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- PDB-6s3w: Solution NMR Structure of TolAIII Bound to a Peptide Derived from... -

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Basic information

Entry
Database: PDB / ID: 6s3w
TitleSolution NMR Structure of TolAIII Bound to a Peptide Derived from the N-terminus of TolB
Components
  • Cell envelope integrity/translocation protein TolA
  • TolBp
KeywordsPROTEIN BINDING / TolA / TolB / Pal / bacterial outer membrane
Function / homology
Function and homology information


bacteriocin transport / toxin transmembrane transporter activity / protein import / periplasmic space / cell cycle / cell division / plasma membrane
Similarity search - Function
TonB C terminal / : / Tol-Pal system, TolA / TolB, N-terminal / Tol-Pal system protein TolB / TolB amino-terminal domain / TonB/TolA, C-terminal / WD40-like beta propeller / WD40-like Beta Propeller Repeat / Six-bladed beta-propeller, TolB-like
Similarity search - Domain/homology
: / Tol-Pal system protein TolA / Tol-Pal system protein TolB
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
MethodSOLUTION NMR / simulated annealing / molecular dynamics
AuthorsKleanthous, C. / Redfield, C. / Rajasekar, K. / Holmes, P.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research CouncilBB/I008691/1 United Kingdom
European Research Council742555 United Kingdom
Wellcome Trust201505/Z/16/Z United Kingdom
CitationJournal: Nat Commun / Year: 2020
Title: The lipoprotein Pal stabilises the bacterial outer membrane during constriction by a mobilisation-and-capture mechanism.
Authors: Szczepaniak, J. / Holmes, P. / Rajasekar, K. / Kaminska, R. / Samsudin, F. / Inns, P.G. / Rassam, P. / Khalid, S. / Murray, S.M. / Redfield, C. / Kleanthous, C.
History
DepositionJun 26, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 25, 2020Provider: repository / Type: Initial release
Revision 1.1Jul 3, 2024Group: Data collection / Database references / Other
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_status
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_nmr_data

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: TolBp
A: Cell envelope integrity/translocation protein TolA


Theoretical massNumber of molelcules
Total (without water)15,0542
Polymers15,0542
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: isothermal titration calorimetry, The 1:1 stoichiometry was determined by ITC. In addition, NMR titrations confirmed this.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area1720 Å2
ΔGint-7 kcal/mol
Surface area8000 Å2
MethodPISA
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 600structures with the lowest energy
RepresentativeModel #1lowest energy

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Components

#1: Protein/peptide TolBp


Mass: 1315.412 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Peptide sequence represents residues 22 to 34 of the TolB sequence. This is the region of TolB that binds to TolAIII.
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Production host: Escherichia coli (E. coli) / References: UniProt: P50601*PLUS
#2: Protein Cell envelope integrity/translocation protein TolA


Mass: 13738.455 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: This is the 3rd domain of the TolA protein. / Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: EFK27_03855 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A454LZ61, UniProt: P50600*PLUS

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
112isotropic13D HNCO
122isotropic13D HNCA
132isotropic13D CBCANH
142isotropic13D CBCA(CO)NH
152isotropic13D H(CCO)NH
162isotropic13D C(CO)NH
172isotropic13D HBHA(CO)NH
182isotropic12D 1H-15N HSQC
191isotropic23D 1H-15N NOESY
1101isotropic23D 1H-15N TOCSY
1111isotropic22D 1H-15N HSQC
1122isotropic12D 1H-13C HSQC
1132isotropic33D 1H-13C NOESY aliphatic
1144isotropic33D 1H-13C NOESY aliphatic
1153isotropic12D HNCO
1163isotropic12D HBHA(CO)NH
1173isotropic12D HNCA
1183isotropic12D CBCA(CO)NH
1193isotropic12D 1H-15N HSQC
1203isotropic12D 1H-13C HSQC
1213isotropic12D HN(CA)CO
1223isotropic12D 1H-13C NOESY aliphatic
1233isotropic12D 1H-15N NOESY
1242isotropic23D (H)CCH-TOCSY
1252isotropic22D 1H-13C HSQC
1261isotropic22D 1H-15N IPAP
1275anisotropic22D 1H-15N IPAP

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Sample preparation

Details
TypeSolution-IDContentsLabelSolvent systemDetails
solution10.6 mM [U-98% 15N] TolAIII, 3 mM TolBp, 20 mM sodium phosphate, 60 mM sodium chloride, 95% H2O/5% D2Osample_195% H2O/5% D2O
solution20.6 mM [U-98% 13C; U-98% 15N] TolAIII, 3 mM TolBp, 20 mM sodium phosphate, 60 mM sodium chloride, 95% H2O/5% D2Osample_295% H2O/5% D2O
solution31.1 mM TolAIII, 0.2 mM [U-98% 13C; U-98% 15N] TolBp, 20 mM sodium phosphate, 60 mM sodium chloride, 95% H2O/5% D2Osample_395% H2O/5% D2O
solution40.6 mM [U-98% 13C; U-98% 15N] TolAIII, 3 mM TolBp, 20 mM sodium phosphate, 60 mM sodium chloride, 100% D2Osample_4100% D2O
solution50.6 mM [U-98% 15N] TolAIII, 3 mM TolBp, 20 mM sodium phosphate, 60 mM sodium chloride, 5 % C12E6/n-hexanol, 95% H2O/5% D2Osample_595% H2O/5% D2OUsed for measurement of residual dipolar couplings. Alignment medium is mixture of C12E6 and n-hexanol as described by Ruckert & Otting (2000).
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
0.6 mMTolAIII[U-98% 15N]1
3 mMTolBpnatural abundance1
20 mMsodium phosphatenatural abundance1
60 mMsodium chloridenatural abundance1
0.6 mMTolAIII[U-98% 13C; U-98% 15N]2
3 mMTolBpnatural abundance2
20 mMsodium phosphatenatural abundance2
60 mMsodium chloridenatural abundance2
1.1 mMTolAIIInatural abundance3
0.2 mMTolBp[U-98% 13C; U-98% 15N]3
20 mMsodium phosphatenatural abundance3
60 mMsodium chloridenatural abundance3
0.6 mMTolAIII[U-98% 13C; U-98% 15N]4
3 mMTolBpnatural abundance4
20 mMsodium phosphatenatural abundance4
60 mMsodium chloridenatural abundance4
0.6 mMTolAIII[U-98% 15N]5
3 mMTolBpnatural abundance5
20 mMsodium phosphatenatural abundance5
60 mMsodium chloridenatural abundance5
5 %C12E6/n-hexanolnatural abundance5
Sample conditionsIonic strength: 140 mM / Ionic strength err: 10 / Label: sample_conditions_1 / pH: 7.0 / PH err: 0.05 / Pressure: 1 atm / Pressure err: 0.01 / Temperature: 293 K / Temperature err: 0.5

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-IDDetails
Bruker AVANCE IIBrukerAVANCE II5001with cryoprobe
Home-built CustomHome-builtCustom7502RT probe
Home-built home buildHome-builthome build9503RT probe

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Processing

NMR software
NameVersionDeveloperClassification
NMRPipeDelaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
CcpNmr AnalysisCCPNchemical shift assignment
ARIA2.3Linge, O'Donoghue and Nilgesstructure calculation
CNS1.2Brunger, Adams, Clore, Gros, Nilges and Readstructure calculation
Refinement
MethodSoftware ordinalDetails
simulated annealing38 rounds of iterative structure calculation using ARIA protocol
molecular dynamics420 lowest energy structures further refined using MD in water via ARIA protocol
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 600 / Conformers submitted total number: 20

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