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

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Basic information

Entry
Database: PDB / ID: 6pts
TitleNMR data-driven model of KRas-GMPPNP:RBD-CRD complex tethered to a nanodisc (state A)
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 / HDL clearance / spherical high-density lipoprotein particle / positive regulation of hydrolase activity / Scavenging by Class B Receptors / negative regulation of response to cytokine stimulus / protein oxidation / regulation of intestinal cholesterol absorption / death-inducing signaling complex assembly ...Defective ABCA1 causes TGD / high-density lipoprotein particle receptor binding / HDL clearance / spherical high-density lipoprotein particle / positive regulation of hydrolase activity / Scavenging by Class B Receptors / negative regulation of response to cytokine stimulus / protein oxidation / regulation of intestinal cholesterol absorption / death-inducing signaling complex assembly / vitamin transport / cholesterol import / blood vessel endothelial cell migration / high-density lipoprotein particle binding / negative regulation of heterotypic cell-cell adhesion / ABC transporters in lipid homeostasis / apolipoprotein A-I receptor binding / negative regulation of cytokine production involved in immune response / apolipoprotein receptor binding / negative regulation of cell adhesion molecule production / HDL assembly / negative regulation of very-low-density lipoprotein particle remodeling / peptidyl-methionine modification / 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 / positive regulation of cholesterol metabolic process / cellular response to lipoprotein particle stimulus / Chylomicron assembly / : / high-density lipoprotein particle clearance / chylomicron / phospholipid efflux / type B pancreatic cell proliferation / high-density lipoprotein particle remodeling / regulation of Rho protein signal transduction / phospholipid homeostasis / reverse cholesterol transport / chemorepellent activity / high-density lipoprotein particle assembly / lipid storage / low-density lipoprotein particle / lipoprotein biosynthetic process / cholesterol transfer activity / cholesterol transport / high-density lipoprotein particle / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / very-low-density lipoprotein particle / Rap1 signalling / regulation of Cdc42 protein signal transduction / triglyceride homeostasis / endothelial cell proliferation / insulin secretion involved in cellular response to glucose stimulus / HDL remodeling / cholesterol efflux / forebrain astrocyte development / Scavenging by Class A Receptors / negative regulation of interleukin-1 beta production / Negative feedback regulation of MAPK pathway / adrenal gland development / negative chemotaxis / cholesterol binding / IFNG signaling activates MAPKs / negative regulation of epithelial cell differentiation / GP1b-IX-V activation signalling / type I pneumocyte differentiation / regulation of synaptic transmission, GABAergic / epithelial tube branching involved in lung morphogenesis / cholesterol biosynthetic process / positive regulation of Rho protein signal transduction / ERBB2-ERBB3 signaling pathway / Rac protein signal transduction / face development / positive regulation of Rac protein signal transduction / skeletal muscle cell differentiation / pseudopodium / regulation of cell differentiation / neurotrophin TRK receptor signaling pathway / amyloid-beta formation / Signaling by RAS GAP mutants / Signaling by RAS GTPase mutants / Activation of RAS in B cells / thyroid gland development / RAS signaling downstream of NF1 loss-of-function variants / somatic stem cell population maintenance / RUNX3 regulates p14-ARF / extrinsic apoptotic signaling pathway via death domain receptors / SOS-mediated signalling / endocytic vesicle / Activated NTRK3 signals through RAS / Activated NTRK2 signals through RAS / MAP kinase kinase kinase activity / positive regulation of cholesterol efflux / SHC1 events in ERBB4 signaling / negative regulation of tumor necrosis factor-mediated signaling pathway
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 family profile. / Ran (Ras-related nuclear proteins) /TC4 subfamily of small GTPases / 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 / 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

