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- PDB-6cc9: NMR data-driven model of GTPase KRas-GMPPNP:Cmpd2 complex tethere... -

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Basic information

Entry
Database: PDB / ID: 6cc9
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 / HDL clearance / peptidyl-methionine modification / 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 / HDL clearance / peptidyl-methionine modification / 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 / high-density lipoprotein particle binding / negative regulation of heterotypic cell-cell adhesion / ABC transporters in lipid homeostasis / apolipoprotein A-I receptor binding / apolipoprotein receptor 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 / chylomicron / phospholipid efflux / high-density lipoprotein particle remodeling / positive regulation of cholesterol metabolic process / lipid storage / reverse cholesterol transport / 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 / endothelial cell proliferation / regulation of Cdc42 protein signal transduction / HDL remodeling / cholesterol efflux / triglyceride homeostasis / GMP binding / response to mineralocorticoid / Scavenging by Class A Receptors / forebrain astrocyte development / negative regulation of interleukin-1 beta production / adrenal gland development / negative chemotaxis / LRR domain binding / cholesterol binding / negative regulation of epithelial cell differentiation / response to isolation stress / cholesterol biosynthetic process / response to gravity / type I pneumocyte differentiation / regulation of synaptic transmission, GABAergic / epithelial tube branching involved in lung morphogenesis / positive regulation of Rho protein signal transduction / amyloid-beta formation / 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 / SOS-mediated signalling / Activated NTRK3 signals through RAS / positive regulation of cholesterol efflux / Activated NTRK2 signals through RAS / endocytic vesicle / 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 / Estrogen-stimulated signaling through PRKCZ / glial cell proliferation / Scavenging of heme from plasma / SHC-mediated cascade:FGFR3 / MET activates RAS signaling / SHC-mediated cascade:FGFR2 / Retinoid metabolism and transport / 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 / Erythropoietin activates RAS
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 6, 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 1.2May 1, 2024Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_nmr_spectrometer
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_nmr_spectrometer.model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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-13C HMQC, 1H-15N TROSY
122isotropic12D 1H-13C HMQC, 1H-15N TROSY
133isotropic13D 15N-separated NOESY
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 and NOE.

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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% D2OND-KRASG12V90% H2O/10% D2O
solution20.2 mM U-15N, Ile, Leu C-delta-13C, Val C-gamma-13C GTPase KRas isoform b, 0.4 mM Membrane Scaffold Protein, 1 mM Cmpd2, 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% D2OND-KRASG12V + Cmpd290% H2O/10% D2O
solution30.2 mM [U-15N] GTPase KRas isoform b, 1 mM Cmpd2, 100 mM sodium chloride, 20 mM TRIS, 5 mM magnesium chloride, 2 mM TCEP, 0.2 mM GMPPNP, 90% H2O/10% D2Ofree KRASG12V + Cmpd290% 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 bU-15N, Ile, Leu C-delta-13C, Val C-gamma-13C2
0.4 mMMembrane Scaffold Proteinnatural abundance2
1 mMCmpd2natural 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
0.2 mMGTPase KRas isoform b[U-15N]3
1 mMCmpd2natural abundance3
100 mMsodium chloridenatural abundance3
20 mMTRISnatural abundance3
5 mMmagnesium chloridenatural abundance3
2 mMTCEPnatural abundance3
0.2 mMGMPPNPnatural abundance3
Sample conditionsIonic strength: 100 mM NaCl mM / Label: condition1 / pH: 7.4 / Pressure: ambient atm / Temperature: 298 K

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NMR measurement

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

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Processing

NMR software
NameDeveloperClassification
CNSBrunger A. T. et.al.refinement
HADDOCKBonvinstructure calculation
SPARKY-NMRFAMGoddard. et. al.chemical 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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