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- PDB-5t1o: Solution-state NMR and SAXS structural ensemble of NPr (1-85) in ... -

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Basic information

Entry
Database: PDB / ID: 5t1o
TitleSolution-state NMR and SAXS structural ensemble of NPr (1-85) in complex with EIN-Ntr (170-424)
Components
  • Phosphocarrier protein NPr
  • Phosphoenolpyruvate-protein phosphotransferase PtsP
KeywordsTRANSFERASE / PTSNtr / phosphotransfer / bacterial / complex
Function / homology
Function and homology information


transferase activity, transferring phosphorus-containing groups / phosphoenolpyruvate-protein phosphotransferase / phosphoenolpyruvate-protein phosphotransferase activity / N-acetylglucosamine transport / phosphoenolpyruvate-dependent sugar phosphotransferase system / kinase activity / metal ion binding / cytoplasm
Similarity search - Function
Phosphotransferase system, enzyme I-like / Phosphotransferase system, enzyme I N-terminal / PtsI, HPr-binding domain superfamily / : / PEP-utilising enzyme, N-terminal / PEP-utilising enzyme, active site / PEP-utilizing enzymes phosphorylation site signature. / PEP-utilising enzyme, conserved site / PEP-utilizing enzymes signature 2. / PEP-utilising enzyme, C-terminal ...Phosphotransferase system, enzyme I-like / Phosphotransferase system, enzyme I N-terminal / PtsI, HPr-binding domain superfamily / : / PEP-utilising enzyme, N-terminal / PEP-utilising enzyme, active site / PEP-utilizing enzymes phosphorylation site signature. / PEP-utilising enzyme, conserved site / PEP-utilizing enzymes signature 2. / PEP-utilising enzyme, C-terminal / PEP-utilising enzyme, PEP-binding domain / Phosphotransferase system, HPr histidine phosphorylation site / PTS HPR domain histidine phosphorylation site signature. / Phosphotransferase system, HPr serine phosphorylation site / PEP-utilising enzyme, mobile domain / Phosphohistidine domain superfamily / PEP-utilising enzyme, mobile domain / 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. / Pyruvate kinase-like domain superfamily / Pyruvate/Phosphoenolpyruvate kinase-like domain superfamily / GAF domain / Domain present in phytochromes and cGMP-specific phosphodiesterases. / GAF domain / GAF-like domain superfamily / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
Phosphocarrier protein NPr / Phosphocarrier protein NPr / Phosphoenolpyruvate-dependent phosphotransferase system
Similarity search - Component
Biological speciesEscherichia coli O157:H7 (bacteria)
Escherichia coli (E. coli)
MethodSOLUTION NMR / SOLUTION SCATTERING / simulated annealing
AuthorsStrickland, M. / Stanley, A.M. / Wang, G. / Schwieters, C.D. / Buchanan, S. / Peterkofsky, A. / Tjandra, N.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI) United States
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK) 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 1.3Sep 20, 2017Group: Author supporting evidence / Data collection / Structure summary
Category: entity / pdbx_audit_support / pdbx_nmr_ensemble
Item: _entity.pdbx_number_of_molecules / _pdbx_audit_support.funding_organization / _pdbx_nmr_ensemble.conformer_selection_criteria
Revision 1.4Nov 1, 2017Group: Author supporting evidence / Structure summary
Category: pdbx_nmr_representative / pdbx_struct_assembly_auth_evidence
Item: _pdbx_nmr_representative.selection_criteria
Revision 1.5May 8, 2019Group: Data collection / Database references / Experimental preparation
Category: exptl / pdbx_database_related / pdbx_nmr_software / Item: _pdbx_nmr_software.name
Revision 1.6Dec 4, 2019Group: Author supporting evidence / Data collection / Category: pdbx_audit_support / pdbx_nmr_spectrometer
Item: _pdbx_audit_support.funding_organization / _pdbx_nmr_spectrometer.model
Revision 1.7Jun 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 1.8May 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: Phosphocarrier protein NPr
B: Phosphoenolpyruvate-protein phosphotransferase PtsP


Theoretical massNumber of molelcules
Total (without water)37,6352
Polymers37,6352
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, SAXS
TypeNameSymmetry operationNumber
identity operation1_5551
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 200structures with the lowest energy
RepresentativeModel #1lowest energy
DetailsSize exclusion gel filtration was used to determine that this is a 1:1 protein interaction and both proteins are monomers. Small angle X-ray scattering confirmed this and was used as a restraint in the structure calculation

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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: Plasmid / Details (production host): 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
#2: Protein Phosphoenolpyruvate-protein phosphotransferase PtsP / Enzyme I-Ntr / Phosphotransferase system / enzyme I


