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- PDB-7bj3: ScpA from Streptococcus pyogenes, S512A active site mutant -

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Basic information

Entry
Database: PDB / ID: 7bj3
TitleScpA from Streptococcus pyogenes, S512A active site mutant
ComponentsC5a peptidase
KeywordsHYDROLASE / C5a peptidase / virulence factor
Function / homology
Function and homology information


C5a peptidase / serine-type endopeptidase activity / proteolysis / membrane / metal ion binding
Similarity search - Function
CHU_C Type IX secretion signal domain / Fn3-like domain / C5a peptidase-like domain / Fn3-like domain / PA domain superfamily / PA domain / PA domain / Peptidase S8, subtilisin, His-active site / Serine proteases, subtilase family, histidine active site. / Serine proteases, subtilase family, aspartic acid active site. ...CHU_C Type IX secretion signal domain / Fn3-like domain / C5a peptidase-like domain / Fn3-like domain / PA domain superfamily / PA domain / PA domain / Peptidase S8, subtilisin, His-active site / Serine proteases, subtilase family, histidine active site. / Serine proteases, subtilase family, aspartic acid active site. / Peptidase S8, subtilisin, Asp-active site / Serine proteases, subtilase family, serine active site. / Peptidase S8, subtilisin, Ser-active site / Serine proteases, subtilase domain profile. / Peptidase S8, subtilisin-related / Peptidase S8/S53 domain superfamily / Subtilase family / Peptidase S8/S53 domain / Immunoglobulin-like fold
Similarity search - Domain/homology
MALONIC ACID / C5a peptidase
Similarity search - Component
Biological speciesStreptococcus pyogenes (bacteria)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 2.6 Å
AuthorsKagawa, T.F. / O'Connell, M.R. / Cooney, J.C.
Funding support Ireland, 3items
OrganizationGrant numberCountry
Irish Research CouncilRS/2005/222 Ireland
Science Foundation Ireland12/RC/2275_P2 Ireland
Enterprise IrelandCF/2013/3336 Ireland
Citation
Journal: Comput Struct Biotechnol J / Year: 2021
Title: Enzyme kinetic and binding studies identify determinants of specificity for the immunomodulatory enzyme ScpA, a C5a inactivating bacterial protease.
Authors: Tecza, M. / Kagawa, T.F. / Jain, M. / Cooney, J.C.
#1: Journal: Eur.J.Biochem. / Year: 2002
Title: Processing, stability, and kinetic parameters of C5a peptidase from Streptococcus pyogenes.
Authors: Anderson, E.T. / Wetherell, M.G. / Winter, L.A. / Olmsted, S.B. / Cleary, P.P. / Matsuka, Y.V.
#2: Journal: Acta Crystallogr D Struct Biol / Year: 2019
Title: Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix.
Authors: Dorothee Liebschner / Pavel V Afonine / Matthew L Baker / Gábor Bunkóczi / Vincent B Chen / Tristan I Croll / Bradley Hintze / Li Wei Hung / Swati Jain / Airlie J McCoy / Nigel W Moriarty ...Authors: Dorothee Liebschner / Pavel V Afonine / Matthew L Baker / Gábor Bunkóczi / Vincent B Chen / Tristan I Croll / Bradley Hintze / Li Wei Hung / Swati Jain / Airlie J McCoy / Nigel W Moriarty / Robert D Oeffner / Billy K Poon / Michael G Prisant / Randy J Read / Jane S Richardson / David C Richardson / Massimo D Sammito / Oleg V Sobolev / Duncan H Stockwell / Thomas C Terwilliger / Alexandre G Urzhumtsev / Lizbeth L Videau / Christopher J Williams / Paul D Adams /
Abstract: Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological ...Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological processes and to develop new therapeutics against diseases. The overall structure-solution workflow is similar for these techniques, but nuances exist because the properties of the reduced experimental data are different. Software tools for structure determination should therefore be tailored for each method. Phenix is a comprehensive software package for macromolecular structure determination that handles data from any of these techniques. Tasks performed with Phenix include data-quality assessment, map improvement, model building, the validation/rebuilding/refinement cycle and deposition. Each tool caters to the type of experimental data. The design of Phenix emphasizes the automation of procedures, where possible, to minimize repetitive and time-consuming manual tasks, while default parameters are chosen to encourage best practice. A graphical user interface provides access to many command-line features of Phenix and streamlines the transition between programs, project tracking and re-running of previous tasks.
History
DepositionJan 13, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 12, 2021Provider: repository / Type: Initial release
Revision 1.1May 19, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jan 31, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: C5a peptidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)111,47716
Polymers110,0151
Non-polymers1,46215
Water3,315184
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: surface plasmon resonance, 1:1 binding with substrate, isothermal titration calorimetry, 1:1 molar ratio with substrate
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2760 Å2
ΔGint-146 kcal/mol
Surface area37040 Å2
MethodPISA
Unit cell
Length a, b, c (Å)167.390, 167.390, 141.840
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number182
Space group name H-MP6322
Space group name HallP6c2c
Symmetry operation#1: x,y,z
#2: x-y,x,z+1/2
#3: y,-x+y,z+1/2
#4: -y,x-y,z
#5: -x+y,-x,z
#6: x-y,-y,-z
#7: -x,-x+y,-z
#8: -x,-y,z+1/2
#9: y,x,-z
#10: -y,-x,-z+1/2
#11: -x+y,y,-z+1/2
#12: x,x-y,-z+1/2

