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- PDB-7am6: Crystal structure of Peptiligase mutant - L217H/M222P/A225N/F189W -

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Basic information

Entry
Database: PDB / ID: 7am6
TitleCrystal structure of Peptiligase mutant - L217H/M222P/A225N/F189W
Components
  • (Subtilisin BPN') x 2
  • LEU-PRO-GLU-GLY-SER-PRO-VAL-THR-ASP-LEU-ARG-TYR
KeywordsLIGASE / subtilisin / peptide ligase
Function / homology
Function and homology information


subtilisin / sporulation resulting in formation of a cellular spore / fibrinolysis / serine-type endopeptidase inhibitor activity / response to wounding / serine-type endopeptidase activity / proteolysis / extracellular region / metal ion binding
Similarity search - Function
Proteinase inhibitor I13, potato inhibitor I / Proteinase inhibitor I13, potato inhibitor I superfamily / Potato inhibitor I family / Potato inhibitor I family signature. / 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. ...Proteinase inhibitor I13, potato inhibitor I / Proteinase inhibitor I13, potato inhibitor I superfamily / Potato inhibitor I family / Potato inhibitor I family signature. / 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
D(-)-TARTARIC ACID / Subtilisin BPN' / Eglin C
Similarity search - Component
Biological speciesBacillus amyloliquefaciens (bacteria)
Hirudo medicinalis (medicinal leech)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 2.7 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

Downloads & links

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Assembly

Deposited unit
A: Subtilisin BPN'
B: Subtilisin BPN'
C: Subtilisin BPN'
P: LEU-PRO-GLU-GLY-SER-PRO-VAL-THR-ASP-LEU-ARG-TYR
hetero molecules


Theoretical massNumber of molelcules
Total (without water)85,05214
Polymers84,0154
Non-polymers1,03710
Water81145
1
A: Subtilisin BPN'
hetero molecules


Theoretical massNumber of molelcules
Total (without water)27,6162
Polymers27,5231
Non-polymers921
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Subtilisin BPN'
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,1187
Polymers27,5071
Non-polymers6116
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
C: Subtilisin BPN'
hetero molecules


Theoretical massNumber of molelcules
Total (without water)27,8584
Polymers27,5231
Non-polymers3343
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
4
P: LEU-PRO-GLU-GLY-SER-PRO-VAL-THR-ASP-LEU-ARG-TYR


  • defined by author
  • 1.46 kDa, 1 polymers
Theoretical massNumber of molelcules
Total (without water)1,4611
Polymers1,4611
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)105.440, 105.440, 192.080
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number91
Space group name H-MP4122
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
12A
22C
13B
23C

NCS domain segments:
Dom-IDComponent-IDEns-IDRefine codeAuth asym-IDAuth seq-ID
1010A1 - 275
2010B1 - 275
1020A1 - 276
2020C1 - 276
1030B1 - 278
2030C1 - 278

NCS ensembles :
ID
1
2
3

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Components

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Protein , 2 types, 3 molecules ACB

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


Mass: 27523.455 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Bacillus amyloliquefaciens (bacteria) / References: UniProt: P00782, subtilisin
#2: Protein Subtilisin BPN' / Alkaline protease / Subtilisin DFE / Subtilisin Novo


Mass: 27507.455 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Bacillus amyloliquefaciens (bacteria) / References: UniProt: P00782, subtilisin

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Protein/peptide , 1 types, 1 molecules P

#3: Protein/peptide LEU-PRO-GLU-GLY-SER-PRO-VAL-THR-ASP-LEU-ARG-TYR


Mass: 1460.652 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: Eglin C fragment / Source: (synth.) Hirudo medicinalis (medicinal leech) / References: UniProt: P01051*PLUS

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Non-polymers , 3 types, 55 molecules

#4: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C3H8O3
#5: Chemical ChemComp-TAR / D(-)-TARTARIC ACID / Tartaric acid


