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- PDB-6ntd: Crystal Structure of G12V HRas-GppNHp bound in complex with the e... -

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Entry
Database: PDB / ID: 6ntd
TitleCrystal Structure of G12V HRas-GppNHp bound in complex with the engineered RBD variant 12 of CRAF Kinase protein
Components
  • GTPase HRasHRAS
  • RAF proto-oncogene serine/threonine-protein kinase
KeywordsTRANSFERASE/PROTEIN BINDING / affinity reagent / engineered protein / RAS inhibitor / TRANSFERASE-PROTEIN BINDING complex
Function / homology
Function and homology information


death-inducing signaling complex assembly / intermediate filament cytoskeleton organization / type B pancreatic cell proliferation / regulation of Rho protein signal transduction / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / Rap1 signalling / regulation of cell motility / GTPase complex / oncogene-induced cell senescence ...death-inducing signaling complex assembly / intermediate filament cytoskeleton organization / type B pancreatic cell proliferation / regulation of Rho protein signal transduction / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / Rap1 signalling / regulation of cell motility / GTPase complex / oncogene-induced cell senescence / insulin secretion involved in cellular response to glucose stimulus / positive regulation of ruffle assembly / regulation of neurotransmitter receptor localization to postsynaptic specialization membrane / negative regulation of GTPase activity / positive regulation of miRNA metabolic process / Negative feedback regulation of MAPK pathway / T-helper 1 type immune response / GP1b-IX-V activation signalling / IFNG signaling activates MAPKs / positive regulation of wound healing / ERBB2-ERBB3 signaling pathway / defense response to protozoan / regulation of cell differentiation / face development / pseudopodium / Signaling by RAS GAP mutants / Signaling by RAS GTPase mutants / somatic stem cell population maintenance / Activation of RAS in B cells / neurotrophin TRK receptor signaling pathway / thyroid gland development / RAS signaling downstream of NF1 loss-of-function variants / positive regulation of protein targeting to membrane / SOS-mediated signalling / Activated NTRK3 signals through RAS / Activated NTRK2 signals through RAS / extrinsic apoptotic signaling pathway via death domain receptors / MAP kinase kinase kinase activity / SHC1 events in ERBB4 signaling / Signalling to RAS / SHC-related events triggered by IGF1R / Activated NTRK2 signals through FRS2 and FRS3 / negative regulation of protein-containing complex assembly / adipose tissue development / Estrogen-stimulated signaling through PRKCZ / SHC-mediated cascade:FGFR3 / MET activates RAS signaling / positive regulation of phospholipase C activity / PTK6 Regulates RHO GTPases, RAS GTPase and MAP kinases / Schwann cell development / Signaling by PDGFRA transmembrane, juxtamembrane and kinase domain mutants / Signaling by PDGFRA extracellular domain mutants / SHC-mediated cascade:FGFR2 / type II interferon-mediated signaling pathway / SHC-mediated cascade:FGFR4 / protein-membrane adaptor activity / Signaling by FGFR4 in disease / SHC-mediated cascade:FGFR1 / Erythropoietin activates RAS / negative regulation of extrinsic apoptotic signaling pathway via death domain receptors / FRS-mediated FGFR3 signaling / Signaling by FLT3 ITD and TKD mutants / FRS-mediated FGFR2 signaling / FRS-mediated FGFR4 signaling / Signaling by FGFR3 in disease / p38MAPK events / FRS-mediated FGFR1 signaling / Tie2 Signaling / Signaling by FGFR2 in disease / GRB2 events in EGFR signaling / EPHB-mediated forward signaling / SHC1 events in EGFR signaling / response to muscle stretch / activation of adenylate cyclase activity / myelination / EGFR Transactivation by Gastrin / Signaling by FLT3 fusion proteins / Ras activation upon Ca2+ influx through NMDA receptor / FLT3 Signaling / Signaling by FGFR1 in disease / GRB2 events in ERBB2 signaling / NCAM signaling for neurite out-growth / CD209 (DC-SIGN) signaling / SHC1 events in ERBB2 signaling / Downstream signal transduction / Insulin receptor signalling cascade / Constitutive Signaling by Overexpressed ERBB2 / intrinsic apoptotic signaling pathway / small monomeric GTPase / G protein activity / insulin-like growth factor receptor signaling pathway / thymus development / Signaling by phosphorylated juxtamembrane, extracellular and kinase domain KIT mutants / VEGFR2 mediated cell proliferation / positive regulation of epithelial cell proliferation / regulation of actin cytoskeleton organization / FCERI mediated MAPK activation / positive regulation of JNK cascade / animal organ morphogenesis / regulation of long-term neuronal synaptic plasticity
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 / Small GTPase, Ras-type / small GTPase Ras family profile. / Phosphatidylinositol 3-kinase Catalytic Subunit; Chain A, domain 1 / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family / Rab subfamily of small GTPases / Ubiquitin-like (UB roll) / Small GTP-binding protein domain / Ubiquitin-like domain superfamily / P-loop containing nucleotide triphosphate hydrolases / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / 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 / P-loop containing nucleoside triphosphate hydrolase / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER / GTPase HRas / RAF proto-oncogene serine/threonine-protein kinase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.15 Å
AuthorsMaisonneuve, P. / Kurinov, I. / Wiechmann, S. / Ernst, A. / Sicheri, F.
CitationJournal: J.Biol.Chem. / Year: 2020
Title: Conformation-specific inhibitors of activated Ras GTPases reveal limited Ras dependency of patient-derived cancer organoids.
Authors: Wiechmann, S. / Maisonneuve, P. / Grebbin, B.M. / Hoffmeister, M. / Kaulich, M. / Clevers, H. / Rajalingam, K. / Kurinov, I. / Farin, H.F. / Sicheri, F. / Ernst, A.
History
DepositionJan 28, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 4, 2020Provider: repository / Type: Initial release
Revision 1.1Apr 15, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Oct 11, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: GTPase HRas
B: RAF proto-oncogene serine/threonine-protein kinase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)29,0814
Polymers28,5342
Non-polymers5472
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)91.761, 91.761, 151.590
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number182
Space group name H-MP6322

