIPROT ID P02511).

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Experimental details

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Experiment

ExperimentMethod: SOLID-STATE NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDType
1112D 13C-13C PDSD
1222D 13C-13C PDSD
1332D 13C-13C CHHC
1432D 13C-13C PDSD
1513D 15N-13C-13C NCACX
1623D 15N-13C-13C NCACX
1713D 15N-13C-13C NCOCX
1823D 15N-13C-13C NCOCX
1942D 15N-13C TEDOR
11052D 15N-13C TEDOR
11142D 15N-13C NHHC
11252D 15N-13C NHHC
11342D 15N-13C PAIN

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Sample preparation

Details
Solution-IDContentsSolvent system
118 mg [1,3-13C]-glycerol; U-100% 15 alphaB-crystallin, 100% H2O100% H2O
217 mg [2-13C]-glycerol; U-100% 15N alphaB-crystallin, 100% H2O100% H2O
34 mg [U-100% 13C; U-100% 15N] alphaB-crystallin, 16 mg alphaB-crystallin, 100% H2O100% H2O
410 mg [U-100% 15N]; [U-100% 12C] 13C depleted alphaB-crystallin, 10 mg [2-13C]-glycerol ; U-100% 15N alphaB-crystallin, 100% H2O100% H2O
510 mg [U-100% 15N]; [U-100% 12C] 13C depleted alphaB-crystallin, 10 mg [1,3-13C]-glycerol ; U-100% 15N alphaB-crystallin, 100% H2O100% H2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
18 mg/mLalphaB-crystallin-1[1,3-13C]-glycerol; U-100% 151
17 mg/mLalphaB-crystallin-2[2-13C]-glycerol ; U-100% 15N2
4 mg/mLalphaB-crystallin-3[U-100% 13C; U-100% 15N]3
16 mg/mLalphaB-crystallin-43
10 mg/mLalphaB-crystallin-5[U-100% 15N]; [U-100% 12C] 13C depleted4
10 mg/mLalphaB-crystallin-6[2-13C]-glycerol ; U-100% 15N4
10 mg/mLalphaB-crystallin-7[U-100% 15N]; [U-100% 12C] 13C depleted5
10 mg/mLalphaB-crystallin-8[1,3-13C]-glycerol ; U-100% 15N5
Sample conditionspH: 7.6 / Temperature: 270 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Bruker AvanceBrukerAvance4001
Bruker AvanceBrukerAvance6002
Bruker AvanceBrukerAvance7003
Bruker AvanceBrukerAvance9004

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Processing

NMR software
NameVersionDeveloperClassification
TOPSPINBruker Biospincollection
TOPSPINBruker Biospinprocessing
SPARKYGoddardchemical shift assignment
SPARKYGoddarddata analysis
SPARKYGoddardpeak picking
X-PLOR_NIHSchwieters, Kuszewski, Tjandra and Clorestructure solution
TALOSCornilescu, Delaglio and Baxdihedral angle determination
ARIA2.2Rieping W, Habeck M, Bardiaux B, Bernard A, Malliavin TE, Nilges M.structure solution
ARIA2.2Rieping W, Habeck M, Bardiaux B, Bernard A, Malliavin TE, Nilges M.refinement
CNS1.2Brunger, Adams, Clore, Gros, Nilges and Readstructure solution
CNS1.2Brunger, Adams, Clore, Gros, Nilges and Readrefinement
SOLARIA1(SOLARIA) Fossi M, Castellani F, Nilges M, Oschkinat H, van Rossum BJ.structure solution
RefinementMethod: simulated annealing, molecular dynamics, torsion angle dynamics
Software ordinal: 1
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 200 / Conformers submitted total number: 10

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