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- PDB-1lu0: Atomic Resolution Structure of Squash Trypsin Inhibitor: Unexpect... -

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Basic information

Entry
Database: PDB / ID: 1lu0
TitleAtomic Resolution Structure of Squash Trypsin Inhibitor: Unexpected Metal Coordination
ComponentsTrypsin inhibitor I
KeywordsHYDROLASE INHIBITOR / serine protease inhibitor / metal coordination
Function / homologyPlant trypsin inhibitors / Proteinase inhibitor I7, squash / Squash family serine protease inhibitor / Squash family of serine protease inhibitors signature. / Proteinase/amylase inhibitor domain superfamily / serine-type endopeptidase inhibitor activity / extracellular region / Trypsin inhibitor 1
Function and homology information
Biological speciesCucurbita maxima (winter squash)
MethodX-RAY DIFFRACTION / SYNCHROTRON / Dual-space recycling / Resolution: 1.03 Å
AuthorsThaimattam, R. / Tykarska, E. / Bierzynski, A. / Sheldrick, G.M. / Jaskolski, M.
Citation
Journal: Acta Crystallogr.,Sect.D / Year: 2002
Title: Atomic resolution structure of squash trypsin inhibitor: unexpected metal coordination.
Authors: Thaimattam, R. / Tykarska, E. / Bierzynski, A. / Sheldrick, G.M. / Jaskolski, M.
#1: Journal: FEBS Lett. / Year: 1989
Title: The refined 2.0 A X-ray crystal structure of the complex formed between bovine beta-trypsin and CMTI-I, a trypsin inhibitor from squash seeds (Cucurbita maxima). Topological similarity of the ...Title: The refined 2.0 A X-ray crystal structure of the complex formed between bovine beta-trypsin and CMTI-I, a trypsin inhibitor from squash seeds (Cucurbita maxima). Topological similarity of the squash seed inhibitors with the carboxypeptidase A inhibitor from potatoes
Authors: Bode, W. / Greyling, H.J. / Huber, R. / Otlewski, J. / Wilusz, T.
#2: Journal: Acta Crystallogr.,Sect.D / Year: 1999
Title: High-Resolution Structures of Three New Trypsin-Squash-Inhibitor Complexes: A Detailed Comparison with Other Trypsins and Their Complexes
Authors: Helland, R. / Berglund, G.I. / Otlewski, J. / Apostoluk, W. / Andersen, O.A. / Willassen, N.P. / Smalas, A.O.
#3: Journal: Protein Sci. / Year: 2000
Title: Conservative mutation Met8 --> Leu Effects Folding and Stability of Squash Trypsin Inhibitor Cmti-I
Authors: Zhukov, I. / Jaroszewski, L. / Bierzynski, A.
#4: Journal: J.Mol.Biol. / Year: 1991
Title: Relaxation matrix refinement of the solution structure of squash trypsin inhibitor
Authors: Nilges, M. / Habazettl, J. / Brunger, A.T. / Holak, T.A.
History
DepositionMay 21, 2002Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 28, 2002Provider: repository / Type: Initial release
Revision 1.1Apr 28, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Derived calculations / Version format compliance
Revision 1.3Jan 20, 2016Group: Derived calculations
Revision 1.4Oct 27, 2021Group: Data collection / Database references / Derived calculations
Category: database_2 / diffrn_source ...database_2 / diffrn_source / pdbx_struct_conn_angle / struct_conn / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _diffrn_source.pdbx_synchrotron_site / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_symmetry / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_symmetry / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _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.ptnr1_symmetry / _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 / _struct_conn.ptnr2_label_seq_id / _struct_conn.ptnr2_symmetry / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Trypsin inhibitor I
B: Trypsin inhibitor I
hetero molecules


Theoretical massNumber of molelcules
Total (without water)6,9887
Polymers6,5242
Non-polymers4645
Water1,36976
1
A: Trypsin inhibitor I
B: Trypsin inhibitor I
hetero molecules

A: Trypsin inhibitor I
B: Trypsin inhibitor I
hetero molecules


Theoretical massNumber of molelcules
Total (without water)13,97514
Polymers13,0484
Non-polymers92810
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_775-x+2,-y+2,z1
Buried area3410 Å2
ΔGint-97 kcal/mol
Surface area8570 Å2
MethodPISA, PQS
Unit cell
Length a, b, c (Å)47.280, 58.630, 19.320
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number18
Space group name H-MP21212
Components on special symmetry positions
IDModelComponents
11A-201-

ZN

21B-205-

SO4

DetailsFour molecules of the inhibitor coordinate a single zinc cation in a tetrahedral fashion via their Glu19 side chains. Two of them (molecules A and B) constitute the contents of the asymmetric unit. The full coordination sphere is generated by the operation: -X, -Y, Z, of a two-fold axis. The zinc cation has a special position (occupancy 0.5) on this two-fold axis.

