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- PDB-2e03: Crystal structure of NQ67E mutant of yeast bleomycin hydrolase -

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Basic information

Entry
Database: PDB / ID: 2000
TitleCrystal structure of NQ67E mutant of yeast bleomycin hydrolase
ComponentsCysteine proteinase 1
KeywordsHYDROLASE / bleomycin hydrolase / thiol protease / C1 proteas
Function / homology
Function and homology information


bleomycin hydrolase / cysteine-type aminopeptidase activity / homocysteine catabolic process / Antigen processing: Ubiquitination & Proteasome degradation / cysteine-type peptidase activity / aminopeptidase activity / response to toxic substance / single-stranded DNA binding / double-stranded DNA binding / RNA polymerase II cis-regulatory region sequence-specific DNA binding ...bleomycin hydrolase / cysteine-type aminopeptidase activity / homocysteine catabolic process / Antigen processing: Ubiquitination & Proteasome degradation / cysteine-type peptidase activity / aminopeptidase activity / response to toxic substance / single-stranded DNA binding / double-stranded DNA binding / RNA polymerase II cis-regulatory region sequence-specific DNA binding / cysteine-type endopeptidase activity / response to antibiotic / mRNA binding / negative regulation of transcription by RNA polymerase II / mitochondrion / proteolysis / cytoplasm
Similarity search - Function
Peptidase C1B, bleomycin hydrolase / Peptidase C1-like family / Cysteine peptidase, histidine active site / Eukaryotic thiol (cysteine) proteases histidine active site. / Cysteine proteinases / Cysteine peptidase, cysteine active site / Eukaryotic thiol (cysteine) proteases cysteine active site. / Cathepsin B; Chain A / Papain-like cysteine peptidase superfamily / Alpha-Beta Complex / Alpha Beta
Similarity search - Domain/homology
Cysteine proteinase 1, mitochondrial
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodX-RAY DIFFRACTION / SYNCHROTRON / isomorphous replacement / Resolution: 2.13 Å
AuthorsO'Farrell, P.A. / Joshua-Tor, L.
CitationJournal: Biochem.J. / Year: 2007
Title: Mutagenesis and crystallographic studies of the catalytic residues of the papain family protease bleomycin hydrolase: new insights into active-site structure
Authors: O'Farrell, P.A. / Joshua-Tor, L.
History
DepositionOct 1, 2006Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 14, 2007Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Derived calculations / Version format compliance
Revision 1.2Nov 10, 2021Group: Database references / Category: database_2 / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details
Revision 1.3Oct 25, 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: Cysteine proteinase 1


Theoretical massNumber of molelcules
Total (without water)52,4281
Polymers52,4281
Non-polymers00
Water3,135174
1
A: Cysteine proteinase 1
x 6


Theoretical massNumber of molelcules
Total (without water)314,5666
Polymers314,5666
Non-polymers00
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_655-y+1,x-y,z1
crystal symmetry operation3_665-x+y+1,-x+1,z1
crystal symmetry operation10_665-y+1,-x+1,-z+1/21
crystal symmetry operation11_655-x+y+1,y,-z+1/21
crystal symmetry operation12_555x,x-y,-z+1/21
Buried area33870 Å2
ΔGint-133 kcal/mol
Surface area104200 Å2
MethodPISA, PQS
Unit cell
Length a, b, c (Å)152.292, 152.292, 89.250
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number182
Space group name H-MP6322

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Components

#1: Protein Cysteine proteinase 1 / / Y3 / Bleomycin hydrolase / BLM hydrolase


Mass: 52427.641 Da / Num. of mol.: 1 / Mutation: Q67E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Production host: Escherichia coli (E. coli) / References: UniProt: Q01532, bleomycin hydrolase
#2: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 174 / 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: 2.85 Å3/Da / Density % sol: 56.82 %
Crystal growTemperature: 290 K / Method: vapor diffusion, hanging drop / pH: 8.5
Details: 14-20% PEG 4K, 100mM Tris-HCL, pH 8.5, VAPOR DIFFUSION, HANGING DROP, temperature 290K

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Data collection

DiffractionMean temperature: 95 K
Diffraction sourceSource: SYNCHROTRON / Site: NSLS / Beamline: X26C / Wavelength: 1.1 Å
DetectorType: ADSC QUANTUM 4 / Detector: CCD
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.1 Å / Relative weight: 1
ReflectionResolution: 2.13→50 Å / Num. obs: 34254 / Biso Wilson estimate: 17.9 Å2

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Processing

Software
NameVersionClassification
CNS1.1refinement
HKL-2000data collection
HKL-2000data reduction
HKL-2000data scaling
RefinementMethod to determine structure: isomorphous replacement
Starting model: 1gcb

1gcb


Resolution: 2.13→42.27 Å / Rfactor Rfree error: 0.006 / Data cutoff high absF: 253082.78 / Data cutoff low absF: 0 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0
RfactorNum. reflection% reflectionSelection details
Rfree0.256 1638 5 %RANDOM
Rwork0.206 ---
obs0.206 32708 94.6 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 54.912 Å2 / ksol: 0.346772 e/Å3
Displacement parametersBiso mean: 31.5 Å2
Baniso -1Baniso -2Baniso -3
1-1.37 Å22.38 Å20 Å2
2--1.37 Å20 Å2
3----2.73 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.31 Å0.23 Å
Luzzati d res low-5 Å
Luzzati sigma a0.23 Å0.14 Å
Refinement stepCycle: LAST / Resolution: 2.13→42.27 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3599 0 0 174 3773
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_bond_d0.02
X-RAY DIFFRACTIONc_angle_deg2
X-RAY DIFFRACTIONc_dihedral_angle_d23
X-RAY DIFFRACTIONc_improper_angle_d1.3
X-RAY DIFFRACTIONc_mcbond_it3.161.5
X-RAY DIFFRACTIONc_mcangle_it3.872
X-RAY DIFFRACTIONc_scbond_it5.162
X-RAY DIFFRACTIONc_scangle_it6.282.5
LS refinement shellResolution: 2.13→2.26 Å / Rfactor Rfree error: 0.02 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.305 228 4.8 %
Rwork0.226 4513 -
obs--83.9 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1protein_rep.paramprotein.top
X-RAY DIFFRACTION2water_rep.param

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