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- PDB-2kt1: Solution NMR Structure of the SH3 Domain from the p85beta subunit... -

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Basic information

Entry
Database: PDB / ID: 2kt1
TitleSolution NMR Structure of the SH3 Domain from the p85beta subunit of Phosphatidylinositol 3-kinase from H.sapiens, Northeast Structural Genomics Consortium Target HR5531E
ComponentsPhosphatidylinositol 3-kinase regulatory subunit beta
KeywordsPROTEIN BINDING / Structural Genomics / NORTHEAST STRUCTURAL GENOMICS CONSORTIUM (NESG) / Target HR5531E / PSI-2 / Protein Structure Initiative / p85beta subunit of PI3K / SH3 domain / Phosphoprotein / Polymorphism
Function / homology
Function and homology information


IRS-mediated signalling / regulation of actin filament polymerization / phosphatidylinositol 3-kinase regulatory subunit binding / 1-phosphatidylinositol-3-kinase regulator activity / RHOF GTPase cycle / regulation of stress fiber assembly / RHOD GTPase cycle / phosphatidylinositol 3-kinase complex / Nephrin family interactions / RND1 GTPase cycle ...IRS-mediated signalling / regulation of actin filament polymerization / phosphatidylinositol 3-kinase regulatory subunit binding / 1-phosphatidylinositol-3-kinase regulator activity / RHOF GTPase cycle / regulation of stress fiber assembly / RHOD GTPase cycle / phosphatidylinositol 3-kinase complex / Nephrin family interactions / RND1 GTPase cycle / Costimulation by the CD28 family / RND2 GTPase cycle / PI3K/AKT activation / RND3 GTPase cycle / phosphatidylinositol 3-kinase complex, class IA / Signaling by ALK / RHOB GTPase cycle / negative regulation of MAPK cascade / RHOJ GTPase cycle / intracellular glucose homeostasis / CD28 dependent PI3K/Akt signaling / Synthesis of PIPs at the plasma membrane / phosphatidylinositol phosphate biosynthetic process / CDC42 GTPase cycle / RHOU GTPase cycle / RET signaling / T cell differentiation / positive regulation of cell adhesion / Interleukin-3, Interleukin-5 and GM-CSF signaling / PI3K Cascade / RHOA GTPase cycle / RAC2 GTPase cycle / RAC3 GTPase cycle / Role of phospholipids in phagocytosis / Role of LAT2/NTAL/LAB on calcium mobilization / Interleukin receptor SHC signaling / regulation of protein localization to plasma membrane / Signaling by PDGFRA transmembrane, juxtamembrane and kinase domain mutants / Signaling by PDGFRA extracellular domain mutants / GPVI-mediated activation cascade / Tie2 Signaling / RAC1 GTPase cycle / Interleukin-7 signaling / phosphotyrosine residue binding / response to endoplasmic reticulum stress / Downstream signal transduction / B cell differentiation / Signaling by phosphorylated juxtamembrane, extracellular and kinase domain KIT mutants / phosphatidylinositol 3-kinase/protein kinase B signal transduction / regulation of autophagy / Regulation of signaling by CBL / Signaling by SCF-KIT / receptor tyrosine kinase binding / VEGFA-VEGFR2 Pathway / positive regulation of protein import into nucleus / cellular response to insulin stimulus / Constitutive Signaling by Aberrant PI3K in Cancer / protein transport / Downstream TCR signaling / PIP3 activates AKT signaling / DAP12 signaling / insulin receptor signaling pathway / PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling / RAF/MAP kinase cascade / G alpha (q) signalling events / protein phosphatase binding / Extra-nuclear estrogen signaling / immune response / protein heterodimerization activity / focal adhesion / positive regulation of transcription by RNA polymerase II / nucleus / cytosol
Similarity search - Function
Phosphatidylinositol 3-kinase regulatory subunit beta, SH3 domain / PI3K p85 subunit, C-terminal SH2 domain / PI3K regulatory subunit p85-related , inter-SH2 domain / PI3K p85 subunit, N-terminal SH2 domain / Phosphatidylinositol 3-kinase regulatory subunit P85 inter-SH2 domain / Rho GTPase-activating protein domain / RhoGAP domain / Rho GTPase-activating proteins domain profile. / GTPase-activator protein for Rho-like GTPases / Rho GTPase activation protein ...Phosphatidylinositol 3-kinase regulatory subunit beta, SH3 domain / PI3K p85 subunit, C-terminal SH2 domain / PI3K regulatory subunit p85-related , inter-SH2 domain / PI3K p85 subunit, N-terminal SH2 domain / Phosphatidylinositol 3-kinase regulatory subunit P85 inter-SH2 domain / Rho GTPase-activating protein domain / RhoGAP domain / Rho GTPase-activating proteins domain profile. / GTPase-activator protein for Rho-like GTPases / Rho GTPase activation protein / SH3 Domains / SH2 domain / Src homology 2 (SH2) domain profile. / Src homology 2 domains / SH2 domain / SH3 type barrels. / Src homology 3 domains / SH2 domain superfamily / SH3-like domain superfamily / Src homology 3 (SH3) domain profile. / SH3 domain / Roll / Mainly Beta
Similarity search - Domain/homology
Phosphatidylinositol 3-kinase regulatory subunit beta
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodSOLUTION NMR / simulated annealing, null
Model detailslowest energy, model 1
AuthorsAramini, J.M. / Ma, L. / Schauder, C. / Everett, J.K. / Montelione, G.T. / Northeast Structural Genomics Consortium (NESG)
CitationJournal: To be Published
Title: Solution NMR Structure of the SH3 Domain from the p85beta subunit of Phosphatidylinositol 3-kinase from H.sapiens, Northeast Structural Genomics Consortium Target HR5531E
Authors: Aramini, J.M. / Ma, L. / Schauder, C. / Everett, J.K. / Montelione, G.T.
History
DepositionJan 15, 2010Deposition site: BMRB / Processing site: RCSB
Revision 1.0Jan 26, 2010Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Feb 26, 2020Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Other
Category: pdbx_database_status / pdbx_nmr_software ...pdbx_database_status / pdbx_nmr_software / pdbx_nmr_spectrometer / pdbx_struct_assembly / pdbx_struct_oper_list / struct_ref_seq_dif
Item: _pdbx_database_status.status_code_cs / _pdbx_nmr_software.name ..._pdbx_database_status.status_code_cs / _pdbx_nmr_software.name / _pdbx_nmr_spectrometer.model / _struct_ref_seq_dif.details
Revision 1.3Jun 14, 2023Group: Database references / Other / Category: database_2 / pdbx_database_status / struct_ref
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 / _struct_ref.pdbx_seq_one_letter_code
Revision 1.4May 8, 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:
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Assembly

