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- PDB-2k1g: Solution NMR structure of lipoprotein spr from Escherichia coli K... -

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Basic information

Entry
Database: PDB / ID: 2k1g
TitleSolution NMR structure of lipoprotein spr from Escherichia coli K12. Northeast Structural Genomics target ER541-37-162
ComponentsLipoprotein spr
KeywordsLIPOPROTEIN / solution NMR structure / bacterial lipoprotein / cysteine peptidase / NplC/P60 family / construct optimized / Membrane / Palmitate / Structural Genomics / PSI-2 / Protein Structure Initiative / Northeast Structural Genomics Consortium / NESG
Function / homology
Function and homology information


muramoyltetrapeptide carboxypeptidase / muramoyltetrapeptide carboxypeptidase activity / capsule polysaccharide biosynthetic process / peptidoglycan metabolic process / peptidoglycan turnover / Hydrolases; Acting on peptide bonds (peptidases) / cysteine-type peptidase activity / cell outer membrane / cell wall organization / endopeptidase activity / proteolysis
Similarity search - Function
: / NlpC/P60 family / NlpC/P60 domain profile. / Endopeptidase, NLPC/P60 domain / endopeptidase domain like (from Nostoc punctiforme) / endopeptidase fold (from Nostoc punctiforme) / Papain-like cysteine peptidase superfamily / Prokaryotic membrane lipoprotein lipid attachment site profile. / Alpha-Beta Complex / Alpha Beta
Similarity search - Domain/homology
Murein DD-endopeptidase MepS/Murein LD-carboxypeptidase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodSOLUTION NMR / simulated annealing
AuthorsAramini, J.M. / Rossi, P. / Zhao, L. / Jiang, M. / Maglaqui, M. / Xiao, R. / Liu, J. / Baran, M.C. / Swapna, G.V.T. / Huang, Y.J. ...Aramini, J.M. / Rossi, P. / Zhao, L. / Jiang, M. / Maglaqui, M. / Xiao, R. / Liu, J. / Baran, M.C. / Swapna, G.V.T. / Huang, Y.J. / Acton, T.B. / Rost, B. / Montelione, G.T. / Northeast Structural Genomics Consortium (NESG)
CitationJournal: Biochemistry / Year: 2008
Title: Solution NMR structure of the NlpC/P60 domain of lipoprotein Spr from Escherichia coli: structural evidence for a novel cysteine peptidase catalytic triad.
Authors: Aramini, J.M. / Rossi, P. / Huang, Y.J. / Zhao, L. / Jiang, M. / Maglaqui, M. / Xiao, R. / Locke, J. / Nair, R. / Rost, B. / Acton, T.B. / Inouye, M. / Montelione, G.T.
History
DepositionMar 3, 2008Deposition site: BMRB / Processing site: RCSB
Revision 1.0Mar 18, 2008Provider: repository / Type: Initial release
SupersessionMar 25, 2008ID: 2JYX
Revision 1.1Jul 13, 2011Group: Source and taxonomy / Version format compliance
Revision 1.2May 1, 2024Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_nmr_software / pdbx_nmr_spectrometer / struct_ref_seq_dif
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 / _struct_ref_seq_dif.details

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Lipoprotein spr


Theoretical massNumber of molelcules
Total (without water)15,5851
Polymers15,5851
Non-polymers00
Water00
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 Lipoprotein spr


Mass: 15584.570 Da / Num. of mol.: 1 / Fragment: Residues 63-188
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K12 / Gene: spr, yeiV, b2175, JW2163 / Plasmid: pET21 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)MGK / References: UniProt: P0AFV4

