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- PDB-7am5: Crystal structure of Peptiligase mutant - L217H/M222P/A225N -

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Basic information

Entry
Database: PDB / ID: 7am5
TitleCrystal structure of Peptiligase mutant - L217H/M222P/A225N
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 / molecular replacement / Resolution: 2.3 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

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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)27,5143
Polymers27,4681
Non-polymers462
Water1,838102
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area250 Å2
ΔGint-21 kcal/mol
Surface area9900 Å2
Unit cell
Length a, b, c (Å)53.744, 59.993, 78.358
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

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


Mass: 27468.420 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus amyloliquefaciens (bacteria) / Plasmid: PBE-S D_1292111488 / Production host: BACILLUS SUBTILIS (bacteria) / Strain (production host): GX4935 / References: UniProt: P00782, subtilisin
#2: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Na
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 102 / 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: 2.2 Å3/Da / Density % sol: 43 %
Crystal growTemperature: 294 K / Method: vapor diffusion, sitting drop / pH: 7.5 / Details: 20% polyacrylic acid 5100

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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: Dec 17, 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: 2.3→44.3 Å / Num. obs: 11252 / % possible obs: 96.9 % / Redundancy: 4 % / CC1/2: 0.973 / Rmerge(I) obs: 0.207 / Rpim(I) all: 0.105 / Rrim(I) all: 0.234 / Net I/σ(I): 3.7
Reflection shellResolution: 2.3→2.38 Å / Redundancy: 3.9 % / Rmerge(I) obs: 0.61 / Num. unique obs: 1083 / CC1/2: 0.802 / Rpim(I) all: 0.33 / Rrim(I) all: 0.699 / % possible all: 96.9

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
XDSdata reduction
Aimless0.7.4data scaling
PHASERphasing
REFMAC5.8.0258refinement
PDB_EXTRACT3.25data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: D_129211609

Resolution: 2.3→44.3 Å / Cor.coef. Fo:Fc: 0.914 / Cor.coef. Fo:Fc free: 0.878 / SU B: 10.221 / SU ML: 0.242 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.475 / ESU R Free: 0.295 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.2867 549 4.9 %RANDOM
Rwork0.23 ---
obs0.2327 10656 95.39 %-
Solvent computationIon probe radii: 0.7 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1 Å / Solvent model: MASK
Displacement parametersBiso max: 76.94 Å2 / Biso mean: 29.531 Å2 / Biso min: 14.09 Å2
Baniso -1Baniso -2Baniso -3
1--0.73 Å2-0 Å2-0 Å2
2---1.26 Å20 Å2
3---1.98 Å2
Refinement stepCycle: final / Resolution: 2.3→44.3 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1897 0 2 102 2001
Biso mean--29.33 31.96 -
Num. residues----268
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0040.0131943
X-RAY DIFFRACTIONr_bond_other_d0.0010.0171725
X-RAY DIFFRACTIONr_angle_refined_deg1.2921.6162652
X-RAY DIFFRACTIONr_angle_other_deg1.2221.5664022
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.4755267
X-RAY DIFFRACTIONr_dihedral_angle_2_deg37.05925.07269
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.85415269
X-RAY DIFFRACTIONr_dihedral_angle_4_deg9.427152
X-RAY DIFFRACTIONr_chiral_restr0.0460.2263
X-RAY DIFFRACTIONr_gen_planes_refined0.0040.022241
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02357
LS refinement shellResolution: 2.3→2.36 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.368 42 -
Rwork0.32 762 -
all-804 -
obs--95.71 %

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