Deposited unit
A: Phosphatidylinositol 3-kinase regulatory subunit beta


Theoretical massNumber of molelcules
Total (without water)9,9591
Polymers9,9591
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 100structures with the lowest energy
RepresentativeModel #1lowest energy

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Components

#1: Protein Phosphatidylinositol 3-kinase regulatory subunit beta / PI3-kinase p85 subunit beta / PtdIns-3-kinase p85-beta


Mass: 9959.187 Da / Num. of mol.: 1 / Fragment: SH3 domain
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PIK3R2 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 (DE3) + Magic / References: UniProt: O00459

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDType
1112D 1H-15N HSQC
1212D 1H-13C HSQC
1313D 1H-15N NOESY
1413D 1H-13C NOESY aliphatic
1513D 1H-13C NOESY aromatic
1613D 1H-13C arom NOESY
1713D HNCO
1813D HN(CA)CO
1913D CBCA(CO)NH
11013D HN(CA)CB
11113D HBHA(CO)NH
11213D HBHANH
11313D (H)CCH-COSY
11413D (H)CCH-TOCSY
11513D CCH-TOCSY
11613D HNCA
11711D 1H-15N T1 and T2
11822D 1H-13C HSQC high res. (L/V methyl stereoassignment)
11922D 1H-15N hetNOE
NMR detailsText: THE PROTEIN IS PREDOMINANTLY MONOMERIC AT 308 K BY 15N T1/T2 RELAXATION. THE STRUCTURE WAS DETERMINED USING TRIPLE RESONANCE NMR SPECTROSCOPY. SPECTRA FOR BACKBONE AND SIDE CHAIN ASSIGNMENTS ...Text: THE PROTEIN IS PREDOMINANTLY MONOMERIC AT 308 K BY 15N T1/T2 RELAXATION. THE STRUCTURE WAS DETERMINED USING TRIPLE RESONANCE NMR SPECTROSCOPY. SPECTRA FOR BACKBONE AND SIDE CHAIN ASSIGNMENTS WERE OBTAINED ON A 1.7-MM MICROCRYOPROBE AT 600 MHZ. ALL NOESY DATA WERE ACQUIRED AT 800 MHZ USING A 5-MM CRYOPROBE. BACKBONE ASSIGNMENTS WERE MADE USING PINE, AND THE SIDE CHAIN ASSIGNMENTS WERE COMPLETED MANUALLY. AUTOMATIC NOESY ASSIGNMENTS WERE DETERMINED USING CYANA 3.0. BACKBONE (PHI/PSI) DIHEDRAL ANGLE CONSTRAINTS WERE OBTAINED FROM TALOSplus. COMPLETENESS OF NMR ASSIGNMENTS (EXCLUDING C-TERMINAL HHHHHH): BACKBONE, 94.5%, SIDE CHAIN, 89.5%, AROMATICS, 86.8%, STEREOSPECIFIC METHYL, 100%, STEREOSPECIFIC SIDE CHAIN NH2: 80.0%. FINAL STRUCTURE QUALITY FACTORS (FOR RESIDUE NUMBERS 1 TO 82, PSVS 1.4), WHERE ORDERED RESIDUES [S(PHI) + S(PSI) > 1.8] COMPRISE: 7-16,18-81: (A) RMSD (ORDERED RESIDUES): BB, 0.7, HEAVY ATOM, 1.3. (B) RAMACHANDRAN STATISTICS FOR ORDERED RESIDUES: MOST FAVORED, 91.8%, ADDITIONALLY ALLOWED, 8.1%, GENEROUSLY ALLOWED, 0.1%, DISALLOWED, 0.0%. (C) PROCHECK SCORES FOR ORDERED RESIDUES (RAW/Z-): PHI-PSI, -0.44/-1.42, ALL, -0.32/-1.89. (D) MOLPROBITY CLASH SCORE (RAW/Z-): 17.41/-1.46 (E) RPF SCORES FOR GOODNESS OF FIT TO NOESY DATA (RESIDUES 1-82): RECALL, 0.969, PRECISION, 0.904, F-MEASURE, 0.935, DP-SCORE, 0.797. (F) NUMBER OF CLOSE CONTACTS PER 20 MODELS: 7. THE C-TERMINAL HIS TAG RESIDUES OF THE PROTEIN (HHHHHH) WERE NOT INCLUDED IN THE STRUCTURE CALCULATIONS AND HAVE BEEN OMITTED FROM THIS DEPOSITION.

