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- PDB-3lq0: Zymogen structure of crayfish astacin metallopeptidase -

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Basic information

Entry
Database: PDB / ID: 3lq0
TitleZymogen structure of crayfish astacin metallopeptidase
ComponentsProAstacin
KeywordsHYDROLASE / metallopeptidase / zymogen activation / proenzyme / protease / Disulfide bond / Metal-binding / Metalloprotease / Zymogen
Function / homology
Function and homology information


astacin / glutamic-type peptidase activity / negative regulation of binding of sperm to zona pellucida / aspartic-type peptidase activity / prevention of polyspermy / cortical granule / positive regulation of protein processing / fertilization / metalloendopeptidase activity / peptidase activity ...astacin / glutamic-type peptidase activity / negative regulation of binding of sperm to zona pellucida / aspartic-type peptidase activity / prevention of polyspermy / cortical granule / positive regulation of protein processing / fertilization / metalloendopeptidase activity / peptidase activity / cell adhesion / proteolysis / zinc ion binding / plasma membrane / cytoplasm
Similarity search - Function
Astacin-like metallopeptidase domain / Astacin-like domain profile. / Peptidase M12A / Astacin (Peptidase family M12A) / Peptidase, metallopeptidase / Zinc-dependent metalloprotease / Collagenase (Catalytic Domain) / Collagenase (Catalytic Domain) / Metallopeptidase, catalytic domain superfamily / Neutral zinc metallopeptidases, zinc-binding region signature. ...Astacin-like metallopeptidase domain / Astacin-like domain profile. / Peptidase M12A / Astacin (Peptidase family M12A) / Peptidase, metallopeptidase / Zinc-dependent metalloprotease / Collagenase (Catalytic Domain) / Collagenase (Catalytic Domain) / Metallopeptidase, catalytic domain superfamily / Neutral zinc metallopeptidases, zinc-binding region signature. / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Biological speciesAstacus astacus (noble crayfish)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.45 Å
AuthorsGuevara, T. / Yiallouros, I. / Kappelhoff, R. / Bissdorf, S. / Stocker, W. / Gomis-Ruth, F.X.
CitationJournal: J.Biol.Chem. / Year: 2010
Title: Proenzyme structure and activation of astacin metallopeptidase
Authors: Guevara, T. / Yiallouros, I. / Kappelhoff, R. / Bissdorf, S. / Stocker, W. / Gomis-Ruth, F.X.
History
DepositionFeb 8, 2010Deposition site: RCSB / Processing site: PDBJ
Revision 1.0Feb 23, 2010Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Sep 19, 2012Group: Refinement description
Revision 1.3Nov 1, 2017Group: Advisory / Refinement description / Category: pdbx_unobs_or_zero_occ_atoms / software / Item: _software.name
Revision 1.4Nov 10, 2021Group: Advisory / Database references / Derived calculations
Category: database_2 / pdbx_struct_conn_angle ...database_2 / pdbx_struct_conn_angle / pdbx_unobs_or_zero_occ_atoms / 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 / _pdbx_struct_conn_angle.ptnr1_PDB_ins_code / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr3_PDB_ins_code / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_ptnr1_PDB_ins_code / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.5Nov 1, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: ProAstacin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)26,8756
Polymers26,4371
Non-polymers4385
Water5,368298
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)55.910, 63.370, 69.190
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 ProAstacin / Crayfish small molecule proteinase


Mass: 26437.385 Da / Num. of mol.: 1 / Mutation: I91L,E93A
Source method: isolated from a genetically manipulated source
Details: Recombinant proastacin Glu93MAla, Ile91MLeu (UniProt Q9U918; numbering is based on the mature enzyme, see below) was expressed in Escherichia coli BL21(DE3) cells as inclusion bodies, ...Details: Recombinant proastacin Glu93MAla, Ile91MLeu (UniProt Q9U918; numbering is based on the mature enzyme, see below) was expressed in Escherichia coli BL21(DE3) cells as inclusion bodies, purified by Ni-NTA-affinity chromatography, and folded by dilution and removal of reducing agents and guanidinium chloride.
Source: (gene. exp.) Astacus astacus (noble crayfish) / Plasmid: pET3a / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P07584, astacin
#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 / Glycerol


Mass: 92.094 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C3H8O3
#4: Chemical ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 298 / Source method: isolated from a natural source / Formula: H2O
Compound detailsTHE FIRST 34 RESIDUES ARE FOR ACTIVATION PEPTIDE.

