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- PDB-2kl6: Solution NMR structure of the CARDB domain of PF1109 from Pyrococ... -

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Basic information

Entry
Database: PDB / ID: 2kl6
TitleSolution NMR structure of the CARDB domain of PF1109 from Pyrococcus furiosus. Northeast Structural Genomics Consortium target PfR193A
ComponentsUncharacterized protein
KeywordsSTRUCTURAL GENOMICS / UNKNOWN FUNCTION / solution NMR structure / CARDB domain / PFAM 07705 / NESG / PSI-2 / Protein Structure Initiative / Northeast Structural Genomics Consortium
Function / homologyCARDB domain / CARDB / Immunoglobulins / Immunoglobulin-like fold / Immunoglobulin-like / Sandwich / Mainly Beta / metal ion binding / CARDB domain-containing protein
Function and homology information
Biological speciesPyrococcus furiosus (archaea)
MethodSOLUTION NMR / simulated annealing
Model detailslowest energy, model 1
AuthorsAramini, J.M. / Lee, D. / Ciccosanti, C. / Hamilton, K. / Nair, R. / Rost, B. / Acton, T.B. / Xiao, R. / Swapna, G.V.T. / Everett, J.K. ...Aramini, J.M. / Lee, D. / Ciccosanti, C. / Hamilton, K. / Nair, R. / Rost, B. / Acton, T.B. / Xiao, R. / Swapna, G.V.T. / Everett, J.K. / Montelione, G.T. / Northeast Structural Genomics Consortium (NESG)
CitationJournal: To be Published
Title: Solution NMR structure of the CARDB domain of PF1109 from Pyrococcus furiosus. Northeast Structural Genomics Consortium target PfR193A
Authors: Aramini, J.M. / Lee, D. / Ciccosanti, C. / Hamilton, K. / Nair, R. / Rost, B. / Acton, T.B. / Xiao, R. / Swapna, G.V.T. / Everett, J.K. / Montelione, G.T.
History
DepositionJun 30, 2009Deposition site: BMRB / Processing site: RCSB
Revision 1.0Aug 25, 2009Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Feb 26, 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 1.3Oct 13, 2021Group: Database references / Category: database_2 / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details
Revision 1.4Jun 14, 2023Group: Other / Category: pdbx_database_status / Item: _pdbx_database_status.status_code_nmr_data
Revision 1.5May 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


Theoretical massNumber of molelcules
Total (without water)12,8681
Polymers12,8681
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 / 100structures with the lowest energy
RepresentativeModel #1lowest energy

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Components

#1: Protein Uncharacterized protein


Mass: 12868.411 Da / Num. of mol.: 1 / Fragment: sequence database residues 436-540 / Mutation: N68S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus (archaea) / Gene: PF1109 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)MGK / References: UniProt: Q8U1U6

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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 1H-15N NOESY
1413D 1H-13C NOESY aliphatic
1513D 1H-13C NOESY aromatic
1622D 1H-13C HSQC high resolution (L/V methyl stereoassignment)
1713D HNCO
1813D HN(CA)CO
1913D HN(CO)CA
11013D HNCA
11113D CBCA(CO)NH
11213D HN(CA)CB
11313D HBHA(CO)NH
11413D (H)CCH-TOCSY
11513D (H)CCH-COSY
11613D CCH-TOCSY
11713D HNHA
11812D 1H-15N hetNOE
11911D 15N T1 and T2
NMR detailsText: THE PROTEIN IS PREDOMINANTLY MONOMERIC BY GEL FILTRATION CHROMATOGRAPHY, STATIC LIGHT SCATTERING AND 15N T1/T2 RELAXATION. THE STRUCTURE WAS DETERMINED USING TRIPLE RESONANCE NMR SPECTROSCOPY. ...Text: THE PROTEIN IS PREDOMINANTLY 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 PINE, AND THE SIDE CHAIN ASSIGNMENTS WERE COMPLETED MANUALLY. COMPLETENESS OF NMR ASSIGNMENTS (EXCLUDING C-TERMINAL HHHHHH): BACKBONE, 98.7%, SIDE CHAIN, 99.0%, AROMATICS, 98.8%, STEREOSPECIFIC METHYL, 88.5%, STEREOSPECIFIC SIDE CHAIN NH2: 100%. THE C-TERMINAL HIS TAG RESIDUES OF THE PROTEIN (HHHHHH) WERE NOT INCLUDED IN THE STRUCTURE CALCULATIONS AND HAVE BEEN OMITTED FROM THIS DEPOSITION.

