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- PDB-2k5d: SOLUTION NMR STRUCTURE OF SAG0934 from Streptococcus agalactiae. ... -

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Basic information

Entry
Database: PDB / ID: 2k5d
TitleSOLUTION NMR STRUCTURE OF SAG0934 from Streptococcus agalactiae. NORTHEAST STRUCTURAL GENOMICS TARGET SaR32[1-108].
Componentsuncharacterized protein SAG0934
Keywordsstructural genomics / unknown function / solution NMR structure / primosomal protein / DUF961 / construct optimized / PSI-2 / Protein Structure Initiative / Northeast Structural Genomics Consortium / NESG
Function / homologyConjugative transposon protein, DUF961 / Protein of unknown function DUF961 / YdcP-like superfamily / Bacterial protein of unknown function (DUF961) / OB fold (Dihydrolipoamide Acetyltransferase, E2P) / Beta Barrel / Mainly Beta / Uncharacterized protein
Function and homology information
Biological speciesStreptococcus agalactiae serogroup V (bacteria)
MethodSOLUTION NMR / simulated annealing
AuthorsAramini, J.M. / Rossi, P. / Zhao, L. / Foote, E.L. / Jiang, M. / Xiao, R. / Sharma, S. / Swapna, G.VT. / Nair, R. / Everett, J.K. ...Aramini, J.M. / Rossi, P. / Zhao, L. / Foote, E.L. / Jiang, M. / Xiao, R. / Sharma, S. / Swapna, G.VT. / Nair, R. / Everett, J.K. / Acton, T.B. / Rost, B. / Montelione, G.T. / Northeast Structural Genomics Consortium (NESG)
CitationJournal: To be Published
Title: SOLUTION NMR STRUCTURE OF SAG0934 from Streptococcus agalactiae. NORTHEAST STRUCTURAL GENOMICS TARGET SaR32[1-108].
Authors: Aramini, J.M. / Rossi, P. / Zhao, L. / Foote, E.L. / Jiang, M. / Xiao, R. / Sharma, S. / Swapna, G.VT. / Nair, R. / Everett, J.K. / Acton, T.B. / Rost, B. / Montelione, G.T.
History
DepositionJun 26, 2008Deposition site: BMRB / Processing site: RCSB
Revision 1.0Aug 26, 2008Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Feb 19, 2020Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Other
Category: database_2 / pdbx_database_status ...database_2 / pdbx_database_status / pdbx_nmr_software / pdbx_nmr_spectrometer / pdbx_struct_assembly / pdbx_struct_oper_list / struct_ref_seq_dif
Item: _pdbx_database_status.status_code_cs / _pdbx_nmr_software.name ..._pdbx_database_status.status_code_cs / _pdbx_nmr_software.name / _pdbx_nmr_spectrometer.model / _struct_ref_seq_dif.details
Revision 1.3Jun 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 1.4May 8, 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: uncharacterized protein SAG0934


Theoretical massNumber of molelcules
Total (without water)13,2261
Polymers13,2261
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 100structures with the lowest energy
RepresentativeModel #1lowest energy

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Components

#1: Protein uncharacterized protein SAG0934


Mass: 13225.892 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus agalactiae serogroup V (bacteria)
Species: agalactiae / Description: pET21 vector / Gene: SAG0934 / Species (production host): coli / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)MGK / References: UniProt: Q8E006

