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- PDB-8pkz: NMR solution structure of PilF-GSPIIB in the c-di-GMP bound state -

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Basic information

Entry
Database: PDB / ID: 8pkz
TitleNMR solution structure of PilF-GSPIIB in the c-di-GMP bound state
ComponentsATP-binding motif-containing protein pilF
KeywordsMOTOR PROTEIN / PilT class / NMR-structure / ligand binding / GSPII
Function / homology
Function and homology information


ATP hydrolysis activity / ATP binding / plasma membrane
Similarity search - Function
Type II secretion system protein GspE, N-terminal / MshEN domain / Type II secretion system protein GspE, N-terminal superfamily / Bacterial type II secretion system protein E signature. / Type II/IV secretion system protein / Type II/IV secretion system protein / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
GUANOSINE-5'-MONOPHOSPHATE / ATP-binding motif-containing protein pilF
Similarity search - Component
Biological speciesThermus thermophilus HB27 (bacteria)
MethodSOLUTION NMR / distance geometry / simulated annealing / torsion angle dynamics
AuthorsNeissner, K. / Woehnert, J.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation (DFG)Wo 901/12-1 Germany
CitationJournal: To Be Published
Title: NMR solution structure of GSPIIB of Thermus thermophilus in the c-di-GMP bound state
Authors: Neissner, K. / Woehnert, J.
History
DepositionJun 27, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 10, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: ATP-binding motif-containing protein pilF
hetero molecules


Theoretical massNumber of molelcules
Total (without water)16,9963
Polymers16,2701
Non-polymers7262
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: NMR Distance Restraints, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area1220 Å2
ΔGint-2 kcal/mol
Surface area9090 Å2
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 100target function
RepresentativeModel #1target function

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Components

#1: Protein ATP-binding motif-containing protein pilF / c-di-GMP binding domain of the ATPase enzyme PilF


Mass: 16269.562 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Each molecule consists of the protein bound to c-di-GMP (residues 147-148).
Source: (gene. exp.) Thermus thermophilus HB27 (bacteria) / Gene: pilF, TT_C1622 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q72H73
#2: Chemical ChemComp-5GP / GUANOSINE-5'-MONOPHOSPHATE


Mass: 363.221 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H14N5O8P / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
111isotropic72D 1H-15N HSQC
121isotropic73D HNCO
131isotropic73D HNCA
141isotropic73D HN(CA)CO
151isotropic73D HN(CA)CB
161isotropic23D HBHA(CO)NH
171isotropic23D C(CO)NH
181isotropic23D H(CCO)NH
191isotropic73D (H)CCH-COSY
1101isotropic13D (H)CCH-TOCSY
1111isotropic13D (H)CCH-TOCSY
1121isotropic42D 1H-13C HSQC aliphatic
1131isotropic22D 1H-13C HSQC aromatic
1141isotropic43D 1H-13C NOESY aliphatic
1151isotropic23D 1H-13C NOESY aromatic
1162isotropic22D 1H-15N HSQC
1172isotropic23D 1H-15N NOESY
1184isotropic22D 1H-13C HSQC aliphatic
1193isotropic72D 1H-13C HSQC aliphatic
1203isotropic73D 1H-13C NOESY aliphatic
1215isotropic72D 1H-31P-SFHMQC
1227isotropic72D HNN-COSY
1236isotropic52D 1H-13C HSQC aliphatic
1246isotropic52D 1H-13C HSQC aromatic
1256isotropic53D 1H-13C NOESY aliphatic
1266isotropic53D 1H-13C NOESY aromatic
1276isotropic12D 1H-15N HSQC NH2 only
1286isotropic13D 1H-15N NOESY NH2 only
1296isotropic72D 1H-13C HSQC aliphatic
1306isotropic72D 1H-13C HSQC aromatic
1316isotropic73D (H)CCH-COSY
1326isotropic72D HCN H1'-C1'-N9
1336isotropic72D HCN H8-C8-N9

