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- PDB-7ry6: Solution NMR structural bundle of the first cyclization domain fr... -

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Basic information

Entry
Database: PDB / ID: 7ry6
TitleSolution NMR structural bundle of the first cyclization domain from yersiniabactin synthetase (Cy1) impacted by dynamics
ComponentsHMWP2 nonribosomal peptide synthetase
KeywordsLIGASE / Heterocyclization / Nonribosomal Peptide Synthetase / Peptide Bond Formation
Function / homologyNonribosomal peptide synthetase, condensation domain / Chloramphenicol acetyltransferase-like domain / Chloramphenicol Acetyltransferase / 2-Layer Sandwich / Alpha Beta / :
Function and homology information
Biological speciesYersinia pestis (bacteria)
MethodSOLUTION NMR / molecular dynamics
AuthorsKancherla, A.K. / Mishra, S.H. / Marincin, K.A. / Nerli, S. / Sgourakis, N.G. / Dowling, D.P. / Bouvignies, G. / Frueh, D.P.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM104257 United States
CitationJournal: Sci Adv / Year: 2022
Title: Global protein dynamics as communication sensors in peptide synthetase domains.
Authors: Mishra, S.H. / Kancherla, A.K. / Marincin, K.A. / Bouvignies, G. / Nerli, S. / Sgourakis, N. / Dowling, D.P. / Frueh, D.P.
History
DepositionAug 24, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 13, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 27, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Aug 3, 2022Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.3Jun 14, 2023Group: Other / Category: pdbx_database_status / Item: _pdbx_database_status.status_code_nmr_data
Revision 1.4May 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: HMWP2 nonribosomal peptide synthetase


Theoretical massNumber of molelcules
Total (without water)51,9151
Polymers51,9151
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 500structures with the lowest energy
RepresentativeModel #1medoid

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Components

#1: Protein HMWP2 nonribosomal peptide synthetase / Yersiniabactin biosynthetic protein / Yersiniabactin non-ribosomal peptide synthetase HMWP2


Mass: 51914.559 Da / Num. of mol.: 1 / Fragment: Cyclization domain 1, residues 101-544
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Yersinia pestis (bacteria) / Gene: irp2, y2399, G4D66_03140, NCTC144_00432 / Plasmid: pET30a / Production host: Escherichia coli (E. coli)
References: UniProt: A0A5P8YEQ8, Ligases; Forming carbon-nitrogen bonds; Acid-amino-acid ligases (peptide synthases)

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
111isotropic11H,15N -TROSY
122isotropic11H,15N -TROSY
133isotropic11H,15N -TROSY
144isotropic11H,15N -TROSY
154isotropic31H,15N -TROSY
165isotropic11H,15N -TROSY
171isotropic23D HNCA
185isotropic13D HNCA
191isotropic23D HNCO
1105isotropic13D HNCO
1111isotropic23D HN(CA)CO
1125isotropic13D HN(CA)CO
1131isotropic23D HN(CA)CB
1145isotropic13D HN(CA)CB
1361isotropic13D HN(CA)CB
1151isotropic23D HN(CO)CA
1165isotropic13D HN(CO)CA
1173isotropic13D HN(COCA)CB
1193isotropic13D Ala-HN(CA)CB
1203isotropic13D Gly-HNCA
1213isotropic13D Ser/Thr- HN(COCA)CB
1373isotropic13D Ser/Thr- HN(CA)CB
1223isotropic13D HMCMCBCA
1233isotropic13D HMCMCGCBCA
1243isotropic13D (H)CC(CO)NH
1383isotropic23D (H)CCH-TOCSY
1256isotropic24D time-shared (TS) 15N/13C HSQC-NOESY-TROSY/ HSQC
1266isotropic13D TS-TROSY/HSQC NOESY
1276isotropic13D TS-NOESY -HSQC/TROSY
1284isotropic33D TS-TROSY/HSQC NOESY
1291isotropic23D 15N TROSY-NOESY
1332anisotropic13D HSQC HNCO
1342anisotropic13D TROSY HNCO
1393isotropic23D hNCAnH
NMR detailsText: The authors state that the structural bundle reflects the fold of the cyclization domain with regions impacted by dynamics as described in the citation associated with this entry. Traditional ...Text: The authors state that the structural bundle reflects the fold of the cyclization domain with regions impacted by dynamics as described in the citation associated with this entry. Traditional analysis of individual conformers in the bundle is not recommended because dynamics may lead to apparent local distortions and because NMR structures of such large proteins rely on restraints involving backbone amides and methyl groups of Ile, Leu, and Val. All other side chains are deuterated and hence not experimentally restrained. The authors recommend that the ensemble is displayed as all states and aligned according to residues in secondary structures.

