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- PDB-6ptw: NMR data-driven model of KRas-GMPPNP:RBD-CRD complex tethered to ... -

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Basic information

Entry
Database: PDB / ID: 6ptw
TitleNMR data-driven model of KRas-GMPPNP:RBD-CRD complex tethered to a nanodisc (state B)
Components
  • Apolipoprotein A-I
  • GTPase KRas
  • RAF proto-oncogene serine/threonine-protein kinase
KeywordsSIGNALING PROTEIN / protein-protein-bilayer complex / small GTPase / RAS-RAF-nanodisc 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 / death-inducing signaling complex assembly / 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 / intermediate filament cytoskeleton organization / 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 / regulation of Rho protein signal transduction / chemorepellent activity / low-density lipoprotein particle / lipoprotein biosynthetic process / cholesterol transfer activity / cholesterol transport / type B pancreatic cell proliferation / high-density lipoprotein particle / very-low-density lipoprotein particle / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / Rap1 signalling / insulin secretion involved in cellular response to glucose stimulus / 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 feedback regulation of MAPK pathway / negative regulation of interleukin-1 beta production / negative chemotaxis / forebrain astrocyte development / IFNG signaling activates MAPKs / cholesterol binding / LRR domain binding / GP1b-IX-V activation signalling / 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 / ERBB2-ERBB3 signaling pathway / neurotrophin TRK receptor signaling pathway / response to gravity / epithelial tube branching involved in lung morphogenesis / type I pneumocyte differentiation / pseudopodium / face development / Rac protein signal transduction / regulation of cell differentiation / thyroid gland development / 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 / extrinsic apoptotic signaling pathway via death domain receptors / endocytic vesicle / SOS-mediated signalling / somatic stem cell population maintenance / Activated NTRK3 signals through RAS
Similarity search - Function
Apolipoprotein A/E / : / Apolipoprotein A1/A4/E 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) ...Apolipoprotein A/E / : / Apolipoprotein A1/A4/E 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 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 / 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 / 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
Chem-17F / PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER / 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / GTPase KRas / Apolipoprotein A-I / RAF proto-oncogene serine/threonine-protein kinase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodSOLUTION NMR / simulated annealing
AuthorsFang, Z. / Lee, K. / Gasmi-Seabrook, G. / Ikura, M. / Marshall, C.B.
Funding support Canada, 1items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR) Canada
CitationJournal: Proc.Natl.Acad.Sci.USA / Year: 2020
Title: Multivalent assembly of KRAS with the RAS-binding and cysteine-rich domains of CRAF on the membrane.
Authors: Fang, Z. / Lee, K.Y. / Huo, K.G. / Gasmi-Seabrook, G. / Zheng, L. / Moghal, N. / Tsao, M.S. / Ikura, M. / Marshall, C.B.
History
DepositionJul 16, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 13, 2020Provider: repository / Type: Initial release
Revision 1.1May 27, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jun 17, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
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

Downloads & links

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Assembly

Deposited unit
A: Apolipoprotein A-I
C: Apolipoprotein A-I
B: GTPase KRas
D: RAF proto-oncogene serine/threonine-protein kinase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)146,20588
Polymers82,5434
Non-polymers63,66284
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, cross-linking, surface plasmon resonance
TypeNameSymmetry operationNumber
identity operation1_5551
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)10 / 3000structures with the lowest energy
RepresentativeModel #1lowest energy

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Components

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Protein , 3 types, 4 molecules ACBD

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


Mass: 23080.129 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 BL21(DE3) (bacteria) / References: UniProt: P02647
#2: Protein GTPase KRas / K-Ras 2 / Ki-Ras / c-K-ras / c-Ki-ras


Mass: 21116.119 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KRAS, KRAS2, RASK2 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P01116
#3: Protein RAF proto-oncogene serine/threonine-protein kinase / Proto-oncogene c-RAF / cRaf / Raf-1


Mass: 15266.923 Da / Num. of mol.: 1 / Fragment: RBD-CRD (UNP residues 56-187)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAF1, RAF / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P04049

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Non-polymers , 5 types, 84 molecules

#4: 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
#5: 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
#6: 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
#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#8: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn

