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

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Basic information

Entry
Database: PDB / ID: 6ntc
TitleCrystal Structure of G12V HRas-GppNHp bound in complex with the engineered RBD variant 1 of CRAF Kinase protein
Components
  • GTPase HRas
  • RAF proto-oncogene serine/threonine-protein kinase
KeywordsTRANSFERASE/PROTEIN BINDING / affinity reagent / 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 / phospholipase C activator activity / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / Rap1 signalling / regulation of cell motility / GTPase complex ...death-inducing signaling complex assembly / intermediate filament cytoskeleton organization / type B pancreatic cell proliferation / regulation of Rho protein signal transduction / phospholipase C activator activity / 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 / positive regulation of ruffle assembly / insulin secretion involved in cellular response to glucose stimulus / negative regulation of GTPase activity / positive regulation of miRNA metabolic process / regulation of neurotransmitter receptor localization to postsynaptic specialization membrane / Negative feedback regulation of MAPK pathway / T-helper 1 type immune response / positive regulation of wound healing / GP1b-IX-V activation signalling / IFNG signaling activates MAPKs / regulation of cell differentiation / ERBB2-ERBB3 signaling pathway / defense response to protozoan / face development / pseudopodium / Signaling by RAS GAP mutants / Signaling by RAS GTPase mutants / Activation of RAS in B cells / neurotrophin TRK receptor signaling pathway / somatic stem cell population maintenance / thyroid gland development / RAS signaling downstream of NF1 loss-of-function variants / SOS-mediated signalling / Activated NTRK3 signals through RAS / Activated NTRK2 signals through RAS / MAP kinase kinase kinase activity / extrinsic apoptotic signaling pathway via death domain receptors / SHC1 events in ERBB4 signaling / positive regulation of protein targeting to membrane / 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 / type II interferon-mediated signaling pathway / Estrogen-stimulated signaling through PRKCZ / SHC-mediated cascade:FGFR3 / MET activates RAS signaling / Signaling by PDGFRA transmembrane, juxtamembrane and kinase domain mutants / Signaling by PDGFRA extracellular domain mutants / SHC-mediated cascade:FGFR2 / PTK6 Regulates RHO GTPases, RAS GTPase and MAP kinases / Schwann cell development / SHC-mediated cascade:FGFR4 / Signaling by FGFR4 in disease / Erythropoietin activates RAS / activation of adenylate cyclase activity / SHC-mediated cascade:FGFR1 / negative regulation of extrinsic apoptotic signaling pathway via death domain receptors / protein-membrane adaptor activity / 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 / Tie2 Signaling / FRS-mediated FGFR1 signaling / Signaling by FGFR2 in disease / GRB2 events in EGFR signaling / SHC1 events in EGFR signaling / response to muscle stretch / EPHB-mediated forward signaling / EGFR Transactivation by Gastrin / Signaling by FLT3 fusion proteins / FLT3 Signaling / Signaling by FGFR1 in disease / myelination / GRB2 events in ERBB2 signaling / CD209 (DC-SIGN) signaling / Ras activation upon Ca2+ influx through NMDA receptor / NCAM signaling for neurite out-growth / SHC1 events in ERBB2 signaling / Downstream signal transduction / intrinsic apoptotic signaling pathway / Constitutive Signaling by Overexpressed ERBB2 / Insulin receptor signalling cascade / : / insulin-like growth factor receptor signaling pathway / positive regulation of GTPase activity / Signaling by phosphorylated juxtamembrane, extracellular and kinase domain KIT mutants / positive regulation of epithelial cell proliferation / small monomeric GTPase / thymus development / VEGFR2 mediated cell proliferation / regulation of actin cytoskeleton organization / animal organ morphogenesis / FCERI mediated MAPK activation / positive regulation of JNK cascade
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) ...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 / P-loop containing nucleotide triphosphate hydrolases / Ubiquitin-like domain superfamily / 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 / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / 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: 2.9 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)29,1925
Polymers28,5532
Non-polymers6393
Water905
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)91.619, 91.619, 151.412
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number182
Space group name H-MP6322
Components on special symmetry positions
IDModelComponents
11A-305-

HOH

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein GTPase 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: 9164.533 Da / Num. of mol.: 1
Mutation: F61L, K65Q, Q66E, R67W, 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

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Non-polymers , 4 types, 8 molecules

#3: Chemical ChemComp-GNP / PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER


Mass: 522.196 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N6O13P3
Comment: GppNHp, GMPPNP, energy-carrying molecule analogue*YM
#4: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Formula: H2O

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Details

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: 3.46 Å3/Da / Density % sol: 64.4 %
Crystal growTemperature: 293.15 K / Method: vapor diffusion, sitting drop / pH: 6.5 / Details: 30% PEG4000, 200mM NH4SO4, 100mM Na CaCo pH 6.5

