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- PDB-7b2b: Solution structure of a non-covalent extended docking domain comp... -

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Basic information

Entry
Database: PDB / ID: 7b2b
TitleSolution structure of a non-covalent extended docking domain complex of the Pax NRPS: PaxA T1-CDD/PaxB NDD
Components
  • Amino acid adenylation domain-containing protein
  • Peptide synthetase PaxA
KeywordsPROTEIN BINDING / PROTEIN
Function / homology
Function and homology information


phenylalanine racemase (ATP-hydrolysing) / phenylalanine racemase (ATP-hydrolyzing) activity / phosphopantetheine binding / catalytic activity
Similarity search - Function
Condensation domain / Condensation domain / Amino acid adenylation domain / ANL, N-terminal domain / AMP-binding enzyme, C-terminal domain / AMP-binding enzyme C-terminal domain / Chloramphenicol acetyltransferase-like domain superfamily / AMP-binding, conserved site / Putative AMP-binding domain signature. / Polyketide synthase, phosphopantetheine-binding domain ...Condensation domain / Condensation domain / Amino acid adenylation domain / ANL, N-terminal domain / AMP-binding enzyme, C-terminal domain / AMP-binding enzyme C-terminal domain / Chloramphenicol acetyltransferase-like domain superfamily / AMP-binding, conserved site / Putative AMP-binding domain signature. / Polyketide synthase, phosphopantetheine-binding domain / Phosphopantetheine attachment site / AMP-dependent synthetase/ligase / AMP-binding enzyme / Phosphopantetheine attachment site / Phosphopantetheine attachment site. / Phosphopantetheine attachment site / ACP-like superfamily / Carrier protein (CP) domain profile. / Phosphopantetheine binding ACP domain
Similarity search - Domain/homology
Amino acid adenylation domain-containing protein / Peptide synthetase PaxA
Similarity search - Component
Biological speciesXenorhabdus cabanillasii (bacteria)
Xenorhabdus cabanillasii JM26 (bacteria)
MethodSOLUTION NMR / torsion angle dynamics / molecular dynamics
AuthorsWatzel, J. / Sarawi, S. / Duchardt-Ferner, E. / Bode, H.B. / Woehnert, J.
CitationJournal: Angew.Chem.Int.Ed.Engl. / Year: 2021
Title: Cooperation between a T Domain and a Minimal C-Terminal Docking Domain to Enable Specific Assembly in a Multiprotein NRPS.
Authors: Watzel, J. / Duchardt-Ferner, E. / Sarawi, S. / Bode, H.B. / Wohnert, J.
History
DepositionNov 26, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 16, 2021Provider: repository / Type: Initial release
Revision 1.1Jun 23, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Jul 3, 2024Group: Data collection / Database references / Other
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_status
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_nmr_data

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: Amino acid adenylation domain-containing protein
A: Peptide synthetase PaxA


Theoretical massNumber of molelcules
Total (without water)15,5932
Polymers15,5932
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: isothermal titration calorimetry, gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area2160 Å2
ΔGint-21 kcal/mol
Surface area8290 Å2
MethodPISA
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 20structures with the least restraint violations
RepresentativeModel #1target function

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Components

#1: Protein/peptide Amino acid adenylation domain-containing protein / Peptide synthetase XpsB (Modular protein)


Mass: 3616.222 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: a non-native tyrosine residue was added to the C-terminus of the PaxB NDD for an accurate determination of the protein sample concentration
Source: (gene. exp.) Xenorhabdus cabanillasii (bacteria) / Gene: BDD26_3339 / Plasmid: pET-11a / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A3D9UGN9
#2: Protein Peptide synthetase PaxA


Mass: 11976.369 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: non-covalent complex of PaxA T1-Cdd 981-1084/PaxB Ndd 1-30
Source: (gene. exp.) Xenorhabdus cabanillasii JM26 (bacteria)
Gene: paxA, XCR1_180002 / Plasmid: pET-11a / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: W1J0W9, phenylalanine racemase (ATP-hydrolysing)