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Sample preparation

Details
TypeSolution-IDContentsDetailsLabelSolvent system
solution1588 uM [U-100% 13C; U-100% 15N; U-100% 2H] Cy1, 20 mM sodium phosphate, 10 mM sodium chloride, 1 mM EDTA, 5 mM DTT, 0.25 mM DSS, 95% H2O/5% D2OUniformly 2H, 13C and 15N labelled protein sample. Used for sequence specific assignmentsCDN95% H2O/5% D2O
filamentous virus2429 uM [U-100% 13C; U-100% 15N; U-100% 2H] Cy1, 20 mM sodium phosphate, 10 mM sodium chloride, 12 mg/mL Pf1 phage, 1 mM EDTA, 5 mM DTT, 0.25 mM DSS, 95% H2O/5% D2OUniformly 2H, 13C and 15N labelled protein sample along with Pf1 phage for partial alignment of the medium for measurement of RDCsCDN_RDC95% H2O/5% D2O
solution3586 uM U-100% 13C; U-100% 15N; U-100% 2H; 1H,13C for methyls of Leu, Val and Ile (delta1) Cy1, 20 mM sodium phosphate, 10 mM sodium chloride, 1 mM EDTA, 5 mM DTT, 0.25 mM DSS, 95% H2O/5% D2OUniformly 2H, 13C and 15N labelled except for methyls of leucine (delta 1 and 2), Valine (gamma 1 and 2), and isoleucine (delta 1) which are 1H and 13C labelledCDN_ILV95% H2O/5% D2O
solution4600 uM U-100% 15N; U-100% 2H; 1H,13C for methyls of Leu delta-2, Val gamma-2, and Ile delta1 Cy1, 20 mM sodium phosphate, 10 mM sodium chloride, 1 mM EDTA, 5 mM DTT, 0.03 % w/v Sodium Azide, 0.25 mM DSS, 95% H2O/5% D2OUniformly 2H, and 15N for backbone and sidechains, except 13C, 1H for isoleucine delta-1, leucine delta-2, and valine gamma-2 methyl groups.DN_ILVstereo95% H2O/5% D2O
solution5986 uM U-100% 13C, U-100% 15N; U-70% 2H; Cy1, 20 mM sodium phosphate, 10 mM sodium chloride, 1 mM EDTA, 5 mM DTT, 0.25 mM DSS, 95% H2O/5% D2O13C, 70% 2H / 30% 1H, 15N backbone and sidechains70DCN95% H2O/5% D2O
solution6350 uM U-100% 2H, U-100% 15N; 1H and 15N for Phe and Tyr; 1H and 13C for methyls of Ile (delta-1), Leu (delta-1 and delta-2), and Val (gamma-1 and gamma-2) Cy1, 20 mM sodium phosphate, 10 mM sodium chloride, 1 mM EDTA, 5 mM DTT, 0.25 mM DSS, 95% H2O/5% D2O12C, 2H, 15N for backbone and sidechains, except 12C, 1H, 15N for phenylalanine and tyrosine, and 13C, 1H for isoleucine delta-1, leucine delta-1 and 2, and valine gamma-1 and 2 methyl groupsDN_FYILV95% H2O/5% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
588 uMCy1[U-100% 13C; U-100% 15N; U-100% 2H]1
20 mMsodium phosphatenatural abundance1
10 mMsodium chloridenatural abundance1
1 mMEDTAnatural abundance1
5 mMDTTnatural abundance1
0.25 mMDSSnatural abundance1
429 uMCy1[U-100% 13C; U-100% 15N; U-100% 2H]2
20 mMsodium phosphatenatural abundance2
10 mMsodium chloridenatural abundance2
12 mg/mLPf1 phagenatural abundance2
1 mMEDTAnatural abundance2
5 mMDTTnatural abundance2
0.25 mMDSSnatural abundance2