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Sample preparation

Details
Solution-IDContentsSolvent system
10.65 mM [U-100% 13C; U-100% 15N] PfR193A, 20 mM MES, 200 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS, 90% H2O/10% D2O90% H2O/10% D2O
20.43 mM [U-5% 13C; U-100% 15N] PfR193A, 20 mM MES, 200 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS, 90% H2O/10% D2O90% H2O/10% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
0.65 mMPfR193A-1[U-100% 13C; U-100% 15N]1
20 mMMES-21
200 mMsodium chloride-31
5 mMcalcium chloride-41
10 mMDTT-51
0.02 %sodium azide-61
50 uMDSS-71
0.43 mMPfR193A-8[U-5% 13C; U-100% 15N]2
20 mMMES-92
200 mMsodium chloride-102
5 mMcalcium chloride-112
10 mMDTT-122
0.02 %sodium azide-132
50 uMDSS-142
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
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
TALOSplusCornilescu, Delaglio and Baxdihedral angle constraints
RefinementMethod: simulated annealing / Software ordinal: 1
Details: THE FINAL STRUCTURES ARE BASED ON A TOTAL OF 2722 CONFORMATIONALLY-RESTRICTING NOE-DERIVED DISTANCE CONSTRAINTS AND 166 DIHEDRAL ANGLE CONSTRAINTS; 0 HYDROGEN BOND CONSTRAINTS (26.7 ...Details: THE FINAL STRUCTURES ARE BASED ON A TOTAL OF 2722 CONFORMATIONALLY-RESTRICTING NOE-DERIVED DISTANCE CONSTRAINTS AND 166 DIHEDRAL ANGLE CONSTRAINTS; 0 HYDROGEN BOND CONSTRAINTS (26.7 CONSTRAINTS PER RESIDUE, 11.5 LONG RANGE CONSTRAINTS PER RESIDUE, COMPUTED FOR RESIDUES 1 TO 108 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. AUTOMATIC NOESY ASSIGNMENTS WERE DETERMINED USING CYANA 3.0. BACKBONE (PHI/PSI) DIHEDRAL ANGLE CONSTRAINTS WERE OBTAINED FROM TALOSplus. FINAL STRUCTURE QUALITY FACTORS (FOR RESIDUES 1 TO 108, PSVS 1.3), WHERE ORDERED RESIDUES [S(PHI) + S(PSI) > 1.8] COMPRISE: 2-107: (A) RMSD (ORDERED RESIDUES): BB, 0.4, HEAVY ATOM, 0.6. (B) RAMACHANDRAN STATISTICS FOR ORDERED RESIDUES: MOST FAVORED, 93.3%, ADDITIONALLY ALLOWED, 6.5%, GENEROUSLY ALLOWED, 0.2%, DISALLOWED, 0.0%. (C) PROCHECK SCORES FOR ORDERED RESIDUES (RAW/Z-): PHI-PSI, -0.42/-1.34, ALL, -0.27/-1.60. (D) MOLPROBITY CLASH SCORE (RAW/Z-): 15.99/-1.22 (E) RPF SCORES FOR GOODNESS OF FIT TO NOESY DATA (RESIDUES 1-108): RECALL, 0.963, PRECISION, 0.951, F-MEASURE, 0.957, DP-SCORE, 0.861. (F) NUMBER OF CLOSE CONTACTS PER 20 MODELS: 4.
NMR constraintsNOE constraints total: 2722 / NOE intraresidue total count: 523 / NOE long range total count: 1239 / NOE medium range total count: 271 / NOE sequential total count: 689 / Protein phi angle constraints total count: 82 / Protein psi angle constraints total count: 84
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: 9.6 ° / Maximum upper distance constraint violation: 0.22 Å
NMR ensemble rmsDistance rms dev: 0.01 Å

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