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
113isotropic12D 1H-15N HSQC
1133isotropic13D HNCO
1123isotropic13D HNCA
1193isotropic33D HBHA(CO)NH
1183isotropic33D CBCA(CO)NH
1173isotropic33D (H)CCH-COSY
1223isotropic23D (H)CCH-TOCSY
1213isotropic23D (H)CCH-TOCSY
1163isotropic12D 1H-13C HSQC aliphatic
1113isotropic13D 1H-13C NOESY aliphatic
1153isotropic22D 1H-13C HSQC aromatic
1103isotropic23D 1H-13C NOESY aromatic
1144isotropic22D 1H-15N HSQC
1304isotropic23D HNCO
1294isotropic23D HN(CA)CB
1274isotropic33D HBHA(CO)NH
1284isotropic33D CBCA(CO)NH
1264isotropic33D (H)CCH-COSY
1254isotropic23D (H)CCH-TOCSY
1244isotropic32D 1H-13C HSQC aliphatic
1314isotropic33D 1H-13C NOESY aliphatic
1234isotropic52D 1H-13C HSQC aromatic
1334isotropic53D 1H-13C NOESY aromatic
125isotropic33D 1H-13C NOESY aliphatic
185isotropic43D 1H-13C-filtered NOESY aliphatic
136isotropic33D 1H-13C NOESY aliphatic
196isotropic43D 1H-13C-filtered NOESY aliphatic
141isotropic13D 1H-15N NOESY
172isotropic23D 1H-15N NOESY
157isotropic12D 1H-13C HSQC aliphatic
168isotropic12D 1H-13C HSQC aliphatic