Mass: 150.087 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C4H6O6
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 45 / 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: 3.18 Å3/Da / Density % sol: 61.3 %
Crystal growTemperature: 294 K / Method: vapor diffusion, hanging drop / pH: 7 / Details: 1.2 -1.8 M ammonium tartrate

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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: 2.7→64.03 Å / Num. obs: 29451 / % possible obs: 96.8 % / Redundancy: 5.5 % / CC1/2: 0.969 / Rmerge(I) obs: 0.282 / Rpim(I) all: 0.122 / Rrim(I) all: 0.309 / Net I/σ(I): 6.1 / Num. measured all: 161792 / Scaling rejects: 126
Reflection shellResolution: 2.7→2.83 Å / Redundancy: 5.4 % / Rmerge(I) obs: 1.137 / Num. unique obs: 3710 / CC1/2: 0.369 / Rpim(I) all: 0.494 / Rrim(I) all: 1.247 / % possible all: 93.9

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Processing

Software
NameVersionClassification
REFMAC5.8.0258refinement
XDSdata reduction
Aimless0.7.4data scaling
PDB_EXTRACT3.25data extraction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: D_1292111626

Resolution: 2.7→58.96 Å / Cor.coef. Fo:Fc: 0.914 / Cor.coef. Fo:Fc free: 0.878 / SU B: 31.932 / SU ML: 0.285 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.667 / ESU R Free: 0.321 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: U VALUES : WITH TLS ADDED HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.2523 1458 5 %RANDOM
Rwork0.2122 ---
obs0.2143 27937 96.22 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 119.81 Å2 / Biso mean: 39.967 Å2 / Biso min: 7.18 Å2
Baniso -1Baniso -2Baniso -3
1--0.14 Å20 Å20 Å2
2---0.14 Å2-0 Å2
3---0.28 Å2
Refinement stepCycle: final / Resolution: 2.7→58.96 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5728 0 68 45 5841
Biso mean--58.97 23.18 -
Num. residues----810
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.010.0135915
X-RAY DIFFRACTIONr_bond_other_d0.0010.0175271
X-RAY DIFFRACTIONr_angle_refined_deg1.6861.628072
X-RAY DIFFRACTIONr_angle_other_deg1.3261.56912289
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.7245805
X-RAY DIFFRACTIONr_dihedral_angle_2_deg40.77425.171205
X-RAY DIFFRACTIONr_dihedral_angle_3_deg16.9615808
X-RAY DIFFRACTIONr_dihedral_angle_4_deg21.31156
X-RAY DIFFRACTIONr_chiral_restr0.0720.2800
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.026761
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021069
Refine LS restraints NCS

Refine-ID: X-RAY DIFFRACTION / Type: interatomic distance / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11A80120.08
12B80120.08
21A76560.12
22C76560.12
31B77080.12
32C77080.12
LS refinement shellResolution: 2.7→2.77 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.391 101 -
Rwork0.338 1959 -
all-2060 -
obs--93.09 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
12.64590.6694-0.89161.3301-0.05511.9176-0.07910.33120.0057-0.11450.14130.0248-0.0913-0.0448-0.06220.1525-0.03010.00680.19620.01680.0053-23.108631.5194-10.7261
21.2166-0.21040.14151.4343-0.09491.74080.0247-0.1205-0.05370.0818-0.0796-0.0552-0.01410.07920.05490.1392-0.0373-0.02350.16030.06210.0255-40.491610.665422.7696
31.868-0.2095-0.0841.83250.06011.99560.07190.1787-0.3496-0.03360.0338-0.25740.21080.2513-0.10570.14250.0427-0.00250.172-0.08390.1706-30.0012-3.2618-11.3915
413.1798-6.57056.120920.7241-9.47775.53760.0313-0.50950.7923-1.7132-0.2785-1.50690.6977-0.03860.24730.26680.04690.01780.2388-0.01510.9755-20.6111-6.9176-1.4445
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A1 - 278
2X-RAY DIFFRACTION2B1 - 278
3X-RAY DIFFRACTION3C1 - 278
4X-RAY DIFFRACTION4P72 - 78

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