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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 HN(COCA)CB
1613D HN(CA)CB
1713D HBHA(CO)NH
1813D (H)CCH-COSY
1913D simultaneous CN NOESY
11013D CCH-TOCSY aromatic
11123D 1H-13C NOESY aromatic
11223D simultaneous CN NOESY
11313D CC(CO)NH TOCSY
11413D (H)CCH-TOCSY
11513D CCH-TOCSY aliphatic
11613D HNHA
11732D 1H-13C high res. (L/V stereo assignment)
11832D 1H-15N hetNOE
NMR detailsText: THE PROTEIN IS MONOMERIC BY GEL FILTRATION CHROMATOGRAPHY AND STATIC LIGHT SCATTERING. THE STRUCTURE WAS DETERMINED USING TRIPLE RESONANCE NMR SPECTROSCOPY. AUTOMATED BACKBONE ASSIGNMENTS WERE ...Text: THE PROTEIN IS MONOMERIC BY GEL FILTRATION CHROMATOGRAPHY AND STATIC LIGHT SCATTERING. 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. 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. COMPLETENESS OF NMR ASSIGNMENTS (EXCLUDING C-TERMINAL HHHHHH): BACKBONE, 97.9%, SIDE CHAIN, 94.2%, AROMATICS, 91.8%, STEREOSPECIFIC METHYL, 100%, STEREOSPECIFIC SIDE CHAIN NH2: 83.3%. FINAL STRUCTURE QUALITY FACTORS (FOR RESIDUES 37 TO 162, PSVS 1.3), WHERE ORDERED RESIDUES [S(PHI) + S(PSI) > 1.8] COMPRISE: 38-58,61-93,96-113,118-124,128-159: (A) RMSD (ORDERED RESIDUES): BB, 0.5, HEAVY ATOM, 1.0. (B) RAMACHANDRAN STATISTICS FOR ORDERED RESIDUES: MOST FAVORED, 88.4%, ADDITIONALLY ALLOWED, 11.5%, GENEROUSLY ALLOWED, 0.1%, DISALLOWED, 0.0%. (C) PROCHECK SCORES FOR ORDERED RESIDUES (RAW/Z-): PHI-PSI, -0.46/-1.49, ALL, -0.27/-1.60. (D) MOLPROBITY CLASH SCORE (RAW/Z-): 18.98/-1.73. (E) RPF SCORES FOR GOODNESS OF FIT TO NOESY DATA (RESIDUES 37-162): RECALL, 0.985, PRECISION, 0.925, F-MEASURE, 0.954, DP-SCORE, 0.816. (F) NUMBER OF CLOSE CONTACTS PER 20 MODELS: 8. 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]: 37,59-60,94-95,114-117,125-127,160-162.

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Sample preparation

Details
Solution-IDContentsSolvent system
11.07 mM [U-100% 13C; U-100% 15N] ER541-37-162, 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
21.07 mM [U-100% 13C; U-100% 15N] ER541-37-162, 20 mM MES, 100 mM sodium chloride, 10 mM DTT, 5 mM calcium chloride, 0.02 % sodium azide, 100% D2O100% D2O
31.03 mM [U-5% 13C; U-100% 15N] ER541-37-162, 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
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
1.07 mMER541-37-162[U-100% 13C; U-100% 15N]1
20 mMMES1
100 mMsodium chloride1
10 mMDTT1
5 mMcalcium chloride1
0.02 %sodium azide1
1.07 mMER541-37-162[U-100% 13C; U-100% 15N]2
20 mMMES2
100 mMsodium chloride2
10 mMDTT2
5 mMcalcium chloride2
0.02 %sodium azide2
1.03 mMER541-37-162[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: 298 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Bruker AvanceBrukerAVANCE8001
Varian INOVAVarianINOVA6002

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Processing

NMR software
NameVersionDeveloperClassification
TopSpin1.3Bruker Biospincollection
VNMR6.1CVariancollection
AutoAssign2.4.0Zimmerman, Moseley, Kulikowski and Montelionechemical shift assignment
NMRPipe2.3Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
Sparky3.11Goddardpeak picking
Sparky3.11Goddarddata analysis
CYANA2.1Guntert, Mumenthaler and Wuthrichstructure solution
CNS1.2Brunger, Adams, Clore, Gros, Nilges and Readrefinement
CNS1.2Brunger, Adams, Clore, Gros, Nilges and Readstructure solution
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 2525 CONFORMATIONALLY-RESTRICTING NOE-DERIVED DISTANCE CONSTRAINTS, 138 DIHEDRAL ANGLE CONSTRAINTS, AND 48 HYDROGEN BOND CONSTRAINTS (21.0 ...Details: THE FINAL STRUCTURES ARE BASED ON A TOTAL OF 2525 CONFORMATIONALLY-RESTRICTING NOE-DERIVED DISTANCE CONSTRAINTS, 138 DIHEDRAL ANGLE CONSTRAINTS, AND 48 HYDROGEN BOND CONSTRAINTS (21.0 CONSTRAINTS PER RESIDUE, 7.1 LONG RANGE CONSTRAINTS PER RESIDUE, COMPUTED FOR RESIDUES 37 TO 162 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, AND USING NEUTRAL HISTIDINES AT POSITIONS 119 and 131.
NMR constraintsNOE constraints total: 2525 / NOE intraresidue total count: 696 / NOE long range total count: 890 / NOE medium range total count: 383 / NOE sequential total count: 556 / Hydrogen bond constraints total count: 48 / Protein phi angle constraints total count: 69 / Protein psi angle constraints total count: 69
NMR representativeSelection criteria: lowest energy
NMR ensembleAverage torsion angle constraint violation: 0.2 °
Conformer selection criteria: structures with the lowest energy
Conformers calculated total number: 100 / Conformers submitted total number: 20 / Maximum torsion angle constraint violation: 2.7 ° / Maximum upper distance constraint violation: 0.41 Å / Torsion angle constraint violation method: PSVS
NMR ensemble rmsDistance rms dev: 0.01 Å

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