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Components

#1: Protein GTPase HRas / HRAS / H-Ras-1 / Ha-Ras / Transforming protein p21 / c-H-ras / p21ras


Mass: 19388.799 Da / Num. of mol.: 1 / Mutation: G12V
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HRAS, HRAS1 / Production host: Escherichia coli (E. coli) / References: UniProt: P01112
#2: Protein RAF proto-oncogene serine/threonine-protein kinase / Proto-oncogene c-RAF / cRaf / Raf-1


Mass: 9145.535 Da / Num. of mol.: 1
Mutation: F61L, K65H, Q66E, N71K, C81S, V88R, R89H, C95S, C96S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAF1, RAF / Production host: Escherichia coli (E. coli)
References: UniProt: P04049, non-specific serine/threonine protein kinase
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#4: Chemical ChemComp-GNP / PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER / 5'-Guanylyl imidodiphosphate


Mass: 522.196 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N6O13P3
Comment: GppNHp, GMPPNP, energy-carrying molecule analogue*YM
Has ligand of interestN

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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: 3.44 Å3/Da / Density % sol: 64.28 %
Crystal growTemperature: 293.15 K / Method: vapor diffusion, sitting drop / pH: 5.6 / Details: 30% PEG4000, 200mM NH4SO4, 100mM Na Citrate

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.9791 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Jul 15, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9791 Å / Relative weight: 1
ReflectionResolution: 3.1→79.47 Å / Num. obs: 7192 / % possible obs: 97.9 % / Redundancy: 12.3 % / CC1/2: 0.998 / Rrim(I) all: 0.147 / Net I/σ(I): 11
Reflection shellResolution: 3.1→3.31 Å / Redundancy: 12.6 % / Mean I/σ(I) obs: 0.4 / Num. unique obs: 1164 / CC1/2: 0.439 / % possible all: 90

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Processing

Software
NameVersionClassification
PHENIX1.17.1_3660refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
Cootmodel building
PDB_EXTRACT3.24data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4G0N