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDType
1112D 1H-15N HSQC
1212D 1H-13C HSQC
1313D HNCO
1413D HN(CA)CO
1513D CBCA(CO)NH
1613D HN(CA)CB
1713D HBHA(CO)NH
1813D (H)CCH-COSY
1913D (H)CCH-TOCSY aliphatic
1101CCH-TOCSY aliphatic
11113D simultaneous CN NOESY
11213D 1H-13C NOESY aromatic
11313D HNHA
1142simultaneous CN NOESY
11522D 1H-13C HSQC
11632D 1H-13C HSQC high res. (L/V stereoassignment)
11732D 1H-15N hetNOE
11831D 1H-15N T1 and T2
NMR detailsText: THE PROTEIN IS MONOMERIC BY GEL FILTRATION CHROMATOGRAPHY, STATIC LIGHT SCATTERING AND 15N T1/T2 RELAXATION. THE STRUCTURE WAS DETERMINED USING TRIPLE RESONANCE NMR SPECTROSCOPY. AUTOMATED ...Text: THE PROTEIN IS MONOMERIC BY GEL FILTRATION CHROMATOGRAPHY, STATIC LIGHT SCATTERING AND 15N T1/T2 RELAXATION. THE STRUCTURE WAS DETERMINED USING TRIPLE RESONANCE NMR SPECTROSCOPY. AUTOMATED BACKBONE ASSIGNMENTS WERE MADE USING AUTOASSIGN, AND THE SIDE CHAIN ASSIGNMENTS WERE COMPLETED MANUALLY. AUTOMATIC NOESY ASSIGNMENTS WERE DETERMINED USING CYANA 2.1. BACKBONE (PHI/PSI) DIHEDRAL ANGLE CONSTRAINTS WERE OBTAINED FROM TALOS. HYDROGEN BOND CONSTRAINTS WERE DETERMINED USING BOTH AUTOSTRUCTURE AND CYANA, AND WERE APPLIED ONLY IN THE FINAL REFINEMENT STAGE (CNS) OF THE STRUCTURE DETERMINATION. ROTAMER STATES OF SPECIFIC ORDERED RESIDUES WERE CONSTRAINED IN THE FINAL STAGE OF THE STRUCTURE REFINEMENT BASED ON PROCHECK. COMPLETENESS OF NMR ASSIGNMENTS (EXCLUDING C-TERMINAL HHHHHH): BACKBONE, 99.5%, SIDE CHAIN, 97.2%, AROMATICS, 100%, STEREOSPECIFIC METHYL, 94.4%, STEREOSPECIFIC SIDE CHAIN NH2: 100%. FINAL STRUCTURE QUALITY FACTORS (FOR RESIDUES 1 TO 110, PSVS 1.3), WHERE ORDERED RESIDUES [S(PHI) + S(PSI) > 1.8] COMPRISE: 18-50,53-67,70-72,74-78,81-83,99-108: (A) RMSD (ORDERED RESIDUES): BB, 0.7, HEAVY ATOM, 1.0. (B) RAMACHANDRAN STATISTICS FOR ORDERED RESIDUES: MOST FAVORED, 90.5%, ADDITIONALLY ALLOWED, 9.5%, GENEROUSLY ALLOWED, 0.0%, DISALLOWED, 0.0%. (C) PROCHECK SCORES FOR ORDERED RESIDUES (RAW/Z-): PHI-PSI, -0.55/-1.85, ALL, -0.28/-1.66. (D) MOLPROBITY CLASH SCORE (RAW/Z-): 17.32/-1.45 (E) RPF SCORES FOR GOODNESS OF FIT TO NOESY DATA (RESIDUES 1-110): RECALL, 0.989, PRECISION, 0.916, F-MEASURE, 0.951, DP-SCORE, 0.751. (F) NUMBER OF CLOSE CONTACTS PER 20 MODELS: 15. THE C-TERMINAL HIS TAG RESIDUES OF THE PROTEIN (HHHHHH) WERE NOT INCLUDED IN THE STRUCTURE CALCULATIONS AND HAVE BEEN OMITTED FROM THIS DEPOSITION. COORDINATES FOR THE FOLLOWING RESIDUES ARE NOT WELL DETERMINED [S(PHI) + S(PSI) < 1.8]: 1-17,51-52,68-69,73,84-98,109-110.