Mass: 28380.070 Da / Num. of mol.: 1 / Mutation: H356Q
Source method: isolated from a genetically manipulated source
Details: This molecule was mutated at position 356 from histidine to glutamine. In E. coli, when the native histidine is present, the sample can be phosphorylated. It was mutated to provide a ...Details: This molecule was mutated at position 356 from histidine to glutamine. In E. coli, when the native histidine is present, the sample can be phosphorylated. It was mutated to provide a homogeneous sample. Additionally, it comprises residues 170-424 of the larger Enzyme I-Ntr molecule. Residue 169 is mutated to glycine due to TEV protease cleavage.
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: ptsP, ygdF, ygdO, b2829, JW2797 / Plasmid: pET28a / Production host: Escherichia coli (E. coli) / Strain (production host): ER2566
References: UniProt: P37177, phosphoenolpyruvate-protein phosphotransferase

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Experimental details

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Experiment

Experiment
Method
SOLUTION NMR
SOLUTION SCATTERING
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
111isotropic13D HN(CO)CACB TROSY
121isotropic13D HN(CA)CO TROSY
131isotropic13D HN(CA)CB TROSY
171isotropic13D HNCO TROSY
181isotropic13D HNCA TROSY
141isotropic23D 1H-15N NOESY TROSY
195isotropic23D HNCA TROSY
1102isotropic23D 1H-15N NOESY TROSY
1112isotropic13D HN(CO)CACB TROSY
1122isotropic13D HN(CA)CB TROSY
1132isotropic13D HNCA TROSY
1146isotropic13D HN(CA)CB
1151isotropic3ARTSY
1162isotropic3ARTSY
1173anisotropic3ARTSY
1184anisotropic3ARTSY
1198anisotropic22D 1H-15N HSQC TROSY
1209isotropic22D 1H-15N HSQC TROSY
12110anisotropic22D 1H-15N HSQC TROSY
12211isotropic22D 1H-15N HSQC TROSY

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Sample preparation

Details
TypeSolution-IDContentsLabelSolvent systemDetails
solution1200 uM [U-13C; U-15N; U-2H] EIN-Ntr, 240 uM [U-2H] NPr, 10 mM TRIS, 100 mM sodium chloride, 0.5 mM EDTA, 93% H2O/7% D2OEINnprIso93% H2O/7% D2O
solution2240 uM [U-2H] EIN-Ntr, 200 uM [U-13C; U-15N; U-2H] NPr, 10 mM TRIS, 100 mM sodium chloride, 0.5 mM EDTA, 93% H2O/7% D2ONPReinIso93% H2O/7% D2O
filamentous virus3200 uM [U-13C; U-15N; U-2H] EIN-Ntr, 240 uM [U-2H] NPr, 10 mM TRIS, 100 mM sodium chloride, 0.5 mM EDTA, 10 mg/mL Pf1 phage, 93% H2O/7% D2OEINnprAniso93% H2O/7% D2O
filamentous virus4240 uM [U-2H] EIN-Ntr, 200 uM [U-13C; U-15N; U-2H] NPr, 10 mM TRIS, 100 mM sodium chloride, 0.5 mM EDTA, 10 mg/mL Pf1 phage, 93% H2O/7% D2ONPReinAniso93% H2O/7% D2OPf1 filamentous phage
solution5200 uM [U-13C; U-15N; U-2H] EIN-Ntr, 10 mM TRIS, 100 mM sodium chloride, 0.5 mM EDTA, 93% H2O/7% D2OEINfree93% H2O/7% D2O
solution6200 uM [U-13C; U-15N; U-2H] NPr, 10 mM TRIS, 100 mM sodium chloride, 0.5 mM na EDTA, 93% H2O/7% D2ONPRfree93% H2O/7% D2O
solution7100 uM [U-13C; U-15N; U-2H] EIN-Ntr, 100 uM [U-2H] NPr, 10 mM TRIS, 100 mM sodium chloride, 0.5 mM EDTA, 93% H2O/7% D2OSAXS93% H2O/7% D2O
solution8200 uM [U-15N; U-2H] EIN-Ntr, 240 uM [U-2H] NPr, 10 mM TRIS, 100 mM sodium chloride, 0.5 mM EDTA, 93% H2O/7% D2OEINnprYb93% H2O/7% D2ONPr was mutated (E45C) to incorporate a covalently bound ytterbium M8-DOTA-SPy tag for the measurement of pseudocontact shifts.
solution9200 uM [U-15N; U-2H] EIN-Ntr, 240 uM [U-2H] NPr, 10 mM TRIS, 100 mM sodium chloride, 0.5 mM EDTA, 93% H2O/7% D2OEINnprLu93% H2O/7% D2ONPr was mutated (E45C) to incorporate a covalently bound lanthanide M8-DOTA-SPy tag for the measurement of pseudocontact shifts.
solution10240 uM [U-2H] EIN-Ntr, 200 uM [U-2H; U-15N] NPr, 10 mM TRIS, 100 mM sodium chloride, 0.5 mM EDTA, 93% H2O/7% D2ONPReinYb93% H2O/7% D2ONPr was mutated (E45C) to incorporate a covalently bound ytterbium M8-DOTA-SPy tag for the measurement of pseudocontact shifts.
solution11240 uM [U-2H] EIN-Ntr, 200 uM [U-2H; U-15N] NPr, 10 mM TRIS, 100 mM sodium chloride, 0.5 mM EDTA, 93% H2O/7% D2ONPReinLu93% H2O/7% D2ONPr was mutated (E45C) to incorporate a covalently bound lanthanide M8-DOTA-SPy tag for the measurement of pseudocontact shifts.
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
200 uMEIN-Ntr[U-13C; U-15N; U-2H]1
240 uMNPr[U-2H]1
10 mMTRISnatural abundance1
100 mMsodium chloridenatural abundance1
0.5 mMEDTAnatural abundance1
240 uMEIN-Ntr[U-2H]2
200 uMNPr[U-13C; U-15N; U-2H]2
10 mMTRISnatural abundance2
100 mMsodium chloridenatural abundance2
0.5 mMEDTAnatural abundance2
200 uMEIN-Ntr[U-13C; U-15N; U-2H]3
240 uMNPr[U-2H]3
10 mMTRISnatural abundance3
100 mMsodium chloridenatural abundance3
0.5 mMEDTAnatural abundance3
10 mg/mLPf1 phagenatural abundance3
240 uMEIN-Ntr[U-2H]4
200 uMNPr[U-13C; U-15N; U-2H]4
10 mMTRISnatural abundance4
100 mMsodium chloridenatural abundance4
0.5 mMEDTAnatural abundance4
10 mg/mLPf1 phagenatural abundance4
200 uMEIN-Ntr[U-13C; U-15N; U-2H]5
10 mMTRISnatural abundance5
100 mMsodium chloridenatural abundance5
0.5 mMEDTAnatural abundance5
200 uMNPr[U-13C; U-15N; U-2H]6
10 mMTRISnatural abundance6
100 mMsodium chloridenatural abundance6
0.5 mMEDTAna6
100 uMEIN-Ntr[U-13C; U-15N; U-2H]7
100 uMNPr[U-2H]7
10 mMTRISnatural abundance7
100 mMsodium chloridenatural abundance7
0.5 mMEDTAnatural abundance7
200 uMEIN-Ntr[U-15N; U-2H]8
240 uMNPr[U-2H]8
10 mMTRISnatural abundance8
100 mMsodium chloridenatural abundance8
0.5 mMEDTAnatural abundance8
200 uMEIN-Ntr[U-15N; U-2H]9
240 uMNPr[U-2H]9
10 mMTRISnatural abundance9
100 mMsodium chloridenatural abundance9
0.5 mMEDTAnatural abundance9
240 uMEIN-Ntr[U-2H]10
200 uMNPr[U-2H; U-15N]10
10 mMTRISnatural abundance10
100 mMsodium chloridenatural abundance10
0.5 mMEDTAnatural abundance10
240 uMEIN-Ntr[U-2H]11
200 uMNPr[U-2H; U-15N]11
10 mMTRISnatural abundance11
100 mMsodium chloridenatural abundance11
0.5 mMEDTAnatural abundance11
Sample conditionsIonic strength: 108 mM / Label: Conditions_1 / pH: 7.5 / Pressure: 1 atm / Temperature: 300 K