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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
121isotropic12D 1H-13C HMQC
132isotropic12D 1H-15N TROSY
142isotropic12D 1H-13C HMQC
153isotropic12D 1H-15N TROSY
1103isotropic12D 1H-13C HMQC
194isotropic12D 1H-15N TROSY
1114isotropic12D 1H-13C HMQC
2125isotropic13D edited NOESY
1136isotropic13D HNCO
1146isotropic13D HN(CA)CO
1156isotropic13D HN(CA)CB
1166isotropic13D CBCA(CO)NH
1176isotropic13D HNCA
1186isotropic13D HN(CO)CA
1197isotropic13D HNCO
1207isotropic13D HN(CA)CO
1217isotropic13D HN(CA)CB
1227isotropic13D CBCA(CO)NH
1237isotropic13D HNCA
1247isotropic13D HN(CO)CA

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Sample preparation

Details
TypeSolution-IDContentsLabelSolvent system
solution10.2 mM [Ile, Leu C-delta-13C; Val C-gamma-13C; U-15N; U-2H] KRAS, 0.2 mM [U-12C; U-14N; U-1H] RBD-CRD, 0.4 mM [U-12C; U-14N; U-1H] MSP, 90% H2O/10% D2OKRAS:RBD-CRD on nanodiscs90% H2O/10% D2O
solution20.2 mM [U-12C; U-14N; U-1H] KRAS, 0.2 mM [Ile, Leu C-delta-13C; Val C-gamma-13C; U-15N; U-2H] RBD-CRD, 0.4 mM [U-12C; U-14N; U-1H] MSP, 90% H2O/10% D2ORBD-CRD:KRAS on nanodiscs90% H2O/10% D2O
solution30.2 mM [Ile, Leu C-delta-13C; Val C-gamma-13C; U-15N] RBD-CRD, 0.2 mM [U-12C; U-14N; U-1H] KRAS Q43C, 90% H2O/10% D2ORBD-CRD:KRAS Q43C TEMPO90% H2O/10% D2O
solution40.2 mM [Ile, Leu C-delta-13C; Val C-gamma-13C; U-15N] RBD-CRD, 0.2 mM [U-12C; U-14N; U-1H] KRAS N-term C, 90% H2O/10% D2ORBD-CRD:KRAS N-term C TEMPO90% H2O/10% D2O
solution50.5 mM [U-15N; Ile C-delta-13C; Met methyl-13C] KRAS, 0.5 mM [Leu C-delta-13C; Val C-gamma-13C] RBD-CRD, 90% H2O/10% D2OKRAS:RBD-CRD NOE90% H2O/10% D2O
solution60.5 mM [U-13C; U-15N] RBD, 90% H2O/10% D2ORBD90% H2O/10% D2O
solution70.5 mM [U-13C; U-15N] CRD, 90% H2O/10% D2OCRD90% H2O/10% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
0.2 mMKRAS[Ile, Leu C-delta-13C; Val C-gamma-13C; U-15N; U-2H]1
0.2 mMRBD-CRD[U-12C; U-14N; U-1H]1
0.4 mMMSP[U-12C; U-14N; U-1H]1
0.2 mMKRAS[U-12C; U-14N; U-1H]2
0.2 mMRBD-CRD[Ile, Leu C-delta-13C; Val C-gamma-13C; U-15N; U-2H]2
0.4 mMMSP[U-12C; U-14N; U-1H]2
0.2 mMRBD-CRD[Ile, Leu C-delta-13C; Val C-gamma-13C; U-15N]3
0.2 mMKRAS Q43C[U-12C; U-14N; U-1H]3
0.2 mMRBD-CRD[Ile, Leu C-delta-13C; Val C-gamma-13C; U-15N]4
0.2 mMKRAS N-term C[U-12C; U-14N; U-1H]4
0.5 mMKRAS[U-15N; Ile C-delta-13C; Met methyl-13C]5
0.5 mMRBD-CRD[Leu C-delta-13C; Val C-gamma-13C]5
0.5 mMRBD[U-13C; U-15N]6
0.5 mMCRD[U-13C; U-15N]7
Sample conditions
Conditions-IDIonic strengthLabelpHPressure (kPa)Temperature (K)
1450 mMDefault condition5.51 atm298 K
2150 mMNOE condition5.51 atm308 K

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NMR measurement

NMR spectrometerType: Bruker AVANCE III / Manufacturer: Bruker / Model: AVANCE III / Field strength: 700 MHz

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Processing

NMR software
NameDeveloperClassification
CNSBrunger A. T. et.al.refinement
TopSpinBruker Biospincollection
SparkyGoddardchemical shift assignment
SparkyGoddardpeak picking
NMRPipeDelaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
NMRDrawDelaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
SparkyGoddarddata analysis
HADDOCKBonvinstructure calculation
RefinementMethod: simulated annealing / Software ordinal: 1
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 3000 / Conformers submitted total number: 10

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