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- PDB-2kif: Solution NMR structure of an O6-methylguanine DNA methyltransfera... -

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Basic information

Entry
Database: PDB / ID: 2kif
TitleSolution NMR structure of an O6-methylguanine DNA methyltransferase family protein from Vibrio parahaemolyticus. Northeast Structural Genomics Consortium target VpR247.
ComponentsO6-methylguanine-DNA methyltransferase
KeywordsTRANSFERASE / Methods Development / solution NMR structure / DNA base repair / O6 methylguanine methyltransferase / NESG / PSI-2 / Methyltransferase / Structural Genomics / Protein Structure Initiative / Northeast Structural Genomics Consortium
Function / homology
Function and homology information


catalytic activity / DNA repair / DNA binding
Similarity search - Function
Methylated-DNA-[protein]-cysteine S-methyltransferase, DNA binding / Methylated DNA-protein cysteine methyltransferase, DNA binding domain / 6-O-methylguanine DNA methyltransferase, DNA binding domain / Winged helix-like DNA-binding domain superfamily/Winged helix DNA-binding domain / Arc Repressor Mutant, subunit A / Winged helix-like DNA-binding domain superfamily / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
DNA base-flipping protein
Similarity search - Component
Biological speciesVibrio parahaemolyticus AQ3810 (bacteria)
MethodSOLUTION NMR / simulated annealing
Model detailslowest energy, model 1
AuthorsAramini, J.M. / Belote, R.L. / Ciccosanti, C.T. / Jiang, M. / Rost, B. / Nair, R. / Swapna, G.V.T. / Acton, T.B. / Xiao, R. / Everett, J.K. ...Aramini, J.M. / Belote, R.L. / Ciccosanti, C.T. / Jiang, M. / Rost, B. / Nair, R. / Swapna, G.V.T. / Acton, T.B. / Xiao, R. / Everett, J.K. / Montelione, G.T. / Northeast Structural Genomics Consortium (NESG)
CitationJournal: J.Biol.Chem. / Year: 2010
Title: Structural basis of O6-alkylguanine recognition by a bacterial alkyltransferase-like DNA repair protein.
Authors: Aramini, J.M. / Tubbs, J.L. / Kanugula, S. / Rossi, P. / Ertekin, A. / Maglaqui, M. / Hamilton, K. / Ciccosanti, C.T. / Jiang, M. / Xiao, R. / Soong, T.T. / Rost, B. / Acton, T.B. / Everett, ...Authors: Aramini, J.M. / Tubbs, J.L. / Kanugula, S. / Rossi, P. / Ertekin, A. / Maglaqui, M. / Hamilton, K. / Ciccosanti, C.T. / Jiang, M. / Xiao, R. / Soong, T.T. / Rost, B. / Acton, T.B. / Everett, J.K. / Pegg, A.E. / Tainer, J.A. / Montelione, G.T.
History
DepositionMay 3, 2009Deposition site: BMRB / Processing site: RCSB
Revision 1.0Jun 30, 2009Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Mar 16, 2022Group: Data collection / Database references / Derived calculations
Category: database_2 / pdbx_nmr_software ...database_2 / pdbx_nmr_software / pdbx_nmr_spectrometer / pdbx_struct_assembly / pdbx_struct_oper_list
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_nmr_software.name / _pdbx_nmr_spectrometer.model
Revision 1.3May 22, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: O6-methylguanine-DNA methyltransferase


Theoretical massNumber of molelcules
Total (without water)12,3411
Polymers12,3411
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 100structures with the lowest energy
RepresentativeModel #1lowest energy

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Components

#1: Protein O6-methylguanine-DNA methyltransferase / Methylated-DNA-protein-cysteine methyltransferase-related protein


Mass: 12341.314 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio parahaemolyticus AQ3810 (bacteria)
Gene: A79_1377, VP0951 / Plasmid: VpR247-21.9 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)MGK / References: UniProt: A6B4U8

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
Details: Structure solved with Bruker 800 MHz NOESY data on a 5-mm cyroprobe
NMR experiment
Conditions-IDExperiment-IDSolution-IDType
1112D 1H-15N HSQC
1212D 1H-13C HSQC
1313D simultaneous CN NOESY
1413D 1H-13C NOESY aromatic
1523D HNCO
1623D HN(CA)CO
1723D HNCA
1823D CBCA(CO)NH
1923D HN(CA)CB
11023D HBHA(CO)NH
11123D (H)CCH-COSY aliphatic
11223D (H)CCH-TOCSY aliphatic
11323D (H)CCH-TOCSY aliphatic
11423D HNHA
11523D HN(CO)CA
11622D 1H-15N hetNOE
11731D 1H-15N T1 and T2
11832D 1H-13C HSQC high res. (L/V methyl stereoassignment)
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. SPECTRA FOR ...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. SPECTRA FOR BACKBONE AND SIDE CHAIN ASSIGNMENTS WERE OBTAINED ON A 1.7-MM MICROCRYOPROBE AT 600 MHZ. ALL NOESY DATA WERE ACQUIRED AT 800 MHZ USING A 5-MM CROYOPROBE. BACKBONE ASSIGNMENTS WERE MADE USING AUTOASSIGN AND PINE, AND THE SIDE CHAIN ASSIGNMENTS WERE COMPLETED MANUALLY. AUTOMATIC NOESY ASSIGNMENTS WERE DETERMINED USING CYANA 3.0. 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 AND MOLPROBITY. COMPLETENESS OF NMR ASSIGNMENTS (EXCLUDING C-TERMINAL HHHHHH): BACKBONE, 97.6%, SIDE CHAIN, 98.3%, AROMATICS, 100%, STEREOSPECIFIC METHYL, 100%, STEREOSPECIFIC SIDE CHAIN NH2: 100%. FINAL STRUCTURE QUALITY FACTORS (FOR RESIDUES 1 TO 102, PSVS 1.3), WHERE ORDERED RESIDUES [S(PHI) + S(PSI) > 1.8] COMPRISE: 2-32,35-47,50-100: (A) RMSD (ORDERED RESIDUES): BB, 0.5, HEAVY ATOM, 0.8. (B) RAMACHANDRAN STATISTICS FOR ORDERED RESIDUES: MOST FAVORED, 91.5%, ADDITIONALLY ALLOWED, 8.5%, GENEROUSLY ALLOWED, 0.0%, DISALLOWED, 0.0%. (C) PROCHECK SCORES FOR ORDERED RESIDUES (RAW/Z-): PHI-PSI, -0.13/-0.20, ALL, -0.02/-0.12. (D) MOLPROBITY CLASH SCORE (RAW/Z-): 18.92/-1.72 (E) RPF SCORES FOR GOODNESS OF FIT TO NOESY DATA (RESIDUES 1-102): RECALL, 0.974, PRECISION, 0.937, F-MEASURE, 0.955, DP-SCORE, 0.835. (F) NUMBER OF CLOSE CONTACTS PER 20 MODELS: 6. 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]: 1,33-34,48-49,101-102.

