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- PDB-6ccx: NMR data-driven model of GTPase KRas-GMPPNP:Cmpd2 complex tethere... -

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Basic information

Entry
Database: PDB / ID: 6ccx
TitleNMR data-driven model of GTPase KRas-GMPPNP:Cmpd2 complex tethered to a nanodisc
Components
  • Apolipoprotein A-I
  • GTPase KRas
KeywordsMEMBRANE PROTEIN/ONCOPROTEIN / protein-bilayer-compound complex / MEMBRANE PROTEIN-ONCOPROTEIN complex
Function / homology
Function and homology information


Defective ABCA1 causes TGD / high-density lipoprotein particle receptor binding / peptidyl-methionine modification / HDL clearance / spherical high-density lipoprotein particle / Scavenging by Class B Receptors / negative regulation of response to cytokine stimulus / protein oxidation / regulation of intestinal cholesterol absorption / vitamin transport ...Defective ABCA1 causes TGD / high-density lipoprotein particle receptor binding / peptidyl-methionine modification / HDL clearance / spherical high-density lipoprotein particle / Scavenging by Class B Receptors / negative regulation of response to cytokine stimulus / protein oxidation / regulation of intestinal cholesterol absorption / vitamin transport / blood vessel endothelial cell migration / cholesterol import / negative regulation of heterotypic cell-cell adhesion / ABC transporters in lipid homeostasis / apolipoprotein A-I receptor binding / apolipoprotein receptor binding / high-density lipoprotein particle binding / negative regulation of cell adhesion molecule production / negative regulation of cytokine production involved in immune response / HDL assembly / negative regulation of very-low-density lipoprotein particle remodeling / phosphatidylcholine biosynthetic process / glucocorticoid metabolic process / acylglycerol homeostasis / phosphatidylcholine-sterol O-acyltransferase activator activity / positive regulation of phospholipid efflux / Chylomicron remodeling / cellular response to lipoprotein particle stimulus / Chylomicron assembly / high-density lipoprotein particle clearance / phospholipid efflux / chylomicron / high-density lipoprotein particle remodeling / positive regulation of cholesterol metabolic process / reverse cholesterol transport / lipid storage / phospholipid homeostasis / high-density lipoprotein particle assembly / chemorepellent activity / low-density lipoprotein particle / lipoprotein biosynthetic process / cholesterol transfer activity / cholesterol transport / high-density lipoprotein particle / very-low-density lipoprotein particle / regulation of Cdc42 protein signal transduction / endothelial cell proliferation / HDL remodeling / cholesterol efflux / Scavenging by Class A Receptors / triglyceride homeostasis / adrenal gland development / response to mineralocorticoid / GMP binding / negative regulation of interleukin-1 beta production / negative chemotaxis / forebrain astrocyte development / cholesterol binding / LRR domain binding / regulation of synaptic transmission, GABAergic / negative regulation of epithelial cell differentiation / cholesterol biosynthetic process / response to isolation stress / amyloid-beta formation / positive regulation of Rho protein signal transduction / response to gravity / epithelial tube branching involved in lung morphogenesis / type I pneumocyte differentiation / Rac protein signal transduction / positive regulation of Rac protein signal transduction / Signaling by RAS GAP mutants / Signaling by RAS GTPase mutants / Activation of RAS in B cells / myoblast proliferation / skeletal muscle cell differentiation / RAS signaling downstream of NF1 loss-of-function variants / RUNX3 regulates p14-ARF / positive regulation of glial cell proliferation / endocytic vesicle / SOS-mediated signalling / Activated NTRK3 signals through RAS / Activated NTRK2 signals through RAS / positive regulation of cholesterol efflux / SHC1 events in ERBB4 signaling / negative regulation of tumor necrosis factor-mediated signaling pathway / cardiac muscle cell proliferation / Signalling to RAS / Activated NTRK2 signals through FRS2 and FRS3 / SHC-related events triggered by IGF1R / Scavenging of heme from plasma / Estrogen-stimulated signaling through PRKCZ / glial cell proliferation / SHC-mediated cascade:FGFR3 / MET activates RAS signaling / SHC-mediated cascade:FGFR2 / cholesterol metabolic process / PTK6 Regulates RHO GTPases, RAS GTPase and MAP kinases / Signaling by PDGFRA transmembrane, juxtamembrane and kinase domain mutants / Signaling by PDGFRA extracellular domain mutants / SHC-mediated cascade:FGFR4
Similarity search - Function
Apolipoprotein A/E / : / Apolipoprotein A1/A4/E domain / Small GTPase, Ras-type / Small GTPase Ras domain profile. / Ran (Ras-related nuclear proteins) /TC4 subfamily of small GTPases / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family ...Apolipoprotein A/E / : / Apolipoprotein A1/A4/E domain / Small GTPase, Ras-type / Small GTPase Ras domain profile. / Ran (Ras-related nuclear proteins) /TC4 subfamily of small GTPases / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family / Rab subfamily of small GTPases / Small GTP-binding protein domain / P-loop containing nucleotide triphosphate hydrolases / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Chem-17F / Chem-EWS / PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER / 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / GTPase KRas / Apolipoprotein A-I
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodSOLUTION NMR / simulated annealing
AuthorsFang, Z. / Marshall, C.B. / Nishikawa, T. / Gossert, A.D. / Jansen, J.M. / Jahnke, W. / Ikura, M.
CitationJournal: Cell Chem Biol / Year: 2018
Title: Inhibition of K-RAS4B by a Unique Mechanism of Action: Stabilizing Membrane-Dependent Occlusion of the Effector-Binding Site.
Authors: Fang, Z. / Marshall, C.B. / Nishikawa, T. / Gossert, A.D. / Jansen, J.M. / Jahnke, W. / Ikura, M.
History
DepositionFeb 7, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 5, 2018Provider: repository / Type: Initial release
Revision 1.1Mar 27, 2019Group: Data collection / Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jun 14, 2023Group: Data collection / Database references / Other
Category: database_2 / pdbx_database_status ...database_2 / pdbx_database_status / pdbx_nmr_software / pdbx_nmr_spectrometer
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
Revision 1.3May 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

