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- PDB-2n0k: Chemical shift assignments and structure of the alpha-crystallin ... -

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Basic information

Entry
Database: PDB / ID: 2n0k
TitleChemical shift assignments and structure of the alpha-crystallin domain from human, HSPB5
ComponentsAlpha-crystallin B chainCRYAB
KeywordsMETAL BINDING PROTEIN / crystallin / human / ACD / Protein
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 / muscle contraction / synaptic membrane / response to hydrogen peroxide / cellular response to gamma radiation / 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)
MethodSOLUTION NMR / simulated annealing
Model detailsclosest to the average, model1
AuthorsRajagopal, P. / Klevit, R.E. / Shi, L. / Baker, D.
Citation
Journal: Elife / Year: 2015
Title: A conserved histidine modulates HSPB5 structure to trigger chaperone activity in response to stress-related acidosis.
Authors: Rajagopal, P. / Tse, E. / Borst, A.J. / Delbecq, S.P. / Shi, L. / Southworth, D.R. / Klevit, R.E.
#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.
#2: 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.
#3: Journal: Proc Natl Acad Sci U S A / Year: 2011
Title: N-terminal domain of alphaB-crystallin provides a conformational switch for multimerization and structural heterogeneity.
Authors: Stefan Jehle / Breanna S Vollmar / Benjamin Bardiaux / Katja K Dove / Ponni Rajagopal / Tamir Gonen / Hartmut Oschkinat / Rachel E Klevit /
Abstract: The small heat shock protein (sHSP) αB-crystallin (αB) plays a key role in the cellular protection system against stress. For decades, high-resolution structural studies on heterogeneous sHSPs have ...The small heat shock protein (sHSP) αB-crystallin (αB) plays a key role in the cellular protection system against stress. For decades, high-resolution structural studies on heterogeneous sHSPs have been confounded by the polydisperse nature of αB oligomers. We present an atomic-level model of full-length αB as a symmetric 24-subunit multimer based on solid-state NMR, small-angle X-ray scattering (SAXS), and EM data. The model builds on our recently reported structure of the homodimeric α-crystallin domain (ACD) and C-terminal IXI motif in the context of the multimer. A hierarchy of interactions contributes to build multimers of varying sizes: Interactions between two ACDs define a dimer, three dimers connected by their C-terminal regions define a hexameric unit, and variable interactions involving the N-terminal region define higher-order multimers. Within a multimer, N-terminal regions exist in multiple environments, contributing to the heterogeneity observed by NMR. Analysis of SAXS data allows determination of a heterogeneity parameter for this type of system. A mechanism of multimerization into higher-order asymmetric oligomers via the addition of up to six dimeric units to a 24-mer is proposed. The proposed asymmetric multimers explain the homogeneous appearance of αB in negative-stain EM images and the known dynamic exchange of αB subunits. The model of αB provides a structural basis for understanding known disease-associated missense mutations and makes predictions concerning substrate binding and the reported fibrilogenesis of αB.
History
DepositionMar 9, 2015Deposition site: BMRB / Processing site: RCSB
Revision 1.0Jun 3, 2015Provider: repository / Type: Initial release
Revision 1.1Jul 22, 2015Group: Database references
Revision 1.2Feb 15, 2017Group: Database references
Revision 1.3Jun 14, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Other
Category: database_2 / pdbx_database_status ...database_2 / pdbx_database_status / pdbx_nmr_software / pdbx_nmr_spectrometer / struct_ref_seq_dif / struct_sheet
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_nmr_data / _pdbx_nmr_software.name / _pdbx_nmr_spectrometer.model / _struct_ref_seq_dif.details / _struct_sheet.number_strands
Revision 1.4May 15, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2 / Item: _database_2.pdbx_DOI

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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)20,4012
Polymers20,4012
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)9 / 100structures with the lowest energy
RepresentativeModel #1closest to the average

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Components

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


Mass: 10200.497 Da / Num. of mol.: 2 / Fragment: unp residues 64-152 / Mutation: N146D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CRYA2, CRYAB / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 (DE3) / References: UniProt: P02511

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDType
1112D 1H-15N HSQC
2222D 1H-15N HSQC
1322D 1H-15N HSQC
1413D 1H-15N NOESY
1542D 1H-13C HSQC aliphatic
1642D 1H-13C HSQC aromatic
1723D HN(CA)CB
1823D HBHA(CO)NH
1923D HNCO
11023D HNCA
11123D HN(CO)CA
11223D HN(COCA)CB
11343D (H)CCH-TOCSY
11453D H(CCO)NH
11553D C(CO)NH
NMR detailsText: Solution structure was determined with NOEs and RDCs.

