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- PDB-7am4: Crystal structure of Peptiligase mutant - L217H/M222P -

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Basic information

Entry
Database: PDB / ID: 7am4
TitleCrystal structure of Peptiligase mutant - L217H/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: 1.81 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)28,1028
Polymers27,4411
Non-polymers6617
Water4,216234
1


  • Idetical with deposited unit
  • defined by software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1370 Å2
ΔGint-48 kcal/mol
Surface area9780 Å2
MethodPISA
Unit cell
Length a, b, c (Å)58.371, 58.371, 125.288
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-628-

HOH

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Components

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


Mass: 27441.396 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-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


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


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


Mass: 18.015 Da / Num. of mol.: 234 / 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: 30 %
Crystal growTemperature: 298 K / Method: vapor diffusion, sitting drop / pH: 6.5 / Details: 1.6 magnesium sulfate, 0.1 M MES

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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 8, 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: 1.81→42.7 Å / Num. obs: 20564 / % possible obs: 99.7 % / Redundancy: 7.6 % / CC1/2: 0.999 / Rmerge(I) obs: 0.057 / Rpim(I) all: 0.022 / Rrim(I) all: 0.062 / Net I/σ(I): 23.8
Reflection shellResolution: 1.81→1.85 Å / Redundancy: 6.8 % / Rmerge(I) obs: 0.288 / Num. unique obs: 1127 / CC1/2: 0.959 / Rpim(I) all: 0.118 / Rrim(I) all: 0.312 / % possible all: 94.9

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Processing

Software
NameVersionClassification
REFMAC5.8.0258refinement
XDSdata reduction
Aimless0.7.3data scaling
PDB_EXTRACT3.25data extraction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: D1292111488

Resolution: 1.81→42.7 Å / Cor.coef. Fo:Fc: 0.968 / Cor.coef. Fo:Fc free: 0.953 / SU B: 3.91 / SU ML: 0.063 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.117 / ESU R Free: 0.108 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: U VALUES : WITH TLS ADDED HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.1718 994 4.8 %RANDOM
Rwork0.136 ---
obs0.1377 19505 99.68 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 81.39 Å2 / Biso mean: 15.941 Å2 / Biso min: 8.31 Å2
Baniso -1Baniso -2Baniso -3
1--0.53 Å20 Å20 Å2
2---0.53 Å20 Å2
3---1.05 Å2
Refinement stepCycle: final / Resolution: 1.81→42.7 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1900 0 38 234 2172
Biso mean--49.43 27.03 -
Num. residues----269
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.010.0132082
X-RAY DIFFRACTIONr_bond_other_d0.0010.0171813
X-RAY DIFFRACTIONr_angle_refined_deg1.5951.6132859
X-RAY DIFFRACTIONr_angle_other_deg1.5591.5664248
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.7445294
X-RAY DIFFRACTIONr_dihedral_angle_2_deg39.85225.27872
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.49315285
X-RAY DIFFRACTIONr_dihedral_angle_4_deg12.309152
X-RAY DIFFRACTIONr_chiral_restr0.0760.2278
X-RAY DIFFRACTIONr_gen_planes_refined0.0090.022442
X-RAY DIFFRACTIONr_gen_planes_other0.0020.02384
LS refinement shellResolution: 1.81→1.855 Å / Rfactor Rfree error: 0
RfactorNum. reflection% reflection
Rfree0.239 76 -
Rwork0.19 1324 -
obs--95.76 %
Refinement TLS params.Method: refined / Origin x: 14.968 Å / Origin y: -12.546 Å / Origin z: 4.093 Å
111213212223313233
T0.0375 Å20.003 Å2-0.0019 Å2-0.0281 Å2-0.003 Å2--0.0027 Å2
L0.5248 °20.2438 °2-0.0558 °2-0.6392 °2-0.1288 °2--0.3246 °2
S-0.0048 Å °-0.0013 Å °-0.0326 Å °-0.0272 Å °0.0118 Å °-0.0081 Å °0.0191 Å °0.0119 Å °-0.0071 Å °

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