4g0n


Resolution: 3.15→79.47 Å / SU ML: 0.42 / Cross valid method: THROUGHOUT / σ(F): 1.33 / Phase error: 35.92 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.2872 367 5.33 %RANDOM
Rwork0.273 6525 --
obs0.2737 6892 98.09 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 330.18 Å2 / Biso mean: 152.6377 Å2 / Biso min: 78.71 Å2
Refinement stepCycle: final / Resolution: 3.15→79.47 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1632 0 33 0 1665
Biso mean--126.91 --
Num. residues----231
LS refinement shellResolution: 3.15→3.31 Å / Rfactor Rfree error: 0 / Total num. of bins used: 3
RfactorNum. reflection% reflection
Rfree0.3713 114 -
Rwork0.371 2059 -
all-2173 -
obs--96 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
13.69810.1170.93553.98451.74335.42710.47711.89810.6251-2.0665-0.1278-1.2284-0.24680.20520.14330.7037-0.0599-0.41480.76660.14281.471439.1083-1.215-15.3503
26.19585.0033-4.62036.3276-2.90794.8591-0.96031.59930.14341.02570.11652.09750.9091-3.0368-0.76471.7840.1048-0.41981.52740.05711.448529.7623-8.6568-19.3492
37.9577-3.0781-2.49153.37251.58936.245-0.25781.54581.28310.3142-0.00440.1113-1.72770.45620.1061.10590.3688-0.03711.04570.27640.89738.58364.6878-15.7323
42.09562.6726-1.62555.741-5.28755.9108-1.7289-0.6807-2.1937-2.16821.32772.78710.50372.062-0.2411.93790.53040.51462.1336-0.03142.582550.0611-6.2674-25.6646
53.9966-1.3231-0.3316.48840.00958.76220.38540.8490.3008-0.41140.3229-0.59630.77310.7285-0.86940.61870.20550.10321.00090.07761.398949.2846-10.7488-13.9319
65.0731-4.9981.11815.49060.3462.34220.440.68751.0377-0.7232-0.14920.1893-0.65990.4022-0.75580.8759-0.01230.23370.87660.01861.601550.6392-4.5645-9.7303
71.76471.3863-0.70381.0995-0.45850.85710.67390.44030.34910.22880.43051.04870.784-0.044-0.23521.26020.0258-0.13690.8453-0.29372.907136.6999-18.9975-4.7438
83.9603-2.8160.83232.650.29392.56430.73061.9054-0.41661.78840.24220.00360.48560.2866-0.67980.97810.1757-0.22510.62420.14561.642744.001-10.1863-3.2399
99.58267.885-8.51596.643-6.63628.19570.9833-2.76540.22310.8269-0.5627-0.1932-2.40580.6573-0.42561.2378-0.0162-0.15680.64170.01191.546543.66193.1153-6.2234
101.96536.637-8.48836.8098-0.98499.9335-1.93741.61522.96-1.9429-2.19740.04273.0797-2.4559-8.02221.48960.133-0.07843.29980.42931.377337.6012-2.3239-34.9684
118.97322.3444-2.55894.29391.12643.223-0.93061.6254-1.3297-0.8221.3261-0.07870.8705-1.5635-0.99821.1522-0.1324-0.27371.7490.31731.199432.1732-2.586-33.2728
127.2221-3.4631-0.56481.68460.2290.0149-0.64771.7471-1.1026-1.2975-2.1520.9937-2.4846-3.1375-6.94982.23440.7961-0.8833.64851.13531.217726.2151.0901-32.4915
136.6593-8.47633.62451.9887-3.3628.90820.22723.7914-6.2693-2.13380.18555.26384.79262.13081.41683.6367-0.09120.15451.6993-0.5381.790431.8586-4.8943-44.521
141.96619.87369.12978.50797.83357.2277-1.37615.2212-1.3411-0.08093.125-3.3979-1.90053.3007-1.7352.2491-0.31970.01242.0279-0.18371.293735.1982.8072-38.2099
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 0 through 24 )A0 - 24
2X-RAY DIFFRACTION2chain 'A' and (resid 25 through 36 )A25 - 36
3X-RAY DIFFRACTION3chain 'A' and (resid 37 through 60 )A37 - 60
4X-RAY DIFFRACTION4chain 'A' and (resid 61 through 74 )A61 - 74
5X-RAY DIFFRACTION5chain 'A' and (resid 75 through 104 )A75 - 104
6X-RAY DIFFRACTION6chain 'A' and (resid 105 through 116 )A105 - 116
7X-RAY DIFFRACTION7chain 'A' and (resid 117 through 126 )A117 - 126
8X-RAY DIFFRACTION8chain 'A' and (resid 127 through 151 )A127 - 151
9X-RAY DIFFRACTION9chain 'A' and (resid 152 through 166 )A152 - 166
10X-RAY DIFFRACTION10chain 'B' and (resid 55 through 62 )B55 - 62
11X-RAY DIFFRACTION11chain 'B' and (resid 63 through 82 )B63 - 82
12X-RAY DIFFRACTION12chain 'B' and (resid 83 through 115 )B83 - 115
13X-RAY DIFFRACTION13chain 'B' and (resid 116 through 121 )B116 - 121
14X-RAY DIFFRACTION14chain 'B' and (resid 122 through 129 )B122 - 129

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