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- PDB-7k7f: Solution Structure of the Corynebacterium diphtheriae SpaA Pilin-... -

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Basic information

Entry
Database: PDB / ID: 7k7f
TitleSolution Structure of the Corynebacterium diphtheriae SpaA Pilin-Signal Peptide Complex
Components
  • Putative surface-anchored fimbrial subunit
  • SpaA sorting signal peptide
KeywordsCELL ADHESION / pili / backbone pilin / sortase / lysine isopeptide bond / Gram-positive bacteria
Function / homology
Function and homology information


carbohydrate metabolic process / membrane / metal ion binding
Similarity search - Function
: / Gram-positive pilin backbone subunit 2, Cna-B-like domain / Gram-positive pilin backbone subunit 2, Cna-B-like domain / Gram-positive pilin subunit D1, N-terminal / Gram-positive pilin subunit D1, N-terminal domain / Fimbrial isopeptide formation D2 domain / Immunoglobulin-like fold
Similarity search - Domain/homology
Surface-anchored fimbrial subunit
Similarity search - Component
Biological speciesCorynebacterium diphtheriae (bacteria)
MethodSOLUTION NMR / simulated annealing
Model detailsSpaA backbone pilin isopeptide complex with sorting signal peptide
AuthorsMcConnell, S.A. / Clubb, R.T.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI52217 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5T32GM007185-43 United States
CitationJournal: Proc.Natl.Acad.Sci.USA / Year: 2021
Title: Sortase-assembled pili in Corynebacterium diphtheriae are built using a latch mechanism.
Authors: McConnell, S.A. / McAllister, R.A. / Amer, B.R. / Mahoney, B.J. / Sue, C.K. / Chang, C. / Ton-That, H. / Clubb, R.T.
History
DepositionSep 22, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 10, 2021Provider: repository / Type: Initial release
Revision 1.1Apr 21, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Putative surface-anchored fimbrial subunit
B: SpaA sorting signal peptide


Theoretical massNumber of molelcules
Total (without water)16,4902
Polymers16,4902
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry, peptide crosslinking was quantified by MS, cross-linking, pilin sortase mediated isopeptide bond formed between peptide and SpaA domain, assay for oligomerization, ...Evidence: mass spectrometry, peptide crosslinking was quantified by MS, cross-linking, pilin sortase mediated isopeptide bond formed between peptide and SpaA domain, assay for oligomerization, peptide crosslinking was quantified by SDS-PAGE and HPLC
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area1390 Å2
ΔGint-11 kcal/mol
Surface area8980 Å2
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 200structures with the lowest energy
RepresentativeModel #1closest to the average

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Components

#1: Protein Putative surface-anchored fimbrial subunit


Mass: 15399.396 Da / Num. of mol.: 1 / Fragment: N-terminal domain, residues 53-195
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Corynebacterium diphtheriae (strain ATCC 700971 / NCTC 13129 / Biotype gravis) (bacteria)
Strain: ATCC 700971 / NCTC 13129 / Biotype gravis / Gene: DIP2013 / Plasmid: pSUMO / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: Q6NF81
#2: Protein/peptide SpaA sorting signal peptide


Mass: 1090.249 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Corynebacterium diphtheriae (bacteria)

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
111isotropic23D 15N-separated NOESY
221isotropic13D (F1) 13C,15N-filtered (F2) 15N-edited NOESY-HSQC
131isotropic12D 1H-15N HSQC
242isotropic12D 1H-13C HSQC
252isotropic12D 1H-13C HSQC aromatic
161isotropic13D HN(CA)CB
171isotropic11H-15N heteronoe
181isotropic13D CBCA(CO)NH
191isotropic1T2/R2 relaxation
1101isotropic1T1/R1 relaxation
1111isotropic13D HN(CA)CO
1121isotropic13D HNCA
1131isotropic13D HNCO
2142isotropic13D (H)CCH-COSY
1171isotropic13D HNHA
1181isotropic23D HBHA(CO)NH
1191isotropic23D 1H-15N TOCSY
1201isotropic13D H(CCO)NH
2212isotropic12D (F1,F2) 13C-filtered NOESY
2222isotropic12D (F1) 13C,15N-filtered TOCSY
2242isotropic13D 13C-separated NOESY aliphatic D2O
1232isotropic13D (F1) 13C,15N-filtered (F2) 13C-edited NOESY-HSQC
1252isotropic13D 13C-separated NOESY aromatic D2O
1261isotropic13D 13C-separated NOESY aliphatic H2O

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Sample preparation

Details
TypeSolution-IDContentsLabelSolvent system
solution11.2 mM [U-13C; U-15N] SpaA backbone pilin for protein, 1.2 mM SpaA sorting signal for peptide, 50 mM NaH2PO4, 100 mM NaCl, 0.01 % NaN3, 92% H2O, 8% D2ONSpaA-signal peptide (H2O)92% H2O, 8% D2O
solution21.2 mM [U-13C; U-15N] SpaA backbone pilin for protein, 1.2 mM SpaA sorting signal for peptide, 50 mM NaH2PO4, 100 mM NaCl, 0.01 % NaN3, 100% D2ONSpaA-signal peptide (D2O)100% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
1.2 mMSpaA backbone pilin for protein[U-13C; U-15N]1
1.2 mMSpaA sorting signal for peptidenatural abundance1
50 mMNaH2PO4natural abundance1
100 mMNaClnatural abundance1
0.01 %NaN3natural abundance1
1.2 mMSpaA backbone pilin for protein[U-13C; U-15N]2
1.2 mMSpaA sorting signal for peptidenatural abundance2
50 mMNaH2PO4natural abundance2
100 mMNaClnatural abundance2
0.01 %NaN3natural abundance2
Sample conditions
Conditions-IDIonic strengthLabelpHPressure (kPa)Temperature (K)
1100mM NaCl mMCond16.0 ambient 298 K
2100mM NaCl mMCond26.0 ambient 298 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Bruker AVANCE III HDBrukerAVANCE III HD6001
Bruker AVANCE NEOBrukerAVANCE NEO8002

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Processing

NMR software
NameVersionDeveloperClassification
X-PLOR NIH2.37Schwieters, Kuszewski, Tjandra and Clorerefinement
X-PLOR NIH2.37Schwieters, Kuszewski, Tjandra and Clorestructure calculation
Xipp1.19Dan Garrettchemical shift assignment
Xipp1.19Dan Garrettpeak picking
UNIOTorsten Herrmannstructure calculation
NMRPipeDelaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
TopSpinBruker Biospincollection
NMRFAM-SPARKYNational Magnetic Resonance Facility at Madison (Wisconsin)data analysis
CARAKeller and Wuthrichpeak picking
TALOS-NCornilescu, Delaglio and Baxgeometry optimization
MOLMOLKoradi, Billeter and Wuthrichrefinement
PROCHECK / PROCHECK-NMRLaskowski, MacArthur, Smith, Jones, Hutchinson, Morris, Moss and Thorntonrefinement
RefinementMethod: simulated annealing / Software ordinal: 1
Details: NOE restraints refined in XIPP, structures were calculated with a simulated annealing protocol using XPLOR-NIH
NMR representativeSelection criteria: closest to the average
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 200 / Conformers submitted total number: 20

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