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Sample preparation

Details
Solution-IDContentsSolvent system
10.6 mM [U-100% 13C; U-100% 15N] HSPB5-ACD, 50 mM sodium phosphate, 100 mM sodium chloride, .1 mM EDTA, 1 mM PMSF, 90% H2O/10% D2O90% H2O/10% D2O
20.6 mM [U-100% 13C; U-100% 15N; U-80% 2H] HSPB5-ACD, 50 mM sodium phosphate, 100 mM sodium chloride, .1 mM EDTA, 1 mM PMSF, 90% H2O/10% D2O90% H2O/10% D2O
30.6 mM [U-100% 13C; U-100% 15N; U-80% 2H] HSPB5-ACD, 50 mM sodium phosphate, 100 mM sodium chloride, .1 mM EDTA, 1 mM PMSF, 14 mg/mL Pf1 phage, 90% H2O/10% D2O90% H2O/10% D2O
41.0 mM [U-100% 13C; U-100% 15N] HSPB5-ACD, 50 mM sodium phosphate, 100 mM sodium chloride, .1 mM EDTA, 1 mM PMSF, 100% D2O100% D2O
51.0 mM [U-100% 13C; U-100% 15N; 50% 2H] HSPB5-ACD, 50 mM sodium phosphate, 100 mM sodium chloride, .1 mM EDTA, 1 mM PMSF, 90% H2O/10% D2O90% H2O/10% D2O
Sample
Conc. (mg/ml)UnitsComponentIsotopic labelingConc. range (mg/ml)Solution-ID
mMHSPB5-ACD-1[U-100% 13C; U-100% 15N]0.2-1.01
50 mMsodium phosphate-21
100 mMsodium chloride-31
.1 mMEDTA-41
1 mMPMSF-51
mMHSPB5-ACD-6[U-100% 13C; U-100% 15N; U-80% 2H]0.2-1.02
50 mMsodium phosphate-72
100 mMsodium chloride-82
.1 mMEDTA-92
1 mMPMSF-102
mMHSPB5-ACD-11[U-100% 13C; U-100% 15N; U-80% 2H]0.2-1.03
50 mMsodium phosphate-123
100 mMsodium chloride-133
.1 mMEDTA-143
1 mMPMSF-153
14 mg/mLPf1 phage-163
1.0 mMHSPB5-ACD-17[U-100% 13C; U-100% 15N]4
50 mMsodium phosphate-184
100 mMsodium chloride-194
.1 mMEDTA-204
1 mMPMSF-214
1.0 mMHSPB5-ACD-22[U-100% 13C; U-100% 15N; 50% 2H]5
50 mMsodium phosphate-235
100 mMsodium chloride-245
.1 mMEDTA-255
1 mMPMSF-265
Sample conditions
Conditions-IDIonic strengthpHPressure (kPa)Temperature (K)
1100 7.5 ambient 295 K
2100 6.5 ambient 295 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Bruker AvanceBrukerAVANCE8001
Bruker AvanceBrukerAVANCE6002
Bruker AvanceBrukerAVANCE5003
Bruker AvanceBrukerAVANCE9004

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Processing

NMR software
NameDeveloperClassification
NMRPipeDelaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxdata analysis
NMRPipeDelaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
NMRPipeDelaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxpeak picking
NMRViewJohnson, One Moon Scientificdata analysis
NMRViewJohnson, One Moon Scientificchemical shift assignment
NMRViewJohnson, One Moon Scientificpeak picking
AutoAssignZimmerman, Moseley, Kulikowski and Montelionechemical shift assignment
AutoAssignZimmerman, Moseley, Kulikowski and Montelionepeak picking
CS-ROSETTAShen, Vernon, Baker and Baxstructure solution
CS-ROSETTAShen, Vernon, Baker and Baxchemical shift assignment
CS-ROSETTAShen, Vernon, Baker and Baxpeak picking
RosettaOligomer(RosettaOligomer)-Sgourakis, Lange, DiMaio, Andre, Fitzkee, Rossi, Montelione, Bax, Bakerstructure solution
RosettaOligomer(RosettaOligomer)-Sgourakis, Lange, DiMaio, Andre, Fitzkee, Rossi, Montelione, Bax, Bakerchemical shift assignment
RosettaOligomer(RosettaOligomer)-Sgourakis, Lange, DiMaio, Andre, Fitzkee, Rossi, Montelione, Bax, Bakerpeak picking
TALOSCornilescu, Delaglio and Baxrefinement
TALOSCornilescu, Delaglio and Baxchemical shift assignment
TALOSCornilescu, Delaglio and Baxpeak picking
GUARDD(GUARDD)-Kleckner, Fosterdata analysis
GUARDD(GUARDD)-Kleckner, Fosterchemical shift assignment
GUARDD(GUARDD)-Kleckner, Fosterpeak picking
RosettaOligomerrefinement
RefinementMethod: simulated annealing / Software ordinal: 1
NMR constraintsNOE constraints total: 838 / NOE intraresidue total count: 310 / NOE long range total count: 188 / NOE medium range total count: 85 / NOE sequential total count: 255 / Protein phi angle constraints total count: 72 / Protein psi angle constraints total count: 72
NMR representativeSelection criteria: closest to the average
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 100 / Conformers submitted total number: 9

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