586 uMCy1U-100% 13C; U-100% 15N; U-100% 2H; 1H,13C for methyls of Leu, Val and Ile (delta1)3
20 mMsodium phosphatenatural abundance3
10 mMsodium chloridenatural abundance3
1 mMEDTAnatural abundance3
5 mMDTTnatural abundance3
0.25 mMDSSnatural abundance3
600 uMCy1U-100% 15N; U-100% 2H; 1H,13C for methyls of Leu delta-2, Val gamma-2, and Ile delta14
20 mMsodium phosphatenatural abundance4
10 mMsodium chloridenatural abundance4
1 mMEDTAnatural abundance4
5 mMDTTnatural abundance4
0.03 % w/vSodium Azidenatural abundance4
0.25 mMDSSnatural abundance4
986 uMCy1U-100% 13C, U-100% 15N; U-70% 2H;5
20 mMsodium phosphatenatural abundance5
10 mMsodium chloridenatural abundance5
1 mMEDTAnatural abundance5
5 mMDTTnatural abundance5
0.25 mMDSSnatural abundance5
350 uMCy1U-100% 2H, U-100% 15N; 1H and 15N for Phe and Tyr; 1H and 13C for methyls of Ile (delta-1), Leu (delta-1 and delta-2), and Val (gamma-1 and gamma-2)6
20 mMsodium phosphatenatural abundance6
10 mMsodium chloridenatural abundance6
1 mMEDTAnatural abundance6
5 mMDTTnatural abundance6
0.25 mMDSSnatural abundance6
Sample conditionsDetails: This sample condition was used for all NMR data acquisition related to assignments, RDCs and initial NOESY experiments. 20 mM sodium phosphate pH 7.0, 10 mM NaCl, 1 mM EDTA, 5 mM DTT
Ionic strength: 10 mM NaCl mM / Label: NMR_Buffer / pH: 7 / Pressure: 1 atm / Temperature: 298 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-IDDetails
Bruker AVANCE IIIBrukerAVANCE III6001QCI Cryo Probe
Varian UNITYVarianUNITY8002Chili Probe
Bruker AVANCE IIIBrukerAVANCE III9503TCI Cryo Probe

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Processing

NMR software
NameVersionDeveloperClassification
CARA1.9.1.4Keller and Wuthrichchemical shift assignment
CYANA3.98.13Guntert, Mumenthaler and Wuthrichstructure calculation
CNS1.3Brunger, Adams, Clore, Gros, Nilges and Readrefinement
NMRPipe9.8Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
NMRFAM-SPARKY3.113T. D. Goddard and D. G. Knellerdata analysis
TALOS-NYang Shen and Ad Baxdata analysis
RefinementMethod: molecular dynamics / Software ordinal: 3
Details: The structures are based on a total of 3468 restraints. 2189 are NOE based distance restraints, 293 are distance restraints based on hydrogen bonds, 810 are backbone dihedral angle ...Details: The structures are based on a total of 3468 restraints. 2189 are NOE based distance restraints, 293 are distance restraints based on hydrogen bonds, 810 are backbone dihedral angle restraints, and 176 are residual dipolar coupling restraints.
NMR representativeSelection criteria: medoid
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 500 / Conformers submitted total number: 20

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