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Sample preparation

Details
TypeSolution-IDContentsLabelSolvent systemDetails
solution1541 uM [U-13C; U-15N] PilF159-302, 812 uM c-di-GMP, 90% H2O/10% D2O13C_15N_PilF159-302_holo90% H2O/10% D2O
solution2460 ug/uL [U-100% 15N] PilF159-302, 707 uM c-di-GMP, 90% H2O/10% D2O15N_PilF159-302_holo90% H2O/10% D2O
solution3545 uM [U-15N]-Leu/Val-13C PilF159-302, 818 uM c-di-GMP, 90% H2O/10% D2O15N_PilF159-302_holo-13C_methylgroups90% H2O/10% D2Oleucine and valine methylgroups (CD1/CD2) (CG1/CG2) were selectively 13C labelled.
solution4522 uM [U-15N]-Leu/Val-13C-stereospecific PilF159-302, 627 uM c-di-GMP, 90% H2O/10% D2O15N_PilF159-302_holo-13C_methylgroups-stereospecific90% H2O/10% D2Oleucine and valine methylgroups (CD1/CD2) (CG1/CG2) were stereospecifically 13C labelled. For expression a mixture of 10%-13C-labelled and 90%-unlabelled glucose was used to achieve stereospecific labelling.
solution5545 uM [U-100% 15N] PilF159-302, 700 uM c-di-GMP, 90% H2O/10% D2O15N_PilF159-302_unlabelled-c-di-GMP90% H2O/10% D2O
solution6420 uM PilF159-302, 420 uM [U-13C; U-15N] c-di-GMP, 90% H2O/10% D2OPilF159-302_13C_15N-c-di-GMP90% H2O/10% D2O
solution7940 uM [U-100% 15N] PilF159-302, 1122 uM [U-13C; U-15N] c-di-GMP, 90% H2O/10% D2O15N_PilF159-302_13C_15N-c-di-GMP90% H2O/10% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
541 uMPilF159-302[U-13C; U-15N]1
812 uMc-di-GMPnatural abundance1
460 ug/uLPilF159-302[U-100% 15N]2
707 uMc-di-GMPnatural abundance2
545 uMPilF159-302[U-15N]-Leu/Val-13C3
818 uMc-di-GMPnatural abundance3
522 uMPilF159-302[U-15N]-Leu/Val-13C-stereospecific4
627 uMc-di-GMPnatural abundance4
545 uMPilF159-302[U-100% 15N]5
700 uMc-di-GMPnatural abundance5
420 uMPilF159-302natural abundance6
420 uMc-di-GMP[U-13C; U-15N]6
940 uMPilF159-302[U-100% 15N]7
1122 uMc-di-GMP[U-13C; U-15N]7
Sample conditionsIonic strength: 200 mM NaCl mM / Label: conditions_1 / pH: 5.8 / Pressure: ambient atm / Temperature: 318 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Bruker AVANCE NEOBrukerAVANCE NEO6001
Bruker AVANCE III HDBrukerAVANCE III HD7002
Bruker AVANCE NEOBrukerAVANCE NEO9005
Bruker AVANCE IIIBrukerAVANCE III9504
Bruker AVANCE IIBrukerAVANCE II6007

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Processing

NMR software
NameDeveloperClassification
CARAKeller and Wuthrichchemical shift assignment
CYANAGuntert, Mumenthaler and Wuthrichstructure calculation
CARAKeller and Wuthrichpeak picking
CcpNmr AnalysisCCPNpeak picking
TopSpinBruker Biospincollection
CYANAGuntert, Mumenthaler and Wuthrichrefinement
Refinement
MethodSoftware ordinal
distance geometry7
simulated annealing8
torsion angle dynamics9
NMR representativeSelection criteria: target function
NMR ensembleConformer selection criteria: target function / Conformers calculated total number: 100 / Conformers submitted total number: 20

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