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- PDB-2kke: Solution NMR Structure of a dimeric protein of unknown function f... -

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Basic information

Entry
Database: PDB / ID: 2kke
TitleSolution NMR Structure of a dimeric protein of unknown function from Methanobacterium thermoautotrophicum, Northeast Structural Genomics Consortium Target TR5
ComponentsUncharacterized protein
KeywordsStructural Genomics / Unknown function / Protein NMR / NORTHEAST STRUCTURAL GENOMICS CONSORTIUM (NESG) / Target TR5 / PSI-2 / Protein Structure Initiative / Methanothermobacter thermautotrophicus / O26567_METTH / Northeast Structural Genomics Consortium / NESG
Function / homologyArc Repressor Mutant - #60 / Arc Repressor Mutant / Orthogonal Bundle / Mainly Alpha / Uncharacterized protein
Function and homology information
Biological speciesMethanothermobacter thermautotrophicus str. Delta H (archaea)
MethodSOLUTION NMR / simulated annealing
Model detailslowest energy, model 1
AuthorsSwapna, G.V.T. / Gunsalus, X. / Huang, L. / Xiao, K. / Everett, J.K. / Acton, T.B. / Montelione, G.T. / Northeast Structural Genomics Consortium (NESG)
CitationJournal: To be Published
Title: NMR Solution Structure of a putative uncharacterized protein from Methanobacterium thermoautotrophicum, Northeast Structural Genomics Consortium Target:TR5
Authors: Swapna, G.V.T. / Gunsalus, X. / Huang, L. / Xiao, K. / Everett, J.K. / Acton, T.B. / Montelione, G.T.
History
DepositionJun 18, 2009Deposition site: BMRB / Processing site: RCSB
Revision 1.0Jul 14, 2009Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Feb 26, 2020Group: Derived calculations / Other
Category: pdbx_database_status / pdbx_struct_assembly / pdbx_struct_oper_list
Item: _pdbx_database_status.status_code_cs
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
B: Uncharacterized protein


Theoretical massNumber of molelcules
Total (without water)11,9462
Polymers11,9462
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


Mass: 5972.941 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Methanothermobacter thermautotrophicus str. Delta H (archaea)
Gene: MTH467, MTH_467 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)+ Magic / References: UniProt: O26567
Sequence detailsNMR ANALYSIS SHOWED THAT THE RESIDUE AT 53 AND 253 IS A GLU AND NOT AN ASP. THE DATABASE HAS TO BE ...NMR ANALYSIS SHOWED THAT THE RESIDUE AT 53 AND 253 IS A GLU AND NOT AN ASP. THE DATABASE HAS TO BE CORRECTED FOR THIS. GLU IS CORRECT.

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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 CBCA(CO)NH
1413D HN(CA)CB
1513D HBHA(CO)NH
1613D (H)CCH-TOCSY
1713D HNCO
1813D 1H-13C NOESY
1913D 1H-15N NOESY
11033D- X-filtered NOESY
11122D 1H-15N HSQC
11222D 1H-13C HSQC
11322D HNOE
NMR detailsText: The structure was determined using triple resonance NMR spectroscopy. Automated backbone assignments were made using AutoAssign software. Sidechain assignments were completed manually. The ...Text: The structure was determined using triple resonance NMR spectroscopy. Automated backbone assignments were made using AutoAssign software. Sidechain assignments were completed manually. The oligomeric state was confirmed to be a dimer from sedimentation-equilibrium analysis (Kd =0.3uM) and static light scattering experiment that reported 90% of the protein in a dimer with a molecular weight of 15.03kD.

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Sample preparation

Details
Solution-IDContentsSolvent system
10.4 mM [U-100% 13C; U-100% 15N] TR5, 95% H2O/5% D2O95% H2O/5% D2O
20.95 mM [U-10% 13C; U-100% 15N] TR5, 95% H2O/5% D2O95% H2O/5% D2O
30.4 mM [U-10% 13C; U-100% 15N] + unlabeled TR5 protein TR5, 95% H2O/5% D2O95% H2O/5% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
0.4 mMTR5-1-1[U-100% 13C; U-100% 15N]1
0.95 mMTR5-2-2[U-10% 13C; U-100% 15N]2
0.4 mMTR5-3-3[U-10% 13C; U-100% 15N] + unlabeled TR5 protein3
Sample conditionsIonic strength: 300 mM NaCl / pH: 6.5 / Pressure: 1 atm / Temperature: 303 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Varian INOVAVarianINOVA6001
Varian UnityVarianUNITY5002

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Processing

NMR software
NameVersionDeveloperClassification
AutoAssign2.2.1Zimmerman, Moseley, Kulikowski and Montelionechemical shift assignment
CYANA2.1Guntert, Mumenthaler and Wuthrichgeometry optimization
CNS2.0.6Brunger, Adams, Clore, Gros, Nilges and Readgeometry optimization
CNS2.0.6Brunger, Adams, Clore, Gros, Nilges and Readrefinement
RefinementMethod: simulated annealing / Software ordinal: 1
Details: Initial NOESY assignments for the monomer were made using AutoStructure and the solution structure of the dimer was obtained using CYANA-2.1. Constraints for the dimer interface were ...Details: Initial NOESY assignments for the monomer were made using AutoStructure and the solution structure of the dimer was obtained using CYANA-2.1. Constraints for the dimer interface were obtained from the x-filtered NOESY data and slow exchanging amides from H/D exchange. 100 structures were calculated and 20 best conformers were then refined in a shell of water using CNS. Initial dihedral angle constraints were obtained from TALOS. Final structure quality factors determined using PSVS software: ordered residues are defined as (10-53,212-236,238-251). (a)RMSD(ordered residues) all Backbone atoms: 0.7A; all heavy atoms: 1.0A. (b) Ramchandran statistics for all ordered residues: Most favoured region: 93.1%; Additionally allowed region: 6.6%; Generously allowed region 0.1% and disallowed region: 0.2%. (c) Procheck scores for all ordered residues (Raw/Z) phi-psi -0.11/-0.12; All: -0.07/-0.41; (d) MolProbity clash score (Raw/Z): 16.97/-1.39. (e) RPF scores for the goodness of fit to NOESY data: Recall :0.913; Precision:0.849; F-measure:0.88; Final DP score :0.703
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 100 / Conformers submitted total number: 20

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