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- PDB-6o44: Insight into subtilisin E-S7 cleavage pattern based on crystal st... -

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Basic information

Entry
Database: PDB / ID: 6o44
TitleInsight into subtilisin E-S7 cleavage pattern based on crystal structure and hydrolysates peptide analysis
ComponentsNattokinase
KeywordsHYDROLASE / subtilisin
Function / homology
Function and homology information


subtilisin / sporulation resulting in formation of a cellular spore / kinase activity / serine-type endopeptidase activity / proteolysis / extracellular region / metal ion binding
Similarity search - Function
Subtilisin Carlsberg-like catalytic domain / Peptidase S8/S53 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 ...Subtilisin Carlsberg-like catalytic domain / Peptidase S8/S53 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 / Peptidase S8, subtilisin-related / Serine proteases, subtilase domain profile. / Peptidase S8/S53 domain superfamily / Subtilase family / Peptidase S8/S53 domain / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Nattokinase / Subtilisin E
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.83 Å
AuthorsTang, H. / Shi, K. / Aihara, H.
Funding support United States, China, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118047 United States
National Natural Science Foundation of China (NSFC)31470160 China
CitationJournal: Biochem. Biophys. Res. Commun. / Year: 2019
Title: Insight into subtilisin E-S7 cleavage pattern based on crystal structure and hydrolysates peptide analysis.
Authors: Tang, H. / Zhang, J. / Shi, K. / Aihara, H. / Du, G.
History
DepositionFeb 28, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 10, 2019Provider: repository / Type: Initial release
Revision 1.1May 1, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Jan 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Oct 11, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Nattokinase
B: Nattokinase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)58,35413
Polymers57,7262
Non-polymers62911
Water8,143452
1
A: Nattokinase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)29,2878
Polymers28,8631
Non-polymers4247
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: Nattokinase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)29,0675
Polymers28,8631
Non-polymers2044
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)74.177, 80.123, 87.729
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein Nattokinase


Mass: 28862.793 Da / Num. of mol.: 2 / Mutation: S327C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (bacteria) / Gene: aprN / Production host: Escherichia coli (E. coli)
References: UniProt: A0A024B5N4, UniProt: P04189*PLUS, subtilisin
#2: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ca
#3: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
#4: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C2H6O2
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 452 / 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: 2.26 Å3/Da / Density % sol: 45.53 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / Details: ammonium sulfate, PEG 4,000, Tris-HCl (pH 8.0).

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.979 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Mar 21, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.979 Å / Relative weight: 1
ReflectionResolution: 1.83→59.162 Å / Num. obs: 46392 / % possible obs: 98.88 % / Redundancy: 5.2 % / CC1/2: 0.994 / Net I/σ(I): 6.62
Reflection shellResolution: 1.83→1.9 Å / Redundancy: 4.9 % / Rmerge(I) obs: 1.57 / Mean I/σ(I) obs: 1.13 / Num. unique obs: 4571 / CC1/2: 0.5 / Rpim(I) all: 0.77 / Rrim(I) all: 1.76 / % possible all: 99.13

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Processing

Software
NameVersionClassification
PHENIX(1.14_3247: ???)refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1SUB