Downloads & links

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Assembly

Deposited unit
A: Apolipoprotein A-I
B: GTPase KRas
C: Apolipoprotein A-I
hetero molecules


Theoretical massNumber of molelcules
Total (without water)131,75086
Polymers67,7713
Non-polymers63,97983
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)10 / 300010 structures for lowest energy
RepresentativeModel #1lowest energy

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Components

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Protein , 2 types, 3 molecules ACB

#1: Protein Apolipoprotein A-I / ApoA-I / Apolipoprotein A1


Mass: 23234.295 Da / Num. of mol.: 2 / Fragment: UNP residues 68-265
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: APOA1 / Production host: Escherichia coli (E. coli) / References: UniProt: P02647
#2: Protein GTPase KRas / K-Ras 2 / Ki-Ras / c-K-ras / c-Ki-ras


Mass: 21302.330 Da / Num. of mol.: 1 / Mutation: G12V
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KRAS, KRAS2, RASK2 / Production host: Escherichia coli (E. coli) / References: UniProt: P01116

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Non-polymers , 5 types, 83 molecules

#3: Chemical...
ChemComp-PCW / 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / (Z,Z)-4-HYDROXY-N,N,N-TRIMETHYL-10-OXO-7-[(1-OXO-9-OCTADECENYL)OXY]-3,5,9-TRIOXA-4-PHOSPHAHEPTACOS-18-EN-1-AMINIUM-4-OXIDE


Mass: 787.121 Da / Num. of mol.: 64 / Source method: obtained synthetically / Formula: C44H85NO8P / Comment: DOPC, phospholipid*YM
#4: Chemical
ChemComp-17F / O-[(S)-({(2R)-2,3-bis[(9Z)-octadec-9-enoyloxy]propyl}oxy)(hydroxy)phosphoryl]-L-serine / 1,2-Dioleoyl-sn-glycero-3-phospho-L-serine


