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- PDB-2k9p: Structure of TM1_TM2 in LPPG micelles -

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Basic information

Entry
Database: PDB / ID: 2k9p
TitleStructure of TM1_TM2 in LPPG micelles
ComponentsPheromone alpha factor receptor
KeywordsMEMBRANE PROTEIN / GPCR / micelle / structurral biology / fragment / G-protein coupled receptor / Glycoprotein / Membrane / Pheromone response / Phosphoprotein / Receptor / Transducer / Transmembrane
Function / homology
Function and homology information


mating-type alpha-factor pheromone receptor activity / cytogamy / mating-type factor pheromone receptor activity / G protein-coupled receptor homodimeric complex / response to pheromone / pheromone-dependent signal transduction involved in conjugation with cellular fusion / membrane => GO:0016020 / plasma membrane
Similarity search - Function
Pheromone alpha factor receptor. / GPCR fungal pheromone mating factor, STE2 / Pheromone alpha factor receptor, double transmembrane domain superfamily / Fungal pheromone mating factor STE2 GPCR / Helix Hairpins / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
Pheromone alpha factor receptor / Pheromone alpha factor receptor
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodSOLUTION NMR / simulated annealing, TORSION ANGLE DYNAMICS
Model detailsStructure of a fragment comprising TM1 and TM2 helices from the yeast Ste2p GPCR in LPPG micelles
AuthorsNeumoin, N. / Zerbe, O. / Naider, F.
CitationJournal: Biophys.J. / Year: 2009
Title: Structure of a double transmembrane fragment of a G-protein-coupled receptor in micelles.
Authors: Neumoin, A. / Cohen, L.S. / Arshava, B. / Tantry, S. / Becker, J.M. / Zerbe, O. / Naider, F.
History
DepositionOct 21, 2008Deposition site: BMRB / Processing site: RCSB
Revision 1.0May 5, 2009Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Feb 19, 2020Group: Data collection / Database references / Other
Category: database_2 / pdbx_database_status / pdbx_nmr_spectrometer
Item: _pdbx_database_status.status_code_cs / _pdbx_nmr_spectrometer.model
Revision 1.3Oct 21, 2020Group: Source and taxonomy / Category: entity_src_gen / Item: _entity_src_gen.host_org_species
Revision 1.4Oct 20, 2021Group: Database references / Category: database_2 / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details
Revision 1.5Jun 14, 2023Group: Database references / Other / Category: pdbx_database_status / struct_ref
Item: _pdbx_database_status.status_code_nmr_data / _struct_ref.pdbx_seq_one_letter_code
Revision 1.6May 8, 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: Pheromone alpha factor receptor


Theoretical massNumber of molelcules
Total (without water)8,7571
Polymers8,7571
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 30structures with the lowest energy
RepresentativeModel #1lowest energy

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Components

#1: Protein Pheromone alpha factor receptor


Mass: 8757.145 Da / Num. of mol.: 1 / Fragment: TM1-TM2 / Mutation: M54L, C59S, M69V, M71I
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Description: arabinose-induced E.coli vector, protein encoded as the delta-Trp fusion, cleaved by CNBr cleavage
Gene: STE2, YFL026W / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 / Variant (production host): AI (arabinose induced) / References: UniProt: P06842, UniProt: D6VTK4*PLUS

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
Details: Structure of a fragment comprising TM1 and TM2 helices from the yeast Ste2p GPCR in LPPG micelles
NMR experiment
Conditions-IDExperiment-IDSolution-IDType
1112D 1H-15N HSQC
1212D 1H-13C HSQC
1313D HNCO
1413D HNCA
1513D HN(CA)CB
1613D HBHA(CO)NH
1713D CBCA(CO)NH
1813D HN(CO)CA
1913D HN(CA)CO
11023D 1H-15N NOESY
11133D 1H-15N NOESY
11243D 1H-13C NOESY
11353D 1H-13C NOESY

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Sample preparation

Details
Solution-IDContentsSolvent system
1200 mM LPPG, 20 mM Na-phosphate buffer, 0.4 mM [U-100% 13C; U-100% 15N] TM1-TM2 peptide, 90% H2O/10% D2O90% H2O/10% D2O
2200 mM LPPG, 20 mM Na-phosphate buffer, 0.4 mM [U-100% 15N] TM1-TM2 peptide, 90% H2O/10% D2O90% H2O/10% D2O
3200 mM LPPG, 20 mM Na-phosphate buffer, 0.4 mM [U-100% 15N; 80% 2H] TM1-TM2 peptide, 90% H2O/10% D2O90% H2O/10% D2O
4200 mM [>85% 2H] LPPG, 20 mM Na-phosphate buffer, 0.4 mM [U-100% 13C; U-100% 15N] TM1-TM2 peptide, 90% H2O/10% D2O90% H2O/10% D2O
5200 mM [>85% 2H] LPPG, 20 mM Na-phosphate buffer, 0.4 mM [U-100% 13C; U-100% 15N; >98% 2H; Me(Ile(Hd1),Leu(Hd),Val(Hg)) 1H] TM1-TM2 peptide, 100% D2O100% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
200 mMLPPG1
20 mMNa-phosphate buffer1
0.4 mMTM1-TM2 peptide[U-100% 13C; U-100% 15N]1
200 mMLPPG2
20 mMNa-phosphate buffer2
0.4 mMTM1-TM2 peptide[U-100% 15N]2
200 mMLPPG3
20 mMNa-phosphate buffer3
0.4 mMTM1-TM2 peptide[U-100% 15N; 80% 2H]3
200 mMLPPG[>85% 2H]4
20 mMNa-phosphate buffer4
0.4 mMTM1-TM2 peptide[U-100% 13C; U-100% 15N]4
200 mMLPPG[>85% 2H]5
20 mMNa-phosphate buffer5
0.4 mMTM1-TM2 peptide[U-100% 13C; U-100% 15N; >98% 2H; Me(Ile(Hd1),Leu(Hd),Val(Hg)) 1H]5
Sample conditionspH: 6.4 / Pressure: AMBIENT / Temperature: 320 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Bruker AvanceBrukerAVANCE7001
Bruker AvanceBrukerAVANCE9002

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Processing

NMR software
NameVersionDeveloperClassification
CYANA3P.GUNTERT ET AL.refinement
XEASY55Bartels et al.structure solution
CARAKeller et al.chemical shift assignment
RefinementMethod: simulated annealing, TORSION ANGLE DYNAMICS / Software ordinal: 1 / Details: AMBER6 energy minimization in explicit solvent
NMR constraintsNOE constraints total: 1247 / NOE intraresidue total count: 439 / NOE long range total count: 24 / NOE medium range total count: 406 / NOE sequential total count: 378
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 30 / Conformers submitted total number: 20

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