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Sample preparation

Details
Solution-IDContentsSolvent system
10.46 mM [U-100% 13C; U-100% 15N] SaR32, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide, 95% H2O/5% D2O95% H2O/5% D2O
20.46 mM [U-100% 13C; U-100% 15N] SaR32, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide, 100% D2O100% D2O
30.57 mM [U-5% 13C; U-100% 15N] SaR32, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide, 100% D2O100% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
0.46 mMSaR32[U-100% 13C; U-100% 15N]1
20 mMMES1
100 mMsodium chloride1
10 mMDTT1
5 mMcalcium chloride1
0.02 %sodium azide1
0.46 mMSaR32[U-100% 13C; U-100% 15N]2
20 mMMES2
100 mMsodium chloride2
10 mMDTT2
5 mMcalcium chloride2
0.02 %sodium azide2
0.57 mMSaR32[U-5% 13C; U-100% 15N]3
20 mMMES3
100 mMsodium chloride3
10 mMDTT3
5 mMcalcium chloride3
0.02 %sodium azide3
Sample conditionsIonic strength: 0.1 / pH: 6.5 / Pressure: ambient / Temperature: 293 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Bruker AvanceBrukerAVANCE8001
Bruker AvanceBrukerAVANCE6002

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Processing

NMR software
NameVersionDeveloperClassification
TopSpin2Bruker Biospincollection
AutoAssign2.4.0Zimmerman, Moseley, Kulikowski and Montelionechemical shift assignment
NMRPipe2.3Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
Sparky3.113Goddardpeak picking
Sparky3.113Goddarddata analysis
CYANA2.1Guntert, Mumenthaler and Wuthrichstructure solution
CNS1.2Brunger, Adams, Clore, Gros, Nilges and Readstructure solution
CNS1.2Brunger, Adams, Clore, Gros, Nilges and Readrefinement
AutoStructure2.2.1Huang, Tejero, Powers and Montelionerpf validation
PSVS1.3Bhattacharya and Montelionestructure validation
PdbStat5Tejero and Montelionepdb analysis
RefinementMethod: simulated annealing / Software ordinal: 1
Details: THE FINAL STRUCTURES ARE BASED ON A TOTAL OF 1629 CONFORMATIONALLY-RESTRICTING NOE-DERIVED DISTANCE CONSTRAINTS, 100 DIHEDRAL ANGLE CONSTRAINTS, AND 38 HYDROGEN BOND CONSTRAINTS (16.2 ...Details: THE FINAL STRUCTURES ARE BASED ON A TOTAL OF 1629 CONFORMATIONALLY-RESTRICTING NOE-DERIVED DISTANCE CONSTRAINTS, 100 DIHEDRAL ANGLE CONSTRAINTS, AND 38 HYDROGEN BOND CONSTRAINTS (16.2 CONSTRAINTS PER RESIDUE, 5.0 LONG RANGE CONSTRAINTS PER RESIDUE, COMPUTED FOR RESIDUES 1 TO 110 BY PSVS 1.3). STRUCTURE DETERMINATION WAS PERFORMED ITERATIVELY USING CYANA 2.1. THE 20 STRUCTURES OUT OF 100 WITH THE LOWEST TARGET FUNCTION WERE FURTHER REFINED BY RESTRAINED MOLECULAR DYNAMICS/ENERGY MINIMIZATION IN EXPLICIT WATER (CNS) WITH PARAM19.
NMR constraintsNOE constraints total: 1629 / NOE intraresidue total count: 514 / NOE long range total count: 509 / NOE medium range total count: 137 / NOE sequential total count: 469 / Hydrogen bond constraints total count: 38 / Protein chi angle constraints total count: 13 / Protein other angle constraints total count: 13 / Protein phi angle constraints total count: 38 / Protein psi angle constraints total count: 38
NMR representativeSelection criteria: lowest energy
NMR ensembleAverage torsion angle constraint violation: 0.49 °
Conformer selection criteria: structures with the lowest energy
Conformers calculated total number: 100 / Conformers submitted total number: 20 / Maximum torsion angle constraint violation: 4 ° / Maximum upper distance constraint violation: 0.24 Å
NMR ensemble rmsDistance rms dev: 0.01 Å

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