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Sample preparation

Details
Solution-IDContentsSolvent system
10.62 mM [U-100% 13C; U-100% 15N] HR5531E, 20 mM sodium phosphate, 100 mM sodium chloride, 50 uM DSS, 10 mM DTT, 95% H2O/5% D2O95% H2O/5% D2O
20.50 mM [U-5% 13C; U-100% 15N] HR5531E, 20 mM sodium phosphate, 100 mM sodium chloride, 50 uM DSS, 10 mM DTT, 95% H2O/5% D2O95% H2O/5% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
0.62 mMHR5531E-1[U-100% 13C; U-100% 15N]1
20 mMsodium phosphate-21
100 mMsodium chloride-31
50 uMDSS-41
10 mMDTT-51
0.50 mMHR5531E-6[U-5% 13C; U-100% 15N]2
20 mMsodium phosphate-72
100 mMsodium chloride-82
50 uMDSS-92
10 mMDTT-102
Sample conditionspH: 6.5 / Pressure: ambient / Temperature: 308 K

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NMR measurement

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

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Processing

NMR software
NameVersionDeveloperClassification
CNS1.2Brunger, Adams, Clore, Gros, Nilges and Readrefinement
CYANA3Guntert, Mumenthaler and Wuthrichstructure solution
AutoStructure2.2.1Huang, Tejero, Powers and Montelionerpf analysis
PINE1Bahrami, Markley, Assadi, and Eghbalniachemical shift assignment
NMRPipe2.3Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
Sparky3.112Goddarddata analysis
Sparky3.112Goddardpeak picking
TopSpin2.1Bruker Biospincollection
TopSpin2.1Bruker Biospindata analysis
PSVS1.4Bhattacharya and Montelionestructure quality analysis
TALOSplusCornilescu, Delaglio and Baxdihedral angle constraints
PdbStat5.1Tejero and Montelionepdb coordinate analysis
RefinementMethod: simulated annealing, null / Software ordinal: 1
Details: THE FINAL STRUCTURES ARE BASED ON A TOTAL OF 972 CONFORMATIONALLY-RESTRICTING NOE-DERIVED DISTANCE CONSTRAINTS AND 108 DIHEDRAL ANGLE CONSTRAINTS (13.5 CONSTRAINTS PER RESIDUE, 5.0 LONG ...Details: THE FINAL STRUCTURES ARE BASED ON A TOTAL OF 972 CONFORMATIONALLY-RESTRICTING NOE-DERIVED DISTANCE CONSTRAINTS AND 108 DIHEDRAL ANGLE CONSTRAINTS (13.5 CONSTRAINTS PER RESIDUE, 5.0 LONG RANGE CONSTRAINTS PER RESIDUE, COMPUTED FOR RESIDUES 1 TO 82 BY PSVS 1.4). STRUCTURE DETERMINATION WAS PERFORMED ITERATIVELY USING CYANA 3.0. THE 20 STRUCTURES OUT OF 100 WITH THE LOWEST TARGET FUNCTION WERE FURTHER REFINED BY RESTRAINED MOLECULAR DYNAMICS/ENERGY MINIMIZATION IN EXPLICIT WATER (CNS) WITH PARAM19.
NMR constraintsNOE constraints total: 972 / NOE intraresidue total count: 214 / NOE long range total count: 397 / NOE medium range total count: 112 / NOE sequential total count: 249
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 100 / Conformers submitted total number: 20

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