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- PDB-5t1n: Solution-state NMR structural ensemble of NPr (1-85) refined with... -

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Basic information

Entry
Database: PDB / ID: 5t1n
TitleSolution-state NMR structural ensemble of NPr (1-85) refined with RDCs and PCS
ComponentsPhosphocarrier protein NPr
KeywordsTRANSFERASE / PTSNtr / phosphotransfer / HPr-like / bacterial
Function / homology
Function and homology information


transferase activity, transferring phosphorus-containing groups / phosphoenolpyruvate-dependent sugar phosphotransferase system / cytoplasm
Similarity search - Function
Phosphotransferase system, HPr histidine phosphorylation site / PTS HPR domain histidine phosphorylation site signature. / Phosphotransferase system, HPr serine phosphorylation site / HPr-like / Phosphocarrier protein HPr-like / HPr-like superfamily / : / PTS HPr component phosphorylation site / PTS HPR domain profile. / Histidine-containing Protein; Chain: A; ...Phosphotransferase system, HPr histidine phosphorylation site / PTS HPR domain histidine phosphorylation site signature. / Phosphotransferase system, HPr serine phosphorylation site / HPr-like / Phosphocarrier protein HPr-like / HPr-like superfamily / : / PTS HPr component phosphorylation site / PTS HPR domain profile. / Histidine-containing Protein; Chain: A; / PTS HPR domain serine phosphorylation site signature. / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
Phosphocarrier protein NPr / Phosphocarrier protein NPr
Similarity search - Component
Biological speciesEscherichia coli O157:H7 (bacteria)
MethodSOLUTION NMR / torsion angle dynamics
AuthorsStrickland, M. / Wang, G. / Peterkofsky, A. / Tjandra, N.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI) United States
CitationJournal: Structure / Year: 2016
Title: Structure of the NPr:EIN(Ntr) Complex: Mechanism for Specificity in Paralogous Phosphotransferase Systems.
Authors: Strickland, M. / Stanley, A.M. / Wang, G. / Botos, I. / Schwieters, C.D. / Buchanan, S.K. / Peterkofsky, A. / Tjandra, N.
History
DepositionAug 19, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 16, 2016Provider: repository / Type: Initial release
Revision 1.1Dec 7, 2016Group: Database references
Revision 1.2Dec 21, 2016Group: Database references
Revision 1.3Jul 26, 2017Group: Author supporting evidence / Database references / Structure summary
Category: entity / pdbx_audit_support / pdbx_database_related
Item: _entity.pdbx_number_of_molecules / _pdbx_audit_support.funding_organization
Revision 1.4Nov 1, 2017Group: Author supporting evidence / Category: pdbx_struct_assembly_auth_evidence
Revision 1.5Dec 4, 2019Group: Author supporting evidence / Data collection
Category: pdbx_audit_support / pdbx_nmr_software / pdbx_nmr_spectrometer
Item: _pdbx_audit_support.funding_organization / _pdbx_nmr_software.name / _pdbx_nmr_spectrometer.model
Revision 1.6May 1, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Phosphocarrier protein NPr


Theoretical massNumber of molelcules
Total (without water)9,2551
Polymers9,2551
Non-polymers00
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)20 / 200structures with the lowest energy
RepresentativeModel #1lowest energy
DetailsMonomer according to Gel filtration

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Components

#1: Protein Phosphocarrier protein NPr / Nitrogen-related HPr


Mass: 9254.570 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli O157:H7 (bacteria) / Gene: ptsO, Z4569, ECs4085 / Plasmid: pETDuet1 / Production host: Escherichia coli (E. coli) / Strain (production host): ER2566
References: UniProt: P0A9N2, UniProt: P0A9N0*PLUS, Transferases; Transferring phosphorus-containing groups; Protein-serine/threonine kinases