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Sample preparation

Details
TypeSolution-IDContentsLabelSolvent system
solution1300 uM [U-15N] PaxA T1-CDD, 360 uM PaxB NDD, 50 mM sodium phosphate, 100 mM sodium chloride, 2 mM beta-mercaptoethanol, 5 % [U-2H] D2O, 100 uM DSS, 95% H2O/5% D2O15N_PaxA T1-CDD/PaxB NDD95% H2O/5% D2O
solution2300 uM [U-15N] PaxB NDD, 360 uM PaxA T1-CDD, 50 mM sodium phosphate, 100 mM sodium chloride, 2 mM beta-mercaptoethanol, 5 % [U-2H] D2O, 100 uM DSS, 95% H2O/5% D2O15N_PaxB NDD/PaxA T1-CDD95% H2O/5% D2O
solution3300 uM [U-13C; U-15N] PaxA T1-CDD, 360 uM PaxB NDD, 50 mM sodium phosphate, 100 mM sodium chloride, 2 mM beta-mercaptoethanol, 5 % [U-2H] D2O, 100 uM DSS, 95% H2O/5% D2O15N,13C_PaxA T1-CDD/PaxB NDD95% H2O/5% D2O
solution7300 uM [U-10% 13C; U-15N] PaxA T1-CDD, 360 uM PaxB NDD, 50 mM sodium phosphate, 100 mM sodium chloride, 2 mM beta-mercaptoethanol, 5 % [U-2H] D2O, 100 uM DSS, 95% H2O/5% D2O15N,13C_stereo_PaxA T1-CDD/PaxB NDD95% H2O/5% D2O
solution5300 uM [U-13C; U-15N] PaxA T1-CDD, 360 uM PaxB NDD, 50 mM sodium phosphate, 100 mM sodium chloride, 2 mM beta-mercaptoethanol, 5 % [U-2H] D2O, 100 uM DSS, 100% D2O15N,13C_D2O_PaxA T1-CDD/PaxB NDD100% D2O
solution4300 uM [U-13C; U-15N] PaxB NDD, 360 uM PaxA T1-CDD, 50 mM sodium phosphate, 100 mM sodium chloride, 2 mM beta-mercaptoethanol, 5 % [U-2H] D2O, 100 uM DSS, 95% H2O/5% D2O15N,13C_PaxB NDD/PaxA T1-CDD95% H2O/5% D2O
solution8150 uM [U-10% 13C; U-15N] PaxB NDD, 180 uM [U-15N] PaxA T1-CDD, 50 mM sodium phosphate, 100 mM sodium chloride, 2 mM beta-mercaptoethanol, 5 % [U-2H] D2O, 100 uM DSS, 95% H2O/5% D2O15N,13C_stereo_PaxB NDD/PaxA T1-CDD95% H2O/5% D2O
solution6300 uM [U-13C; U-15N] PaxB NDD, 360 uM PaxA T1-CDD, 50 mM sodium phosphate, 100 mM sodium chloride, 2 mM beta-mercaptoethanol, 5 % [U-2H] D2O, 100 uM DSS, 100% D2O15N,13C_D2O_PaxB NDD/PaxA T1-CDD100% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
300 uMPaxA T1-CDD[U-15N]1
360 uMPaxB NDDnatural abundance1
50 mMsodium phosphatenatural abundance1
100 mMsodium chloridenatural abundance1
2 mMbeta-mercaptoethanolnatural abundance1
5 %D2O[U-2H]1
100 uMDSSnatural abundance1
300 uMPaxB NDD[U-15N]2
360 uMPaxA T1-CDDnatural abundance2
50 mMsodium phosphatenatural abundance2
100 mMsodium chloridenatural abundance2
2 mMbeta-mercaptoethanolnatural abundance2
5 %D2O[U-2H]2
100 uMDSSnatural abundance2
300 uMPaxA T1-CDD[U-13C; U-15N]3
360 uMPaxB NDDnatural abundance3
50 mMsodium phosphatenatural abundance3
100 mMsodium chloridenatural abundance3
2 mMbeta-mercaptoethanolnatural abundance3
5 %D2O[U-2H]3
100 uMDSSnatural abundance3
300 uMPaxA T1-CDD[U-10% 13C; U-15N]7
360 uMPaxB NDDnatural abundance7
50 mMsodium phosphatenatural abundance7
100 mMsodium chloridenatural abundance7
2 mMbeta-mercaptoethanolnatural abundance7
5 %D2O[U-2H]7
100 uMDSSnatural abundance7
300 uMPaxA T1-CDD[U-13C; U-15N]5
360 uMPaxB NDDnatural abundance5
50 mMsodium phosphatenatural abundance5
100 mMsodium chloridenatural abundance5
2 mMbeta-mercaptoethanolnatural abundance5
5 %D2O[U-2H]5
100 uMDSSnatural abundance5
300 uMPaxB NDD[U-13C; U-15N]4
360 uMPaxA T1-CDDnatural abundance4
50 mMsodium phosphatenatural abundance4
100 mMsodium chloridenatural abundance4
2 mMbeta-mercaptoethanolnatural abundance4
5 %D2O[U-2H]4
100 uMDSSnatural abundance4
150 uMPaxB NDD[U-10% 13C; U-15N]8
180 uMPaxA T1-CDD[U-15N]8
50 mMsodium phosphatenatural abundance8
100 mMsodium chloridenatural abundance8
2 mMbeta-mercaptoethanolnatural abundance8
5 %D2O[U-2H]8
100 uMDSSnatural abundance8
300 uMPaxB NDD[U-13C; U-15N]6
360 uMPaxA T1-CDDnatural abundance6
50 mMsodium phosphatenatural abundance6
100 mMsodium chloridenatural abundance6
2 mMbeta-mercaptoethanolnatural abundance6
5 %D2O[U-2H]6
100 uMDSSnatural abundance6
Sample conditionsIonic strength: 100 mM / Label: conditions_1 / pH: 6.5 / Pressure: AMBIENT bar / Temperature: 293 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Bruker AVANCE III HDBrukerAVANCE III HD8001
Bruker AVANCE IIIBrukerAVANCE III6002
Bruker AVANCE IIBrukerAVANCE II6003
Bruker AVANCE NEOBrukerAVANCE NEO9004
Bruker AVANCE IIIBrukerAVANCE III9505

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Processing

NMR software
NameVersionDeveloperClassification
TopSpin3.6.2Bruker Biospindata analysis
CARA1.8.4.2Keller and Wuthrichchemical shift assignment
CcpNmr Analysis2.4.2CCPNpeak picking
CYANA3.98Guntert, Mumenthaler and Wuthrichstructure calculation
OPALpKoradi, Billeter and Guntertrefinement
Refinement
MethodSoftware ordinal
torsion angle dynamics4
molecular dynamics5
NMR representativeSelection criteria: target function
NMR ensembleConformer selection criteria: structures with the least restraint violations
Conformers calculated total number: 20 / Conformers submitted total number: 20

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