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- PDB-2l05: Solution NMR Structure of the Ras-binding domain of Serine/threon... -

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Entry
Database: PDB / ID: 2l05
TitleSolution NMR Structure of the Ras-binding domain of Serine/threonine-protein kinase B-raf from Homo sapiens, Northeast Structural Genomics Consortium Target HR4694F
ComponentsSerine/threonine-protein kinase B-raf
KeywordsTRANSFERASE / Structural Genomics / NORTHEAST STRUCTURAL GENOMICS CONSORTIUM (NESG) / PSI-2 / Protein Structure Initiative
Function / homology
Function and homology information


trehalose metabolism in response to stress / CD4-positive, alpha-beta T cell differentiation / CD4-positive or CD8-positive, alpha-beta T cell lineage commitment / negative regulation of synaptic vesicle exocytosis / head morphogenesis / Signalling to p38 via RIT and RIN / myeloid progenitor cell differentiation / ARMS-mediated activation / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling ...trehalose metabolism in response to stress / CD4-positive, alpha-beta T cell differentiation / CD4-positive or CD8-positive, alpha-beta T cell lineage commitment / negative regulation of synaptic vesicle exocytosis / head morphogenesis / Signalling to p38 via RIT and RIN / myeloid progenitor cell differentiation / ARMS-mediated activation / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / endothelial cell apoptotic process / negative regulation of fibroblast migration / positive regulation of glucose transmembrane transport / establishment of protein localization to membrane / mitogen-activated protein kinase kinase binding / regulation of T cell differentiation / Negative feedback regulation of MAPK pathway / positive regulation of axonogenesis / Frs2-mediated activation / stress fiber assembly / positive regulation of axon regeneration / face development / synaptic vesicle exocytosis / somatic stem cell population maintenance / MAP kinase kinase activity / thyroid gland development / MAP kinase kinase kinase activity / negative regulation of endothelial cell apoptotic process / positive regulation of substrate adhesion-dependent cell spreading / positive regulation of stress fiber assembly / response to cAMP / cellular response to calcium ion / ERK1 and ERK2 cascade / substrate adhesion-dependent cell spreading / cellular response to nerve growth factor stimulus / thymus development / long-term synaptic potentiation / animal organ morphogenesis / Spry regulation of FGF signaling / RAF activation / Signaling by high-kinase activity BRAF mutants / visual learning / MAP2K and MAPK activation / epidermal growth factor receptor signaling pathway / response to peptide hormone / Negative regulation of MAPK pathway / Signaling by RAF1 mutants / Signaling by moderate kinase activity BRAF mutants / Paradoxical activation of RAF signaling by kinase inactive BRAF / Signaling downstream of RAS mutants / MAPK cascade / Signaling by BRAF and RAF1 fusions / cellular response to xenobiotic stimulus / presynapse / positive regulation of peptidyl-serine phosphorylation / T cell differentiation in thymus / T cell receptor signaling pathway / regulation of cell population proliferation / cell body / scaffold protein binding / negative regulation of neuron apoptotic process / Ras protein signal transduction / positive regulation of ERK1 and ERK2 cascade / non-specific serine/threonine protein kinase / protein kinase activity / neuron projection / protein phosphorylation / intracellular membrane-bounded organelle / protein serine kinase activity / protein serine/threonine kinase activity / calcium ion binding / protein-containing complex binding / positive regulation of gene expression / negative regulation of apoptotic process / mitochondrion / ATP binding / identical protein binding / nucleus / plasma membrane / cytosol
Similarity search - Function
Raf-like Ras-binding domain / Raf-like Ras-binding / Ras-binding domain (RBD) profile. / Raf-like Ras-binding domain / Diacylglycerol/phorbol-ester binding / Phorbol esters/diacylglycerol binding domain (C1 domain) / Zinc finger phorbol-ester/DAG-type signature. / Zinc finger phorbol-ester/DAG-type profile. / Protein kinase C conserved region 1 (C1) domains (Cysteine-rich domains) / Protein kinase C-like, phorbol ester/diacylglycerol-binding domain ...Raf-like Ras-binding domain / Raf-like Ras-binding / Ras-binding domain (RBD) profile. / Raf-like Ras-binding domain / Diacylglycerol/phorbol-ester binding / Phorbol esters/diacylglycerol binding domain (C1 domain) / Zinc finger phorbol-ester/DAG-type signature. / Zinc finger phorbol-ester/DAG-type profile. / Protein kinase C conserved region 1 (C1) domains (Cysteine-rich domains) / Protein kinase C-like, phorbol ester/diacylglycerol-binding domain / C1-like domain superfamily / Phosphatidylinositol 3-kinase Catalytic Subunit; Chain A, domain 1 / Ubiquitin-like (UB roll) / Protein tyrosine and serine/threonine kinase / Serine-threonine/tyrosine-protein kinase, catalytic domain / Ubiquitin-like domain superfamily / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Serine/Threonine protein kinases, catalytic domain / Roll / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily / Alpha Beta
Similarity search - Domain/homology
Serine/threonine-protein kinase B-raf
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodSOLUTION NMR / simulated annealing
Model detailslowest energy, model 1
AuthorsAramini, J.M. / Janjua, H. / Ciccosanti, C. / Shastry, R. / Huang, Y.J. / Acton, T.B. / Xiao, R. / Everett, J.K. / Montelione, G.T. / Northeast Structural Genomics Consortium (NESG)
CitationJournal: To be Published
Title: Solution NMR Structure of the Ras-binding domain of Serine/threonine-protein kinase B-raf from Homo sapiens, Northeast Structural Genomics Consortium Target HR4694F
Authors: Aramini, J.M. / Janjua, H. / Ciccosanti, C. / Shastry, R. / Huang, Y.J. / Acton, T.B. / Xiao, R. / Everett, J.K. / Montelione, G.T.
History
DepositionJun 30, 2010Deposition site: BMRB / Processing site: RCSB
Revision 1.0Jul 14, 2010Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Feb 5, 2020Group: Data collection / Database references / Other
Category: database_2 / pdbx_database_status ...database_2 / pdbx_database_status / pdbx_nmr_software / pdbx_nmr_spectrometer / 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
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_nmr_data
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: Serine/threonine-protein kinase B-raf