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Components

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Protein , 1 types, 1 molecules A

#1: Protein C5a peptidase / SCP


Mass: 110014.945 Da / Num. of mol.: 1 / Mutation: S512A
Source method: isolated from a genetically manipulated source
Details: Prior to crystallization, ScpA S512A was subjected to limited proteolysis by SpeB (cysteine protease from S. pyogenes) as described in Anderson et al. (2002). This removes the propepide, ...Details: Prior to crystallization, ScpA S512A was subjected to limited proteolysis by SpeB (cysteine protease from S. pyogenes) as described in Anderson et al. (2002). This removes the propepide, leaving K90 as the N-terminal residue.
Source: (gene. exp.) Streptococcus pyogenes (bacteria) / Gene: scpA / Plasmid: pGEX-6P-3 / Production host: Escherichia coli (E. coli) / Strain (production host): DH5alpha / References: UniProt: B6ETQ5, C5a peptidase

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Non-polymers , 6 types, 199 molecules

#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#3: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#4: Chemical ChemComp-EPE / 4-(2-HYDROXYETHYL)-1-PIPERAZINE ETHANESULFONIC ACID / HEPES


Mass: 238.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H18N2O4S / Comment: pH buffer*YM
#5: Chemical ChemComp-MLA / MALONIC ACID / DICARBOXYLIC ACID C3 / PROPANEDIOLIC ACID / METHANEDICARBOXYLIC ACID


Mass: 104.061 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H4O4
#6: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: SO4
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 184 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.61 Å3/Da / Density % sol: 52.82 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 7.5 / Details: 2.0 M Ammonium sulfate, 0.2 M Hepes/KOH pH 7.5

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Data collection

DiffractionMean temperature: 110 K / Serial crystal experiment: N
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU MICROMAX-007 / Wavelength: 1.5418 Å
DetectorType: RIGAKU RAXIS IV++ / Detector: IMAGE PLATE / Date: Jul 13, 2010
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 2.6→36.24 Å / Num. obs: 36554 / % possible obs: 100 % / Redundancy: 5.6 % / Biso Wilson estimate: 25.22 Å2 / CC1/2: 0.968 / Rpim(I) all: 0.125 / Rrim(I) all: 0.299 / Net I/σ(I): 7.5
Reflection shellResolution: 2.6→2.72 Å / Redundancy: 5.6 % / Mean I/σ(I) obs: 1.6 / Num. unique obs: 4386 / CC1/2: 0.499 / Rpim(I) all: 0.509 / Rrim(I) all: 1.213 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX1.17.1_3660refinement
MOSFLMdata reduction
Aimlessdata scaling
PHASERphasing
Cootmodel building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3EIF

3eif


Resolution: 2.6→36.24 Å / SU ML: 0.3634 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 25.1321
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflectionSelection details
Rfree0.2646 1825 5 %random
Rwork0.2063 34699 --
obs0.2092 36524 99.99 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 24.89 Å2
Refinement stepCycle: LAST / Resolution: 2.6→36.24 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6998 0 79 184 7261
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00227207
X-RAY DIFFRACTIONf_angle_d0.50759822
X-RAY DIFFRACTIONf_chiral_restr0.04061096
X-RAY DIFFRACTIONf_plane_restr0.00321290
X-RAY DIFFRACTIONf_dihedral_angle_d11.13722538
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.6-2.670.38071330.29882636X-RAY DIFFRACTION100
2.67-2.750.30651570.27732573X-RAY DIFFRACTION100
2.75-2.840.31451160.27162661X-RAY DIFFRACTION100
2.84-2.940.32521560.26652602X-RAY DIFFRACTION100
2.94-3.060.33491310.25752631X-RAY DIFFRACTION100
3.06-3.20.29931480.24392617X-RAY DIFFRACTION99.96
3.2-3.360.27171340.21572660X-RAY DIFFRACTION100
3.36-3.570.26021490.1962639X-RAY DIFFRACTION100
3.57-3.850.25091330.17762672X-RAY DIFFRACTION100
3.85-4.240.20311250.15772675X-RAY DIFFRACTION100
4.24-4.850.19341410.14552704X-RAY DIFFRACTION100
4.85-6.10.24391480.17392736X-RAY DIFFRACTION100
6.11-36.240.24421540.19622893X-RAY DIFFRACTION99.87

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