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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: 2.9→151.41 Å / Num. obs: 8883 / % possible obs: 99.8 % / Redundancy: 18 % / CC1/2: 0.999 / Rmerge(I) obs: 0.14 / Rrim(I) all: 0.144 / Net I/σ(I): 15.1
Reflection shellResolution: 2.9→3.08 Å / Redundancy: 18.7 % / Mean I/σ(I) obs: 0.4 / Num. unique obs: 1366 / CC1/2: 0.423 / % possible all: 98.7

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Processing

Software
NameVersionClassification
PHENIX1.16_3549refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
Cootmodel building
PDB_EXTRACT3.24data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4G0N

4g0n


Resolution: 2.9→79.344 Å / SU ML: 0.58 / Cross valid method: THROUGHOUT / σ(F): 1.33 / Phase error: 37.29 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.2781 449 5.29 %RANDOM
Rwork0.2517 8041 --
obs0.2529 8490 95.64 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 292.6 Å2 / Biso mean: 145.1335 Å2 / Biso min: 73.81 Å2
Refinement stepCycle: final / Resolution: 2.9→79.344 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1614 0 39 5 1658
Biso mean--122.49 98.11 -
Num. residues----231
LS refinement shellResolution: 2.9002→3.08 Å / Rfactor Rfree error: 0
RfactorNum. reflection% reflection
Rfree0.5067 138 -
Rwork0.3942 2342 -
obs--87 %
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)
12.5751-1.3359-0.2755.7572-0.5146.56840.49571.2493-0.4926-0.9446-0.0173-1.04220.1184-0.55490.2190.7220.1902-0.12530.98360.04320.988936.0374-3.7549-17.0784
28.19982.5207-1.48919.1159-2.90729.3024-0.24320.6502-0.3173-1.466-0.084-0.2744-0.12280.27960.22820.81480.2417-0.00160.89370.12340.957842.7703-1.9536-16.815
33.5993-2.6146-3.30582.77232.56625.16810.41081.3752-0.7492-0.67090.4418-1.57-0.06221.0389-0.99861.00670.0150.19331.0862-0.01471.82252.5051-13.1778-14.8529
42.5139-3.08610.34349.791.7237.03950.52150.0669-0.49770.57730.1384-0.13860.1390.1097-0.40410.75410.0716-0.11110.71760.07741.288544.2318-6.8352-5.6474
55.30752.7163-1.3068.1121.19659.3137-0.1983-0.6492-0.03490.485-0.99460.36490.4054-0.7344-0.35390.92290.463-0.27091.4830.18021.112536.51880.9812-30.3902
66.32891.6312-4.53745.7989-1.65613.29311.0505-1.0093-1.11841.09020.3362-0.87741.7785-1.55680.38691.74770.0017-0.09951.78220.28930.529428.5522-5.2402-31.973
72.9690.4773.0864.8254-0.92383.6320.790.75551.4213-0.78440.44563.2375-0.9802-3.204-0.24821.98820.9961-0.37512.6980.4852.971922.35291.8711-29.518
80.1127-0.0193-0.78380.43730.20025.4753-0.2381-0.96480.03260.03390.13370.5707-0.4194-1.8078-0.49842.57881.5121-0.42773.2691-0.13151.371826.23626.4322-35.9136
94.4232-1.1883-4.9571.99898.320720.36352.62110.2772-2.7151-1.76551.31081.5939-2.54970.91443.93870.4848-0.5522.9890.08851.290428.03626.7844-42.8976
101.9983-3.2242-5.42837.40256.64116.6405-0.14064.7695-0.35980.48390.37760.6015-0.0612-0.19751.63821.46880.31460.08451.5617-0.49941.02631.7868-0.2066-40.3969
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid -1 through 37 )A-1 - 37
2X-RAY DIFFRACTION2chain 'A' and (resid 38 through 86 )A38 - 86
3X-RAY DIFFRACTION3chain 'A' and (resid 87 through 103 )A87 - 103
4X-RAY DIFFRACTION4chain 'A' and (resid 104 through 166 )A104 - 166
5X-RAY DIFFRACTION5chain 'B' and (resid 56 through 70 )B56 - 70
6X-RAY DIFFRACTION6chain 'B' and (resid 71 through 89 )B71 - 89
7X-RAY DIFFRACTION7chain 'B' and (resid 90 through 94 )B90 - 94
8X-RAY DIFFRACTION8chain 'B' and (resid 95 through 99 )B95 - 99
9X-RAY DIFFRACTION9chain 'B' and (resid 109 through 113 )B109 - 113
10X-RAY DIFFRACTION10chain 'B' and (resid 114 through 130 )B114 - 130

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