Theoretical massNumber of molelcules
Total (without water)10,8831
Polymers10,8831
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)20 / 100structures with the lowest energy
RepresentativeModel #1lowest energy

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Components

#1: Protein Serine/threonine-protein kinase B-raf / Proto-oncogene B-Raf / p94 / v-Raf murine sarcoma viral oncogene homolog B1


Mass: 10882.762 Da / Num. of mol.: 1 / Fragment: RBD domain residues 149-232
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: BRAF, BRAF1, RAFB1 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)pMgK
References: UniProt: P15056, non-specific serine/threonine protein kinase

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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 15N-NOESY
1413D 1H-13C NOESY aliphatic
1513D 1H-13C NOESY aromatic
1622D 1H-13C HSQC high resolution (L/V methyl stereoassignment)
1713D HNCO
1813D HN(CA)CO
1913D HN(CO)CA
11013D HNCA
11113D CBCA(CO)NH
11213D HN(CA)CB
11313D HBHA(CO)NH
11413D (H)CCH-TOCSY
11513D (H)CCH-COSY
11613D CCH-TOCSY
11713D HNHA
11812D 1H-15N hetNOE
11911D 15N T1 and T2
NMR detailsText: THE PROTEIN IS MONOMERIC BY GEL FILTRATION CHROMATOGRAPHY, STATIC LIGHT SCATTERING AND 15N T1/T2 RELAXATION. THE STRUCTURE WAS DETERMINED USING TRIPLE RESONANCE NMR SPECTROSCOPY. SPECTRA FOR ...Text: THE PROTEIN IS MONOMERIC BY GEL FILTRATION CHROMATOGRAPHY, STATIC LIGHT SCATTERING AND 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 CROYOPROBE. 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 N-TERMINAL TAG): BACKBONE, 98.3%, SIDE CHAIN, 98.1%, AROMATICS, 100%, STEREOSPECIFIC METHYL, 100%, STEREOSPECIFIC SIDE CHAIN NH2: 100%. FINAL STRUCTURE QUALITY FACTORS (FOR RESIDUES 147 TO 232, PSVS 1.4), WHERE ORDERED RESIDUES [S(PHI) + S(PSI) > 1.8] COMPRISE: 154-228: (A) RMSD (ORDERED RESIDUES): BB, 0.6, HEAVY ATOM, 1.0. (B) RAMACHANDRAN STATISTICS FOR ORDERED RESIDUES: MOST FAVORED, 91.9%, ADDITIONALLY ALLOWED, 8.0%, GENEROUSLY ALLOWED, 0.1%, DISALLOWED, 0.0%. (C) PROCHECK SCORES FOR ORDERED RESIDUES (RAW/Z-): PHI-PSI, -0.36/-1.10, ALL, -0.15/-0.89. (D) MOLPROBITY CLASH SCORE (RAW/Z-): 14.54/-0.97 (E) RPF SCORES FOR GOODNESS OF FIT TO NOESY DATA (RESIDUES 147 TO 232): RECALL, 0.979, PRECISION, 0.933, F-MEASURE, 0.956, DP-SCORE, 0.808. (F) NUMBER OF CLOSE CONTACTS PER 20 MODELS: 2. THE MAJORITY OF THE N-TERMINAL HIS TAG RESIDUES OF THE PROTEIN (MGHHHHHHS) WERE NOT INCLUDED IN THE STRUCTURE CALCULATIONS AND HAVE BEEN OMITTED FROM THIS DEPOSITION.