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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: surface plasmon resonance, gel filtration, cross-linking
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-13C HMQC
122isotropic12D 1H-13C HMQC
132isotropic12D 1H-15N TROSY
143isotropic12D 1H-13C HMQC
153isotropic12D 1H-15N TROSY
164isotropic12D 1H-13C HMQC
174isotropic12D 1H-15N TROSY
285isotropic13D 5N edited NOESY
196isotropic13D HN(CA)CB
1106isotropic13D CBCA(CO)NH
1116isotropic13D HNCA
1126isotropic13D HN(CO)CA
1166isotropic13D HNCO
1156isotropic13D HN(CA)CO
1147isotropic13D HN(CA)CB
1137isotropic13D CBCA(CO)NH
1227isotropic13D HNCA
1217isotropic13D HN(CO)CA
1207isotropic13D HNCO
1197isotropic13D HN(CA)CO

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Sample preparation

Details
TypeSolution-IDContentsLabelSolvent systemDetails
solution10.2 mM U-2H; U-15N; Ile Leu C-delta-13C, Val C-gamma-13C KRAS, 0.2 mM U-12C, 14N, 1H RBD-CRD, 0.4 mM U-12C, 14N, 1H MSP, 90% H2O/10% D2OKRAS:RBD-CRD on nanodiscs90% H2O/10% D2O
solution20.2 mM U-12C, 14N, 1H KRAS, 0.2 mM U-2H; U-15N; Ile Leu C-delta-13C, Val C-gamma-13C RBD-CRD, 0.4 mM U-12C, 14N, 1H MSP, 90% H2O/10% D2ORBD-CRD:KRAS on nanodiscs90% H2O/10% D2O
solution30.2 mM U-12C, 14N, 1H KRAS, 0.2 mM U-2H; U-15N; Ile Leu C-delta-13C, Val C-gamma-13C RBD-CRD, 90% H2O/10% D2ORBD-CRD:KRAS Q43C TEMPO90% H2O/10% D2OKRAS Q43C is crosslinked to spin label TEMPO.
solution40.2 mM U-12C, 14N, 1H KRAS, 0.2 mM U-2H; U-15N; Ile Leu C-delta-13C, Val C-gamma-13C RBD-CRD, 90% H2O/10% D2ORBD-CRD:KRAS N-term C TEMPO90% H2O/10% D2OKRAS N-terminal Cys is crosslinked to spin label TEMPO.
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-99% 15N]; [U-13C]; RBD, 90% H2O/10% D2ORBD 3D assignment90% H2O/10% D2O
solution70.3 mM [U-99% 15N]; [U-13C]; CRD, 90% H2O/10% D2OCRD 3D assignment90% H2O/10% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
0.2 mMKRASU-2H; U-15N; Ile Leu C-delta-13C, Val C-gamma-13C1
0.2 mMRBD-CRDU-12C, 14N, 1H1
0.4 mMMSPU-12C, 14N, 1H1
0.2 mMKRASU-12C, 14N, 1H2
0.2 mMRBD-CRDU-2H; U-15N; Ile Leu C-delta-13C, Val C-gamma-13C2
0.4 mMMSPU-12C, 14N, 1H2
0.2 mMKRASU-12C, 14N, 1H3
0.2 mMRBD-CRDU-2H; U-15N; Ile Leu C-delta-13C, Val C-gamma-13C3
0.2 mMKRASU-12C, 14N, 1H4
0.2 mMRBD-CRDU-2H; U-15N; Ile Leu C-delta-13C, Val C-gamma-13C4
0.5 mMKRASU-15N; Ile C-delta-13C, Met methyl-13C5
0.5 mMRBD-CRDLeu C-delta-13C, Val C-gamma-13C,5
0.5 mMRBD[U-99% 15N]; [U-13C];6
0.3 mMCRD[U-99% 15N]; [U-13C];7
Sample conditions
Conditions-IDDetailsIonic strengthLabelpHPressure (kPa)Temperature (K)
1Conditions used in most of experiments450 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
NMRPipeDelaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
NMRDrawDelaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
SparkyGoddarddata analysis
SparkyGoddardpeak picking
HADDOCKBonvinstructure calculation
SparkyGoddardchemical shift assignment
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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