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Data collection

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-IDDetails
Bruker AVANCEBrukerAVANCE6001Cryoprobe
Bruker AVANCEBrukerAVANCE8002Cryoprobe
Bruker AVANCEBrukerAVANCE9003Cryoprobe

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Processing

NMR software
NameVersionDeveloperClassification
X-PLOR NIH2.37.7SCHWIETERS, KUSZEWSKI, TJANDRA AND CLORErefinement
ANALYSIS2.4.2structure solution
TopSpin3structure solution
Gaussian9structure solution
NMRPipe8.2structure solution
TALOS-N4.12structure solution
RefinementMethod: simulated annealing / Software ordinal: 1
Details: DOCKING WAS FIRST CARRIED OUT BETWEEN THE TWO PROTEINS USING RIGID BODY SIMULATED ANNEALING (USING THE FREE FORM STRUCTURES - 5T1N AND 5T12). THE TOP TWENTY STRUCTURES WERE THEN TAKEN ...Details: DOCKING WAS FIRST CARRIED OUT BETWEEN THE TWO PROTEINS USING RIGID BODY SIMULATED ANNEALING (USING THE FREE FORM STRUCTURES - 5T1N AND 5T12). THE TOP TWENTY STRUCTURES WERE THEN TAKEN THROUGH FOR THE REFINEMENT STEP. TORSION ANGLE DYNAMICS REFINEMENT. IN THE REFINEMENT STEP ALL RESIDUES IN NPR (AS WELL AS THE PCS TAG) EXCEPT FOR RESIDUES 14-18 WERE RIGID. EIN WAS FLEXIBLE. NCS RESTRAINTS WERE USED TO GUIDE DOMAIN ORIENTATION OF EIN AND TO AID IN THE CONVERGENCE OF THE COMPLEX 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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