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Sample preparation

Details
Solution-IDContentsSolvent system
10.94 mM [U-100% 13C; U-100% 15N] VpR247, 20 mM MES, 200 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS, 5 mM Shigemi, 90% H2O/10% D2O90% H2O/10% D2O
20.94 mM [U-100% 13C; U-100% 15N] VpR247, 20 mM MES, 200 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS, 1.7 mM microtube, 90% H2O/10% D2O90% H2O/10% D2O
30.92 mM [U-5% 13C; U-100% 15N] VpR247, 20 mM MES, 200 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS, 1.7 mM microtube, 90% H2O/10% D2O90% H2O/10% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
0.94 mMVpR247[U-100% 13C; U-100% 15N]1
20 mMMES1
200 mMsodium chloride1
5 mMcalcium chloride1
10 mMDTT1
0.02 %sodium azide1
50 uMDSS1
5 mMShigemi1
0.94 mMVpR247[U-100% 13C; U-100% 15N]2
20 mMMES2
200 mMsodium chloride2
5 mMcalcium chloride2
10 mMDTT2
0.02 %sodium azide2
50 uMDSS2
1.7 mMmicrotube2
0.92 mMVpR247[U-5% 13C; U-100% 15N]3
20 mMMES3
200 mMsodium chloride3
5 mMcalcium chloride3
10 mMDTT3
0.02 %sodium azide3
50 uMDSS3
1.7 mMmicrotube3
Sample conditionsIonic strength: 0.2 / pH: 6.5 / Pressure: ambient / Temperature: 298 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
TopSpin2.1Bruker Biospincollection
TopSpin2.1Bruker Biospindata analysis
NMRPipe2.3Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
Sparky3.112Goddarddata analysis
Sparky3.112Goddardpeak picking
PINE1Bahrami, Markley, Assadi, and Eghbalniachemical shift assignment
AutoAssign2.4.0Zimmerman, Moseley, Kulikowski and Montelionechemical shift assignment
AutoAssign2.4.0Zimmerman, Moseley, Kulikowski and Montelionevalidation
CYANA3Guntert, Mumenthaler and Wuthrichstructure solution
CNS1.2Brunger, Adams, Clore, Gros, Nilges and Readrefinement
AutoStructure2.2.1Huang, Tejero, Powers and Montelionerpf analysis
PSVS1.3Bhattacharya and Montelionestructure quality analysis
MolProbity3.15Richardsonstructure quality analysis
PdbStat5.1Tejero and Montelionepdb coordinate analysis
RefinementMethod: simulated annealing / Software ordinal: 1
Details: THE FINAL STRUCTURES ARE BASED ON A TOTAL OF 2448 CONFORMATIONALLY-RESTRICTING NOE-DERIVED DISTANCE CONSTRAINTS, 125 DIHEDRAL ANGLE CONSTRAINTS, AND 62 HYDROGEN BOND CONSTRAINTS (26.1 ...Details: THE FINAL STRUCTURES ARE BASED ON A TOTAL OF 2448 CONFORMATIONALLY-RESTRICTING NOE-DERIVED DISTANCE CONSTRAINTS, 125 DIHEDRAL ANGLE CONSTRAINTS, AND 62 HYDROGEN BOND CONSTRAINTS (26.1 CONSTRAINTS PER RESIDUE, 7.9 LONG RANGE CONSTRAINTS PER RESIDUE, COMPUTED FOR RESIDUES 1 TO 102 BY PSVS 1.3). STRUCTURE DETERMINATION WAS PERFORMED ITERATIVELY USING CYANA 3.0. 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, AND USING NEUTRAL HISTIDINE TAUTOMERS (NE2H FORM) AT POSITIONS 13 AND 38.
NMR constraintsNOE constraints total: 2448 / NOE intraresidue total count: 621 / NOE long range total count: 787 / NOE medium range total count: 493 / NOE sequential total count: 547 / Hydrogen bond constraints total count: 62 / Protein chi angle constraints total count: 5 / Protein other angle constraints total count: 11 / Protein phi angle constraints total count: 54 / Protein psi angle constraints total count: 55
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 100 / Conformers submitted total number: 20 / Maximum torsion angle constraint violation: 4.6 ° / Maximum upper distance constraint violation: 0.31 Å
NMR ensemble rmsDistance rms dev: 0.01 Å

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