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- PDB-2kpn: Solution NMR structure of a Bacterial Ig-like (Big_3) domain from... -

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Basic information

Entry
Database: PDB / ID: 2kpn
TitleSolution NMR structure of a Bacterial Ig-like (Big_3) domain from Bacillus cereus. Northeast Structural Genomics Consortium target BcR147A
ComponentsBacillolysin
KeywordsHYDROLASE / solution NMR structure / Big_3 domain / PF07523 / PSI BIG-OPEN target / NESG / Cell wall / Peptidoglycan-anchor / Structural Genomics / Protein Structure Initiative / Northeast Structural Genomics Consortium
Function / homology
Function and homology information


bacillolysin / cell wall / metalloendopeptidase activity / membrane => GO:0016020 / extracellular region
Similarity search - Function
Pesticidal crystal protein Cry22Aa, Ig-like domain / Bacterial surface protein, Ig-like domain / : / Peptidase M4, C-terminal / FTP domain / Peptidase M4 domain / Peptidase M4 / Thermolysin metallopeptidase, catalytic domain / Thermolysin metallopeptidase, alpha-helical domain / Fungalysin/Thermolysin Propeptide Motif ...Pesticidal crystal protein Cry22Aa, Ig-like domain / Bacterial surface protein, Ig-like domain / : / Peptidase M4, C-terminal / FTP domain / Peptidase M4 domain / Peptidase M4 / Thermolysin metallopeptidase, catalytic domain / Thermolysin metallopeptidase, alpha-helical domain / Fungalysin/Thermolysin Propeptide Motif / LPXTG cell wall anchor motif / Gram-positive cocci surface proteins LPxTG motif profile. / LPXTG cell wall anchor domain / Peptidase M4/M1, CTD superfamily / Immunoglobulins / Immunoglobulin-like fold / Immunoglobulin-like / Sandwich / Mainly Beta
Similarity search - Domain/homology
Biological speciesBacillus cereus ATCC 14579 (bacteria)
MethodSOLUTION NMR / simulated annealing
Model detailslowest energy, model 1
AuthorsAramini, J.M. / Wang, D. / Ciccosanti, C.T. / Janjua, H. / 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 a Bacterial Ig-like (Big_3) domain from Bacillus cereus. Northeast Structural Genomics Consortium target BcR147A
Authors: Aramini, J.M. / Wang, D. / Ciccosanti, C.T. / Janjua, H. / Rost, B. / Acton, T.B. / Xiao, R. / Swapna, G.V.T. / Everett, J.K. / Montelione, G.T.
History
DepositionOct 16, 2009Deposition site: BMRB / Processing site: RCSB
Revision 1.0Jan 12, 2010Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Nov 23, 2011Group: Structure summary
Revision 1.3Jun 27, 2018Group: Data collection / Database references / Category: struct_ref_seq_dif / Item: _struct_ref_seq_dif.details

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Bacillolysin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)11,2852
Polymers11,2451
Non-polymers401
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 Bacillolysin /


Mass: 11244.577 Da / Num. of mol.: 1 / Fragment: sequence database residues 658-753
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus cereus ATCC 14579 (bacteria) / Strain: ATCC 14579 / DSM 31 / Gene: BC_2506 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)MGK / References: UniProt: Q81D73, bacillolysin
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca

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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)
1722D 1H-15N hetNOE
1813D HNCO
1913D HN(CA)CO
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
11711D 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. 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 CRYOPROBE. BACKBONE ASSIGNMENTS WERE MADE USING 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 TALOSplus. COMPLETENESS OF NMR ASSIGNMENTS (EXCLUDING C-TERMINAL HHHHHH): BACKBONE, 97.5%, SIDE CHAIN, 99.2%, AROMATICS, 100%, STEREOSPECIFIC METHYL, 90%, STEREOSPECIFIC SIDE CHAIN NH2: 66.7%. FINAL STRUCTURE QUALITY FACTORS (FOR RESIDUE NUMBERS 1 TO 97, PSVS 1.3), WHERE ORDERED RESIDUES [S(PHI) + S(PSI) > 1.8] COMPRISE: 7-85: (A) RMSD (ORDERED RESIDUES): BB, 0.5, HEAVY ATOM, 0.8. (B) RAMACHANDRAN STATISTICS FOR ORDERED RESIDUES: MOST FAVORED, 96.3%, ADDITIONALLY ALLOWED, 3.7%, GENEROUSLY ALLOWED, 0.0%, DISALLOWED, 0.0%. (C) PROCHECK SCORES FOR ORDERED RESIDUES (RAW/Z-): PHI-PSI, -0.45/-1.46, ALL, -0.27/-1.60. (D) MOLPROBITY CLASH SCORE (RAW/Z-): 14.17/-0.91 (E) RPF SCORES FOR GOODNESS OF FIT TO NOESY DATA (RESIDUES 1-97): RECALL, 0.979, PRECISION, 0.939, F-MEASURE, 0.959, DP-SCORE, 0.854. (F) NUMBER OF CLOSE CONTACTS PER 20 MODELS: 3. THE C-TERMINAL HIS TAG RESIDUES OF THE PROTEIN (HHHHHH) WERE NOT INCLUDED IN THE STRUCTURE CALCULATIONS AND HAVE BEEN OMITTED FROM THIS DEPOSITION. THE LIGANDS INVOLVED IN THE CALCIUM ION COORDINATION SPHERE ARE CONSISTENT WITH CHANGES IN THE NH-HSQC SPECTRUM UPON REMOVAL/ADDITION OF CA2+.

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Sample preparation

Details
Solution-IDContentsSolvent system
10.94 mM [U-100% 13C; U-100% 15N] BcR147A, 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
20.78 mM [U-5% 13C; U-100% 15N] BcR147A, 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
0.94 mMBcR147A-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
0.78 mMBcR147A-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 1806 CONFORMATIONALLY-RESTRICTING NOE-DERIVED DISTANCE CONSTRAINTS, 132 DIHEDRAL ANGLE CONSTRAINTS, AND 9 METAL-OXYGEN DISTANCE CONSTRAINTS (20.8 ...Details: THE FINAL STRUCTURES ARE BASED ON A TOTAL OF 1806 CONFORMATIONALLY-RESTRICTING NOE-DERIVED DISTANCE CONSTRAINTS, 132 DIHEDRAL ANGLE CONSTRAINTS, AND 9 METAL-OXYGEN DISTANCE CONSTRAINTS (20.8 CONSTRAINTS PER RESIDUE, 7.3 LONG RANGE CONSTRAINTS PER RESIDUE, COMPUTED FOR RESIDUES 1 TO 97 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.
NMR constraintsNOE constraints total: 1806 / NOE intraresidue total count: 419 / NOE long range total count: 683 / NOE medium range total count: 179 / NOE sequential total count: 525 / Protein chi angle constraints total count: 1 / Protein other angle constraints total count: 1 / Protein phi angle constraints total count: 64 / Protein psi angle constraints total count: 66
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: 6.2 ° / Maximum upper distance constraint violation: 0.26 Å
NMR ensemble rmsDistance rms dev: 0.01 Å

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