1sub


Resolution: 1.83→59.162 Å / SU ML: 0.22 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 27.03
RfactorNum. reflection% reflection
Rfree0.2217 2243 4.85 %
Rwork0.1867 --
obs0.1885 46276 98.88 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å
Refinement stepCycle: LAST / Resolution: 1.83→59.162 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3908 0 33 452 4393
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0034022
X-RAY DIFFRACTIONf_angle_d0.6415484
X-RAY DIFFRACTIONf_dihedral_angle_d9.1582346
X-RAY DIFFRACTIONf_chiral_restr0.049634
X-RAY DIFFRACTIONf_plane_restr0.004716
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.83-1.86980.33241400.32282709X-RAY DIFFRACTION99
1.8698-1.91330.29251430.28592748X-RAY DIFFRACTION99
1.9133-1.96120.35891250.27632715X-RAY DIFFRACTION99
1.9612-2.01420.31721260.25282745X-RAY DIFFRACTION99
2.0142-2.07350.28841300.23982735X-RAY DIFFRACTION99
2.0735-2.14040.28881360.21642708X-RAY DIFFRACTION99
2.1404-2.21690.21981240.20342769X-RAY DIFFRACTION100
2.2169-2.30560.23671430.19952736X-RAY DIFFRACTION100
2.3056-2.41060.23341300.18732748X-RAY DIFFRACTION99
2.4106-2.53770.25431420.17592746X-RAY DIFFRACTION99
2.5377-2.69670.22481340.16672753X-RAY DIFFRACTION99
2.6967-2.90490.20751500.16352759X-RAY DIFFRACTION100
2.9049-3.19720.19071670.16442759X-RAY DIFFRACTION99
3.1972-3.65980.18951650.1552741X-RAY DIFFRACTION99
3.6598-4.61070.15411420.14152799X-RAY DIFFRACTION98
4.6107-59.1930.2181460.18682863X-RAY DIFFRACTION96
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)
11.3875-1.96360.1263.9083-2.26963.8930.19470.0255-0.3541-0.0237-0.2264-0.00080.2707-0.0048-0.01070.26850.0089-0.09790.2079-0.00720.17475.0931-7.4207-36.4483
20.32520.3057-0.27012.13161.22631.9346-0.0568-0.05640.1806-0.0328-0.02010.1884-0.1281-0.16160.07970.2120.0137-0.11020.18170.03850.23841.52986.7682-26.6307
31.0494-0.4337-1.1091.17950.5831.4860.02810.01180.1074-0.0369-0.0205-0.115-0.10640.03-0.01140.1814-0.0132-0.11410.15270.01510.25727.4256.8745-25.4319
48.3494-0.20752.41524.1442-0.93026.10210.1599-0.35010.1540.2972-0.3103-0.0152-0.02370.49870.1420.2056-0.0097-0.08350.16460.01490.3067.40547.7409-15.3567
50.352-0.0161-0.95182.9437-0.81142.9876-0.0597-0.0246-0.06910.01070.05160.1393-0.07360.0788-0.03710.1479-0.0016-0.05470.1491-0.010.2028-1.08291.1196-10.086
60.8335-0.0126-0.2291.0584-0.15930.4885-0.0606-0.0101-0.07490.03250.04080.01090.0333-0.00970.02230.18030.0119-0.09910.1737-0.00450.22625.649-9.2607-20.2085
76.4111-2.5034.48013.0996-1.73796.16680.0177-0.0264-0.5267-0.1430.1410.2711-0.0201-0.1122-0.22070.1853-0.0136-0.0970.128-0.00150.25763.0154-19.196-24.6425
81.2790.5696-0.9760.8229-0.69612.38480.15960.03230.1410.1391-0.0804-0.1433-0.04080.1676-0.04540.154-0.0029-0.110.188-0.00330.2055-31.9983-5.0273-7.4003
90.9339-0.4321-0.25151.74741.42142.90730.0383-0.0714-0.24780.2281-0.10940.01330.27980.12270.06450.1872-0.0474-0.07770.16540.03040.1935-35.5635-19.1866-17.3131
101.3113-0.0530.31321.20030.59891.84570.0526-0.0146-0.14160.01270.0343-0.15460.12420.0276-0.05380.19590.0005-0.08980.14630.02340.2197-29.6829-19.2909-18.5039
116.4454-0.4864-3.83132.74370.32015.7659-0.08830.0443-0.2447-0.1406-0.0617-0.2760.28870.40630.10770.2224-0.004-0.07250.18310.01610.2032-29.7363-20.1894-28.6259
121.5833-0.67290.36161.87860.0922.60990.05850.1620.0698-0.2426-0.13120.1384-0.15960.21330.06930.23190.011-0.10880.21250.00970.2149-37.9235-13.5956-33.8302
131.2524-0.1094-0.00610.49220.13820.7148-0.0060.04320.1378-0.0672-0.0286-0.0603-0.04810.02480.02890.19880.0059-0.10310.1626-0.00330.2534-31.4877-3.1925-23.5096
145.70074.0426-5.57083.9215-3.74145.87430.14550.26080.3105-0.03660.02640.0371-0.1358-0.2028-0.13050.16490.0155-0.12330.17460.02740.2787-34.05026.7991-19.1455
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A' and (resid 1 through 19 )
2X-RAY DIFFRACTION2chain 'A' and (resid 20 through 49 )
3X-RAY DIFFRACTION3chain 'A' and (resid 50 through 88 )
4X-RAY DIFFRACTION4chain 'A' and (resid 89 through 103 )
5X-RAY DIFFRACTION5chain 'A' and (resid 104 through 144 )
6X-RAY DIFFRACTION6chain 'A' and (resid 145 through 252 )
7X-RAY DIFFRACTION7chain 'A' and (resid 253 through 276 )
8X-RAY DIFFRACTION8chain 'B' and (resid 1 through 19 )
9X-RAY DIFFRACTION9chain 'B' and (resid 20 through 49 )
10X-RAY DIFFRACTION10chain 'B' and (resid 50 through 88 )
11X-RAY DIFFRACTION11chain 'B' and (resid 89 through 103 )
12X-RAY DIFFRACTION12chain 'B' and (resid 104 through 144 )
13X-RAY DIFFRACTION13chain 'B' and (resid 145 through 252 )
14X-RAY DIFFRACTION14chain 'B' and (resid 253 through 276 )

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