Mass: 788.043 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: C42H78NO10P / Comment: phospholipid*YM
#5: 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
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#7: Chemical ChemComp-EWS / (2R,4S)-4-[(5-bromo-1H-indole-3-carbonyl)amino]-2-[(4-chlorophenyl)methyl]piperidin-1-ium


Mass: 447.776 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H22BrClN3O

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
111isotropic12D 1H-15N TROSY
122isotropic12D 1H-15N TROSY
141isotropic12D 1H-13C HMQC
132isotropic12D 1H-13C HMQC
NMR detailsText: HADDOCK modelling consists of (I) rigid-body docking, (II) a semi-flexible refinement stage, and (III) final refinement in explicit solvent (final gentle water). The starting structure is 2MSE. ...Text: HADDOCK modelling consists of (I) rigid-body docking, (II) a semi-flexible refinement stage, and (III) final refinement in explicit solvent (final gentle water). The starting structure is 2MSE. Distance restraints are based on PRE, NOE and CSP.

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Sample preparation

Details
TypeSolution-IDContentsLabelSolvent system
solution10.2 mM U-15N, Ile, Leu C-delta-13C, Val C-gamma-13C GTPase KRas isoform b, 0.4 mM Membrane Scaffold Protein, 100 mM sodium chloride, 20 mM TRIS, 5 mM magnesium chloride, 2 mM TCEP, 0.2 mM GMPPNP, 12 mM DOPC, 3.2 mM DOPS, 0.8 mM PE-MCC, 90% H2O/10% D2ONanodisc-KRAS-G12V + Cmpd290% H2O/10% D2O
solution20.2 mM GTPase KRas isoform b, 0.4 mM Membrane Scaffold Protein, 100 mM sodium chloride, 20 mM TRIS, 5 mM magnesium chloride, 2 mM TCEP, 0.2 mM GMPPNP, 12 mM DOPC, 3.2 mM DOPS, 0.8 mM PE-MCC, 0.4 mM PE-DTPA-Gd, 90% H2O/10% D2ONanodisc-KRAS-G12V + Cmpd2 + Gd90% H2O/10% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
0.2 mMGTPase KRas isoform bU-15N, Ile, Leu C-delta-13C, Val C-gamma-13C1
0.4 mMMembrane Scaffold Proteinnatural abundance1
100 mMsodium chloridenatural abundance1
20 mMTRISnatural abundance1
5 mMmagnesium chloridenatural abundance1
2 mMTCEPnatural abundance1
0.2 mMGMPPNPnatural abundance1
12 mMDOPCnatural abundance1
3.2 mMDOPSnatural abundance1
0.8 mMPE-MCCnatural abundance1
0.2 mMGTPase KRas isoform bnatural abundance2
0.4 mMMembrane Scaffold Proteinnatural abundance2
100 mMsodium chloridenatural abundance2
20 mMTRISnatural abundance2
5 mMmagnesium chloridenatural abundance2
2 mMTCEPnatural abundance2
0.2 mMGMPPNPnatural abundance2
12 mMDOPCnatural abundance2
3.2 mMDOPSnatural abundance2
0.8 mMPE-MCCnatural abundance2
0.4 mMPE-DTPA-Gdnatural abundance2
Sample conditionsIonic strength: 105 mM / Label: condition1 / pH: 7.4 / Pressure: 1 atm / Temperature: 298 K

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NMR measurement

NMR spectrometerType: Bruker AVANCE II / Manufacturer: Bruker / Model: AVANCE II / Field strength: 800 MHz

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Processing

NMR software
NameDeveloperClassification
TopSpinBruker Biospincollection
NMRDrawDelaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
NMRPipeDelaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
SparkyGoddarddata analysis
HADDOCKBonvinstructure calculation
HADDOCKBonvinrefinement
CNSBrunger, Adams, Clore, Gros, Nilges and Readrefinement
SparkyGoddardchemical shift assignment
RefinementMethod: simulated annealing / Software ordinal: 1
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: 10 structures for lowest energy
Conformers calculated total number: 3000 / Conformers submitted total number: 10

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