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- PDB-5ox2: Crystal structure of thymoligase, a substrate-tailored peptiligas... -

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Basic information

Entry
Database: PDB / ID: 5ox2
TitleCrystal structure of thymoligase, a substrate-tailored peptiligase variant
Components
  • Fragment of prodomain
  • Subtilisin BPN'
KeywordsLIGASE / peptide ligase / subtilisin
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
: / Fervidolysin N-terminal prodomain / Subtilisin Carlsberg-like catalytic domain / Peptidase S8/S53 domain / : / 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 ...: / Fervidolysin N-terminal prodomain / Subtilisin Carlsberg-like catalytic domain / Peptidase S8/S53 domain / : / 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 / Peptidase S8, subtilisin-related / Serine proteases, subtilase domain profile. / Peptidase S8/S53 domain superfamily / Peptidase S8/S53 domain / Subtilase family / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Biological speciesBacillus amyloliquefaciens (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.24 Å
AuthorsRozeboom, H.J. / Janssen, D.B.
CitationJournal: Org. Biomol. Chem. / Year: 2018
Title: Design of a substrate-tailored peptiligase variant for the efficient synthesis of thymosin-alpha1.
Authors: Schmidt, M. / Toplak, A. / Rozeboom, H.J. / Wijma, H.J. / Quaedflieg, P.J.L.M. / van Maarseveen, J.H. / Janssen, D.B. / Nuijens, T.
History
DepositionSep 5, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 10, 2018Provider: repository / Type: Initial release
Revision 1.1Apr 18, 2018Group: Data collection / 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 / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Subtilisin BPN'
P: Fragment of prodomain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,8616
Polymers28,4762
Non-polymers3844
Water93752
1


  • Idetical with deposited unit
  • defined by software
  • Evidence: gel filtration, and dynamic light scattering
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1490 Å2
ΔGint-45 kcal/mol
Surface area10190 Å2
MethodPISA
Unit cell
Length a, b, c (Å)47.478, 40.061, 64.250
Angle α, β, γ (deg.)90.00, 106.04, 90.00
Int Tables number4
Space group name H-MP1211

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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
Details: Mutations: Q2K, S3C, P5S, S9A, I31L, K43N, M50F, G74A, DELTA75-83, E156N, G166D, G169A, S188P. F189W, Q206C, N212G, Y217R, N218S, S221C, M222G, P225N, T254A, Q271E
Source: (gene. exp.) Bacillus amyloliquefaciens (bacteria) / Gene: apr / Plasmid: pBE-S / Production host: Bacillus subtilis (bacteria) / Strain (production host): GX4935 / References: UniProt: P00782, subtilisin
#2: Protein/peptide Fragment of prodomain


Mass: 1008.042 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus amyloliquefaciens (bacteria) / Gene: apr / Plasmid: pBE-S / Production host: Bacillus subtilis (bacteria) / Strain (production host): GX4935 / References: UniProt: P00782*PLUS
#3: Chemical
ChemComp-SO4 / SULFATE ION


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


Mass: 18.015 Da / Num. of mol.: 52 / Source method: isolated from a natural source / Formula: H2O

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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: 2.1 Å3/Da / Density % sol: 43 %
Crystal growTemperature: 294 K / Method: vapor diffusion, sitting drop / pH: 5.5 / Details: 1.0 M Ammonium Sulfate, 0.1 M Bis-tris, 1% PEG3350

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, DESY / Beamline: P11 / Wavelength: 1.0332 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Apr 17, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.0332 Å / Relative weight: 1
ReflectionResolution: 2.24→45.6 Å / Num. obs: 10355 / % possible obs: 91.6 % / Redundancy: 3.5 % / Biso Wilson estimate: 21.8 Å2 / CC1/2: 0.99 / Rmerge(I) obs: 0.102 / Rpim(I) all: 0.071 / Net I/σ(I): 7.8
Reflection shellResolution: 2.24→2.32 Å / Rmerge(I) obs: 0.479 / Num. unique obs: 950 / CC1/2: 0.78 / Rpim(I) all: 0.35 / % possible all: 92.1

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Processing

Software
NameVersionClassification
XDSdata reduction
Aimlessdata scaling
PHASERphasing
REFMAC5.8.0158refinement
XDSdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.24→45.6 Å / Cor.coef. Fo:Fc: 0.951 / Cor.coef. Fo:Fc free: 0.923 / SU B: 8.911 / SU ML: 0.205 / Cross valid method: THROUGHOUT / ESU R: 0.47 / ESU R Free: 0.245 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.22998 513 5 %RANDOM
Rwork0.1877 ---
obs0.19004 9833 91.17 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 24.68 Å2
Baniso -1Baniso -2Baniso -3
1-1.79 Å2-0 Å2-1.41 Å2
2---2.03 Å2-0 Å2
3---0.9 Å2
Refinement stepCycle: 1 / Resolution: 2.24→45.6 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1942 0 20 52 2014
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0090.0191998
X-RAY DIFFRACTIONr_bond_other_d0.0020.021774
X-RAY DIFFRACTIONr_angle_refined_deg1.3561.9412731
X-RAY DIFFRACTIONr_angle_other_deg0.92834128
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.45273
X-RAY DIFFRACTIONr_dihedral_angle_2_deg40.02625.71470
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.92815275
X-RAY DIFFRACTIONr_dihedral_angle_4_deg10.75153
X-RAY DIFFRACTIONr_chiral_restr0.0690.2317
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.0212288
X-RAY DIFFRACTIONr_gen_planes_other0.0020.02363
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it1.0682.4431098
X-RAY DIFFRACTIONr_mcbond_other1.0692.4431097
X-RAY DIFFRACTIONr_mcangle_it1.743.6581369
X-RAY DIFFRACTIONr_mcangle_other1.7393.6591370
X-RAY DIFFRACTIONr_scbond_it1.4812.673900
X-RAY DIFFRACTIONr_scbond_other1.4012.611885
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other2.2143.8461339
X-RAY DIFFRACTIONr_long_range_B_refined3.57329.9732188
X-RAY DIFFRACTIONr_long_range_B_other3.47829.9442184
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.244→2.302 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.336 30 -
Rwork0.31 730 -
obs--90.26 %

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