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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: 2.32 Å3/Da / Density % sol: 46.94 %
Crystal growTemperature: 277 K / Method: vapor diffusion, sitting drop / pH: 5.6
Details: For crystallization, reservoir solutions were prepared by a Tecan robot and 200-nL crystallization drops were dispensed on 96x2-well MRC plates (Innovadyne) by a Cartesian (Genomic Solutions) ...Details: For crystallization, reservoir solutions were prepared by a Tecan robot and 200-nL crystallization drops were dispensed on 96x2-well MRC plates (Innovadyne) by a Cartesian (Genomic Solutions) nanodrop robot at the High-Throughput Crystallography Platform of the Barcelona Science Park. Best crystals appeared in a Bruker steady-temperature crystal farm at 4C with protein solution (10 mg/mL in 50mM AMPSO pH9.0) and 20% PEG 8000, 0.1M (NH4)2SO4, 0.01M MgCl2, 0.05M MES pH5.6 as reservoir solution. These conditions were efficiently scaled up to the microliter range with 24-well Cryschem crystallization dishes (Hampton Research). Crystals were cryo-protected with 16% PEG 8000, 20% glycerol, 0.1M (NH4)2SO4, 0.01M MgCl2, 0.05M MES pH5.6. , VAPOR DIFFUSION, SITTING DROP, temperature 277K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID23-2 / Wavelength: 0.873 Å
DetectorType: MAR CCD 165 mm / Detector: CCD / Date: Oct 6, 2009
RadiationMonochromator: horizontally diffracting Si (111) monochromator
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.873 Å / Relative weight: 1
ReflectionResolution: 1.45→46.73 Å / Num. all: 44316 / Num. obs: 44227 / % possible obs: 99.8 % / Observed criterion σ(F): 1.44 / Observed criterion σ(I): 2 / Redundancy: 7.1 % / Biso Wilson estimate: 14.6 Å2 / Rmerge(I) obs: 0.044 / Rsym value: 0.044 / Net I/σ(I): 27.3
Reflection shellResolution: 1.45→1.54 Å / Redundancy: 6.2 % / Rmerge(I) obs: 0.474 / Mean I/σ(I) obs: 3.8 / Num. unique all: 6285 / Rsym value: 0.474 / % possible all: 98.9

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Processing

Software
NameVersionClassification
ProDCdata collection
PHASERphasing
REFMAC5.5.0046refinement
XDSdata reduction
SCALAdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 1AST

1ast


Resolution: 1.45→41.92 Å / Cor.coef. Fo:Fc: 0.972 / Cor.coef. Fo:Fc free: 0.957 / SU B: 2.29 / SU ML: 0.039 / Cross valid method: THROUGHOUT / σ(F): 0 / σ(I): 0 / ESU R: 0.069 / ESU R Free: 0.06 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS.; THE NUMBERING USED IN THE PRIMARY CITATION IS: RESIDUES 1 TO 34 AND CHAIN P FOR THE PROPEPTIDE AND RESIDUES 1 TO 201 AND CHAIN M FOR THE ...Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS.; THE NUMBERING USED IN THE PRIMARY CITATION IS: RESIDUES 1 TO 34 AND CHAIN P FOR THE PROPEPTIDE AND RESIDUES 1 TO 201 AND CHAIN M FOR THE MATURE PROTEASE MOIETY.
RfactorNum. reflection% reflectionSelection details
Rfree0.18046 768 1.7 %RANDOM
Rwork0.15169 ---
all0.15218 44284 --
obs0.15218 43398 99.73 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: BABINET MODEL WITH MASK
Displacement parametersBiso mean: 7.029 Å2
Baniso -1Baniso -2Baniso -3
1--0.69 Å20 Å20 Å2
2---0.87 Å20 Å2
3---1.56 Å2
Refinement stepCycle: LAST / Resolution: 1.45→41.92 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1859 0 24 298 2181
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0160.0211998
X-RAY DIFFRACTIONr_bond_other_d0.0010.021312
X-RAY DIFFRACTIONr_angle_refined_deg1.5321.9432707
X-RAY DIFFRACTIONr_angle_other_deg0.95833176
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.2135242
X-RAY DIFFRACTIONr_dihedral_angle_2_deg31.38223.96101
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.50415308
X-RAY DIFFRACTIONr_dihedral_angle_4_deg19.9771512
X-RAY DIFFRACTIONr_chiral_restr0.090.2285
X-RAY DIFFRACTIONr_gen_planes_refined0.0080.022251
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02429
X-RAY DIFFRACTIONr_mcbond_it1.2191.51199
X-RAY DIFFRACTIONr_mcbond_other0.5081.5496
X-RAY DIFFRACTIONr_mcangle_it1.79121934
X-RAY DIFFRACTIONr_scbond_it2.6943799
X-RAY DIFFRACTIONr_scangle_it3.6754.5773
X-RAY DIFFRACTIONr_rigid_bond_restr1.35633310
X-RAY DIFFRACTIONr_sphericity_free5.3323300
X-RAY DIFFRACTIONr_sphericity_bonded2.08533260
LS refinement shellResolution: 1.45→1.488 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.273 48 -
Rwork0.242 3107 -
obs--97.5 %
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)
14.8161.2244-1.31211.6195-0.61372.25320.10360.2890.58180.08540.04360.0286-0.2799-0.0621-0.14730.09270.0147-0.00750.09020.03190.1170.396-0.588-10.271
21.5124-0.46580.41471.1497-0.06570.58960.04030.11430.169-0.0543-0.0198-0.1494-0.02390.0108-0.02040.0414-0.00940.0110.0320.02050.03964.478-9.052-14.13
31.3651-0.33150.33220.946-0.05030.66970.0115-0.00610.07970.00180.0134-0.0976-0.0129-0.006-0.02490.164-0.01170.00870.14210.00810.1134.923-10.076-10.479
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A1 - 34
2X-RAY DIFFRACTION2A35 - 201
3X-RAY DIFFRACTION2A999
4X-RAY DIFFRACTION3A501 - 504
5X-RAY DIFFRACTION3A505 - 802

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