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Sample preparation

Details
Solution-IDContentsSolvent system
10.84 mM [U-100% 13C; U-100% 15N] HR4694F, 20 mM ammonium acetate, 100 mM sodium chloride-3, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS, 90% H2O/10% D2O90% H2O/10% D2O
20.69 mM [U-5% 13C; U-100% 15N] HR4694F, 20 mM ammonium acetate, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS-14, 90% H2O/10% D2O90% H2O/10% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
0.84 mMHR4694F-1[U-100% 13C; U-100% 15N]1
20 mMammonium acetate-21
100 mMsodium chloride-31
5 mMcalcium chloride-41
10 mMDTT-51
0.02 %sodium azide-61
50 uMDSS-71
0.69 mMHR4694F-8[U-5% 13C; U-100% 15N]2
20 mMammonium acetate-92
100 mMsodium chloride-102
5 mMcalcium chloride-112
10 mMDTT-122
0.02 %sodium azide-132
50 uMDSS-142
Sample conditionsIonic strength: 0.1 / pH: 4.5 / Pressure: ambient / Temperature: 298 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 Wuthrichrefinement
CYANA3Guntert, Mumenthaler and Wuthrichstructure solution
AutoStructure2.2.1Huang, Tejero, Powers and Montelionerpf analysis
PSVS1.4Bhattacharya and Montelionestructure quality analysis
NMRPipe2.3Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
MolProbity3.17Richardsonstructure quality analysis
TopSpin2.1Bruker Biospincollection
TopSpin2.1Bruker Biospindata analysis
PdbStat5.1Tejero & Montelionepdb coordinate analysis
PINE1Bahrami, Markley, Assadi, and Eghbalniachemical shift assignment
Sparky3.112Goddarddata analysis
Sparky3.112Goddardpeak picking
TALOS+Shen, Cornilescu, Delaglio and Baxdihedral angle constraints
RefinementMethod: simulated annealing / Software ordinal: 1
Details: THE FINAL STRUCTURES ARE BASED ON A TOTAL OF 1750 CONFORMATIONALLY-RESTRICTING NOE-DERIVED DISTANCE CONSTRAINTS AND 125 DIHEDRAL ANGLE CONSTRAINTS; 0 HYDROGEN BOND CONSTRAINTS (22.1 ...Details: THE FINAL STRUCTURES ARE BASED ON A TOTAL OF 1750 CONFORMATIONALLY-RESTRICTING NOE-DERIVED DISTANCE CONSTRAINTS AND 125 DIHEDRAL ANGLE CONSTRAINTS; 0 HYDROGEN BOND CONSTRAINTS (22.1 CONSTRAINTS PER RESIDUE, 6.4 LONG RANGE CONSTRAINTS PER RESIDUE, COMPUTED FOR RESIDUES 147 TO 232 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: 1750 / NOE intraresidue total count: 455 / NOE long range total count: 546 / NOE medium range total count: 256 / NOE sequential total count: 493
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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