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
111isotropic33D 1H-15N NOESY
121isotropic33D 1H-13C NOESY
131isotropic34D HNHC NOESY
143isotropic13D HN(CA)CB
2153isotropic12D IPAP
2162anisotropic12D IPAP
2173isotropic13D HN(CA)CB
2204isotropic22D 1H-15N HSQC
2185anisotropic22D 1H-15N HSQC

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Sample preparation

Details
TypeSolution-IDContentsDetailsLabelSolvent system
solution11 mM [U-13C; U-15N] NPr, 25 mM Tris, 93% H2O/7% D2OUsed for NOE and hydrogen bond restraints.NPr_Wang93% H2O/7% D2O
filamentous virus20.6 mM [U-13C; U-15N; U-2H] NPr, 10 mM TRIS, 100 mM sodium chloride, 0.5 mM EDTA, 93% H2O/7% D2OPf1 filamentous phage was used to induce partial alignment.NPr_rdc93% H2O/7% D2O
solution30.6 mM [U-13C; U-15N; U-2H] NPr, 10 mM TRIS, 100 mM sodium chloride, 0.5 mM EDTA, 93% H2O/7% D2OUsed for assignment and for RDC isotropic sample.NPr_assign93% H2O/7% D2O
solution40.6 mM [U-15N; U-2H] NPr, 10 mM TRIS, 100 mM sodium chloride, 0.5 mM EDTA, 93% H2O/7% D2OThe sample was mutated (E45C) to accommodate a Lu-M8-SPy tag, which was the control sample for pseudocontact shifts.NPr_PCSiso93% H2O/7% D2O
solution50.6 mM [U-15N; U-2H] NPr, 10 mM TRIS, 100 mM sodium chloride, 0.5 mM EDTA, 93% H2O/7% D2OThe sample was mutated (E45C) to accommodate a Yb-M8-SPy tag, which was used to induce pseudocontact shifts.NPr_PCSaniso93% H2O/7% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
1 mMNPr[U-13C; U-15N]1
25 mMTrisnatural abundance1
0.6 mMNPr[U-13C; U-15N; U-2H]2
10 mMTRISnatural abundance2
100 mMsodium chloridenatural abundance2
0.5 mMEDTAnatural abundance2
0.6 mMNPr[U-13C; U-15N; U-2H]3
10 mMTRISnatural abundance3
100 mMsodium chloridenatural abundance3
0.5 mMEDTAnatural abundance3
0.6 mMNPr[U-15N; U-2H]4
10 mMTRISnatural abundance4
100 mMsodium chloridenatural abundance4
0.5 mMEDTAnatural abundance4
0.6 mMNPr[U-15N; U-2H]5
10 mMTRISnatural abundance5
100 mMsodium chloridenatural abundance5
0.5 mMEDTAnatural abundance5
Sample conditions
Conditions-IDIonic strengthLabelpHPressure (kPa)Temperature (K)
125 mMconditions_171 atm308 K
2108 mMconditions_27.51 atm300 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-IDDetails
Bruker AVANCEBrukerAVANCE6001Cryoprobe
Bruker AVANCEBrukerAVANCE8002Cryoprobe
Varian INOVAVarianINOVA6003

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Processing

NMR software
NameVersionDeveloperClassification
Analysis2.4.2CCPNchemical shift assignment
PIPPGarrettchemical shift assignment
PIPPGarrettpeak picking
Analysis2.4.2CCPNpeak picking
VNMRVariancollection
TopSpin3Bruker Biospincollection
NMRPipe8.2Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
X-PLOR NIH2.37.7Schwieters, Kuszewski, Tjandra and Clorestructure calculation
X-PLOR NIH2.37.7Schwieters, Kuszewski, Tjandra and Clorerefinement
TALOS-N4.12Cornilescu, Delaglio and Baxdata analysis
Gaussian9Gaussian, Inc.geometry optimization
RefinementMethod: torsion angle dynamics / Software ordinal: 8
Details: Structure was refined using PDB ID 5T17 as a starting structure.
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 200 / Conformers submitted total number: 20

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