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Components

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Protein/peptide , 1 types, 2 molecules AB

#1: Protein/peptide Trypsin inhibitor I / CMTI-I


Mass: 3261.881 Da / Num. of mol.: 2 / Mutation: M8L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Cucurbita maxima (winter squash) / Plasmid: pAED4 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)-pLys(S) / References: UniProt: P01074

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Non-polymers , 5 types, 81 molecules

#2: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#3: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O3
#4: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
#5: Chemical ChemComp-MRD / (4R)-2-METHYLPENTANE-2,4-DIOL


Mass: 118.174 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H14O2 / Comment: precipitant*YM
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 76 / 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: 2.23 Å3/Da / Density % sol: 44.8 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: PEG 8000, zinc sulfate, cacodylate, MPD, pH 6.5, VAPOR DIFFUSION, HANGING DROP, temperature 293.0K
Crystal grow
*PLUS
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDChemical formulaDetails
15 mM1reservoirZnSO4
250 mMcacodylate1reservoirpH6.5
314 %MPD1reservoir
48 %PEG80001reservoir
512 mg/mlprotein1drop

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: EMBL/DESY, HAMBURG / Beamline: BW7A / Wavelength: 0.8919 Å
DetectorType: MARRESEARCH / Detector: CCD / Date: Apr 7, 2000
RadiationMonochromator: Si, double crystal, tunable / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.8919 Å / Relative weight: 1
ReflectionResolution: 1.03→15 Å / Num. all: 27045 / Num. obs: 27045 / % possible obs: 99.2 % / Observed criterion σ(F): 0 / Observed criterion σ(I): -3 / Redundancy: 9.3 % / Biso Wilson estimate: 12.9 Å2 / Rmerge(I) obs: 0.047 / Net I/σ(I): 30.7
Reflection shellResolution: 1.03→1.07 Å / Redundancy: 3.5 % / Rmerge(I) obs: 0.151 / Mean I/σ(I) obs: 2.73 / Num. unique all: 2637 / % possible all: 98.3
Reflection
*PLUS
Lowest resolution: 15 Å / Num. measured all: 252201
Reflection shell
*PLUS
% possible obs: 98.3 %

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Processing

Software
NameClassification
DENZOdata reduction
SCALEPACKdata scaling
SHELXDphasing
SHELXL-97refinement
RefinementMethod to determine structure: Dual-space recycling / Resolution: 1.03→10 Å / Num. parameters: 5179 / Num. restraintsaints: 6000 / Isotropic thermal model: Anisotropic / Cross valid method: FREE R / σ(F): 0 / σ(I): -3 / StereochEM target val spec case: CSD / Stereochemistry target values: ENGH AND HUBER
Details: Anisotropic refinement without stereochemical restraints on main chain atoms with low Beq (<15 A**2). In both molecules (A and B) there is a salt bridge between the C-terminus and the Arg1 side chain. The Arg5 side chain in molecule A is modeled in two conformations. The Val21 and Tyr27 side chains in both molecules are modeled in double conformation. His25 has different protonation and conformation in the two molecules. The zinc and sulfate ions lie on a common two fold axis. One MPD and two glycerol molecules are modeled in the solvent region. Blocked full-matix least-squares calculations were performed at the conclusion of the refinement. In remark 500, the atoms in all cases represent complementary pairs with partial occupancies.
RfactorNum. reflection% reflectionSelection details
Rfree0.1449 1345 5 %RANDOM
Rwork0.12 ---
all0.1206 26990 --
obs0.1206 26990 99.1 %-
Solvent computationSolvent model: MOEWS & KRETSINGER, J.MOL.BIOL.91(1973)201-228
Refine analyzeNum. disordered residues: 5 / Occupancy sum hydrogen: 432 / Occupancy sum non hydrogen: 517.49
Refinement stepCycle: LAST / Resolution: 1.03→10 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms444 0 24 76 544
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONs_bond_d0.025
X-RAY DIFFRACTIONs_angle_d0.039
X-RAY DIFFRACTIONs_similar_dist0
X-RAY DIFFRACTIONs_from_restr_planes0.031
X-RAY DIFFRACTIONs_zero_chiral_vol0.106
X-RAY DIFFRACTIONs_non_zero_chiral_vol0.13
X-RAY DIFFRACTIONs_anti_bump_dis_restr0
X-RAY DIFFRACTIONs_rigid_bond_adp_cmpnt0.006
X-RAY DIFFRACTIONs_similar_adp_cmpnt0.056
X-RAY DIFFRACTIONs_approx_iso_adps0.091
Software
*PLUS
Name: SHELXL / Version: 97 / Classification: refinement
Refinement
*PLUS
Lowest resolution: 10 Å / Rfactor all: 0.12 / Rfactor Rfree: 0.1395 / Rfactor Rwork: 0.1141
Solvent computation
*PLUS
Displacement parameters
*PLUS
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONs_angle_d
X-RAY DIFFRACTIONs_angle_deg2.7
X-RAY DIFFRACTIONs_chiral_restr0.106

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