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- PDB-8adt: Rational design of a calcium-independent trypsin variant -

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Basic information

Entry
Database: PDB / ID: 8adt
TitleRational design of a calcium-independent trypsin variant
ComponentsSerine protease 1
KeywordsHYDROLASE / trypsin artificial disulfide bond enzyme engineering
Function / homology
Function and homology information


trypsin / serpin family protein binding / serine protease inhibitor complex / digestion / endopeptidase activity / serine-type endopeptidase activity / proteolysis / extracellular space / metal ion binding
Similarity search - Function
: / Serine proteases, trypsin family, histidine active site / Serine proteases, trypsin family, serine active site / Serine proteases, trypsin family, histidine active site. / Peptidase S1A, chymotrypsin family / Serine proteases, trypsin family, serine active site. / Serine proteases, trypsin domain profile. / Trypsin-like serine protease / Serine proteases, trypsin domain / Trypsin ...: / Serine proteases, trypsin family, histidine active site / Serine proteases, trypsin family, serine active site / Serine proteases, trypsin family, histidine active site. / Peptidase S1A, chymotrypsin family / Serine proteases, trypsin family, serine active site. / Serine proteases, trypsin domain profile. / Trypsin-like serine protease / Serine proteases, trypsin domain / Trypsin / Trypsin-like serine proteases / Thrombin, subunit H / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan / Beta Barrel / Mainly Beta
Similarity search - Domain/homology
Biological speciesBos taurus (domestic cattle)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 1.4 Å
AuthorsSimon, A.H. / Liebscher, S. / Kattner, A. / Kattner, C. / Bordusa, F.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Catalysts / Year: 2022
Title: Rational Design of a Calcium-Independent Trypsin Variant
Authors: Simon, A.H. / Liebscher, S. / Kattner, A. / Kattner, C. / Bordusa, F.
History
DepositionJul 11, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 14, 2022Provider: repository / Type: Initial release
Revision 1.1Jan 31, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
Revision 1.2Nov 6, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Serine protease 1


Theoretical massNumber of molelcules
Total (without water)23,2561
Polymers23,2561
Non-polymers00
Water3,405189
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area0 Å2
ΔGint0 kcal/mol
Surface area9360 Å2
MethodPISA
Unit cell
Length a, b, c (Å)57.473, 57.473, 105.010
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number152
Space group name H-MP3121

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Components

#1: Protein Serine protease 1 / Anionic trypsin I / Anionic trypsin-I / Beta-trypsin / Cationic trypsin / Pretrypsinogen I / ...Anionic trypsin I / Anionic trypsin-I / Beta-trypsin / Cationic trypsin / Pretrypsinogen I / Trypsin I / Trypsin-I


Mass: 23256.346 Da / Num. of mol.: 1 / Mutation: E70C; E80C; S195A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (domestic cattle) / Gene: PRSS1, TRP1, TRY1, TRYP1 / Details (production host): pet28a / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P00760, trypsin
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 189 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationY

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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: 2.22 Å3/Da / Density % sol: 44.65 % / Description: trigonal crystal
Crystal growTemperature: 293.2 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: 10 mg/ml Trypsin 0.2 M KNO3 22 % (w/v) PEG 3350) 14 days

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Data collection

DiffractionMean temperature: 101.2 K / Serial crystal experiment: N
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU MICROMAX-007 / Wavelength: 1.5418 Å
DetectorType: RIGAKU SATURN 944+ / Detector: CCD / Date: Jun 12, 2014
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 1.4→49.9766 Å / Num. obs: 39855 / % possible obs: 99.57 % / Redundancy: 9.12533 % / Biso Wilson estimate: 16.744 Å2 / Rsym value: 0.069 / Net I/σ(I): 0.4088
Reflection shellResolution: 1.4→1.43 Å / Num. unique obs: 2442 / Rsym value: 0.421

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Processing

Software
NameVersionClassification
REFMAC5.8.0049refinement
HKL-2000data reduction
HKL-2000data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1MTS

1mts


Resolution: 1.4→49.77 Å / Cor.coef. Fo:Fc: 0.96 / Cor.coef. Fo:Fc free: 0.957 / Cross valid method: THROUGHOUT / ESU R: 0.066 / ESU R Free: 0.063 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.20525 1997 5 %RANDOM
Rwork0.19302 ---
obs0.19362 37858 99.57 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 18.941 Å2
Baniso -1Baniso -2Baniso -3
1-0.23 Å20.12 Å20 Å2
2--0.23 Å2-0 Å2
3----0.75 Å2
Refinement stepCycle: 1 / Resolution: 1.4→49.77 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1571 0 0 189 1760
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0060.0191608
X-RAY DIFFRACTIONr_bond_other_d00.021487
X-RAY DIFFRACTIONr_angle_refined_deg1.1591.9462184
X-RAY DIFFRACTIONr_angle_other_deg3.69333441
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.8575214
X-RAY DIFFRACTIONr_dihedral_angle_2_deg40.57525.47253
X-RAY DIFFRACTIONr_dihedral_angle_3_deg11.15115258
X-RAY DIFFRACTIONr_dihedral_angle_4_deg2.323152
X-RAY DIFFRACTIONr_chiral_restr0.0410.2245
X-RAY DIFFRACTIONr_gen_planes_refined0.0040.0211851
X-RAY DIFFRACTIONr_gen_planes_other0.0040.02353
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_it1.8981.736862
X-RAY DIFFRACTIONr_mcbond_other1.8981.734861
X-RAY DIFFRACTIONr_mcangle_it2.5812.5991074
X-RAY DIFFRACTIONr_mcangle_other2.582.6011075
X-RAY DIFFRACTIONr_scbond_it2.5781.92746
X-RAY DIFFRACTIONr_scbond_other2.5751.92746
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other3.5152.7941110
X-RAY DIFFRACTIONr_long_range_B_refined4.25215.171929
X-RAY DIFFRACTIONr_long_range_B_other4.21514.7011853
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.403→1.439 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.259 129 -
Rwork0.239 2675 -
obs--95.73 %

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