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- PDB-7am3: Crystal structure of Peptiligase mutant - M222P -

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Basic information

Entry
Database: PDB / ID: 7am3
TitleCrystal structure of Peptiligase mutant - M222P
ComponentsSubtilisin BPN'
KeywordsLIGASE / subtilisin / peptide ligase
Function / homology
Function and homology information


subtilisin / sporulation resulting in formation of a cellular spore / fibrinolysis / serine-type endopeptidase activity / proteolysis / extracellular region / metal ion binding
Similarity search - Function
Subtilisin Carlsberg-like catalytic domain / Peptidase S8 propeptide/proteinase inhibitor I9 / Peptidase inhibitor I9 / Peptidase S8 propeptide/proteinase inhibitor I9 superfamily / 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 ...Subtilisin Carlsberg-like catalytic domain / Peptidase S8 propeptide/proteinase inhibitor I9 / Peptidase inhibitor I9 / Peptidase S8 propeptide/proteinase inhibitor I9 superfamily / 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
Similarity search - Domain/homology
Biological speciesBacillus amyloliquefaciens (bacteria)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 1.61 Å
AuthorsRozeboom, H.J. / Janssen, D.J.
CitationJournal: Comput Struct Biotechnol J / Year: 2021
Title: From thiol-subtilisin to omniligase: Design and structure of a broadly applicable peptide ligase.
Authors: Toplak, A. / Teixeira de Oliveira, E.F. / Schmidt, M. / Rozeboom, H.J. / Wijma, H.J. / Meekels, L.K.M. / de Visser, R. / Janssen, D.B. / Nuijens, T.
History
DepositionOct 8, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 17, 2021Provider: repository / Type: Initial release
Revision 1.1Mar 24, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
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

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Assembly

Deposited unit
A: Subtilisin BPN'
hetero molecules


Theoretical massNumber of molelcules
Total (without water)27,9777
Polymers27,4161
Non-polymers5616
Water5,819323
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1100 Å2
ΔGint-22 kcal/mol
Surface area9950 Å2
Unit cell
Length a, b, c (Å)58.489, 58.489, 126.005
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number92
Space group name H-MP41212
Components on special symmetry positions
IDModelComponents
11A-710-

HOH

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Components

#1: Protein Subtilisin BPN' / Alkaline protease / Subtilisin DFE / Subtilisin Novo


Mass: 27416.408 Da / Num. of mol.: 1
Mutation: Q2K S3C P5S S9A I31L S212C P216A M50F A73L DELTA75-83 E156S G166S G169A S188P Q206C N212G K217L N218S S221A T254A Q271E M222P
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus amyloliquefaciens (bacteria) / Gene: apr / Plasmid: PBE-S D_1292111488 / Production host: BACILLUS SUBTILIS (bacteria) / Strain (production host): GX4935 / References: UniProt: P00782, subtilisin
#2: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C3H8O3
#3: Chemical ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: SO4
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 323 / Source method: isolated from a natural source / Formula: H2O
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: 1.8 Å3/Da / Density % sol: 37.42 %
Crystal growTemperature: 294 K / Method: vapor diffusion, hanging drop / pH: 6.5 / Details: 1.4 M MgSO4 , 0.1 M MES pH 6.5.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: ROTATING ANODE / Type: BRUKER AXS MICROSTAR-H / Wavelength: 1.5418 Å
DetectorType: MARRESEARCH / Detector: IMAGE PLATE / Date: Jan 7, 2019
RadiationMonochromator: HELIOS MX MIRRORS / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 1.61→42.86 Å / Num. obs: 29222 / % possible obs: 99.6 % / Redundancy: 7.6 % / CC1/2: 0.999 / Rmerge(I) obs: 0.05 / Rpim(I) all: 0.019 / Rrim(I) all: 0.054 / Net I/σ(I): 26
Reflection shellResolution: 1.61→1.64 Å / Redundancy: 6.6 % / Rmerge(I) obs: 0.197 / Num. unique obs: 1315 / CC1/2: 0.982 / Rpim(I) all: 0.081 / Rrim(I) all: 0.214 / % possible all: 91.6

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Processing

Software
NameVersionClassification
Aimless0.7.3data scaling
REFMAC5.8.0258refinement
PDB_EXTRACT3.25data extraction
XDSdata reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5OX2

5ox2


Resolution: 1.61→42.86 Å / Cor.coef. Fo:Fc: 0.972 / Cor.coef. Fo:Fc free: 0.962 / SU B: 2.286 / SU ML: 0.042 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.078 / ESU R Free: 0.075 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: U VALUES : WITH TLS ADDED HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.1591 1443 5 %RANDOM
Rwork0.134 ---
obs0.1352 27701 99.61 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 59.08 Å2 / Biso mean: 13.749 Å2 / Biso min: 5.85 Å2
Baniso -1Baniso -2Baniso -3
1-0.02 Å2-0 Å2-0 Å2
2--0.02 Å2-0 Å2
3----0.05 Å2
Refinement stepCycle: final / Resolution: 1.61→42.86 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1898 0 34 323 2255
Biso mean--40.21 25.85 -
Num. residues----269
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.010.0132042
X-RAY DIFFRACTIONr_bond_other_d0.0010.0171803
X-RAY DIFFRACTIONr_angle_refined_deg1.5851.6132799
X-RAY DIFFRACTIONr_angle_other_deg1.6051.5664218
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.8695289
X-RAY DIFFRACTIONr_dihedral_angle_2_deg38.76725.07567
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.25915278
X-RAY DIFFRACTIONr_dihedral_angle_4_deg13.827152
X-RAY DIFFRACTIONr_chiral_restr0.0770.2274
X-RAY DIFFRACTIONr_gen_planes_refined0.0090.022385
X-RAY DIFFRACTIONr_gen_planes_other0.0030.02377
LS refinement shellResolution: 1.61→1.65 Å / Rfactor Rfree error: 0
RfactorNum. reflection% reflection
Rfree0.213 109 -
Rwork0.171 1903 -
obs--94.73 %
Refinement TLS params.Method: refined / Origin x: 14.93 Å / Origin y: -12.704 Å / Origin z: 4.107 Å
111213212223313233
T0.0085 Å2-0.0011 Å2-0.0017 Å2-0.0011 Å2-0.0006 Å2--0.0191 Å2
L0.6229 °20.2067 °2-0.1051 °2-0.654 °2-0.1065 °2--0.3072 °2
S-0.015 Å °0.0011 Å °-0.0423 Å °-0.023 Å °0.0174 Å °-0.0104 Å °0.0117 Å °0.0092 Å °-0.0025 Å °

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