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- PDB-7jl7: Zebrafish Caspase N213T -

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Basic information

Entry
Database: PDB / ID: 7jl7
TitleZebrafish Caspase N213T
Components
  • (Caspase 3, apoptosis-related cysteine protease a) x 2
  • ASP-GLU-VAL-ASP peptide
KeywordsCELL CYCLE / enzyme specificity / apoptosis / caspase / zebrafish / protein evolution
Function / homology
Function and homology information


regulation of collateral sprouting in absence of injury / Activation of caspases through apoptosome-mediated cleavage / Apoptotic cleavage of cellular proteins / SMAC, XIAP-regulated apoptotic response / Apoptosis induced DNA fragmentation / Signaling by Hippo / Caspase-mediated cleavage of cytoskeletal proteins / : / Caspase activation via Dependence Receptors in the absence of ligand / Other interleukin signaling ...regulation of collateral sprouting in absence of injury / Activation of caspases through apoptosome-mediated cleavage / Apoptotic cleavage of cellular proteins / SMAC, XIAP-regulated apoptotic response / Apoptosis induced DNA fragmentation / Signaling by Hippo / Caspase-mediated cleavage of cytoskeletal proteins / : / Caspase activation via Dependence Receptors in the absence of ligand / Other interleukin signaling / : / Pyroptosis / : / : / Apoptotic cleavage of cell adhesion proteins / Regulation of TNFR1 signaling / cysteine-type endopeptidase activity involved in apoptotic process / cellular response to antibiotic / cellular response to retinoic acid / keratinocyte differentiation / erythrocyte differentiation / neuron differentiation / cellular response to xenobiotic stimulus / peptidase activity / positive regulation of apoptotic process / apoptotic process / proteolysis
Similarity search - Function
Peptidase C14 family / Peptidase family C14A, His active site / Caspase family histidine active site. / Peptidase C14, caspase non-catalytic subunit p10 / Peptidase family C14A, cysteine active site / Caspase family cysteine active site. / Caspase family p10 domain profile. / Peptidase C14A, caspase catalytic domain / Caspase, interleukin-1 beta converting enzyme (ICE) homologues / Peptidase C14, p20 domain ...Peptidase C14 family / Peptidase family C14A, His active site / Caspase family histidine active site. / Peptidase C14, caspase non-catalytic subunit p10 / Peptidase family C14A, cysteine active site / Caspase family cysteine active site. / Caspase family p10 domain profile. / Peptidase C14A, caspase catalytic domain / Caspase, interleukin-1 beta converting enzyme (ICE) homologues / Peptidase C14, p20 domain / Caspase family p20 domain profile. / : / Caspase domain / Caspase-like domain superfamily
Similarity search - Domain/homology
Caspase 3, apoptosis-related cysteine peptidase a / Caspase 3, apoptosis-related cysteine peptidase a
Similarity search - Component
Biological speciesDanio rerio (zebrafish)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.05 Å
AuthorsClark, A.C. / Swartz, P.D.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM127654 United States
CitationJournal: Biosci.Rep. / Year: 2021
Title: Remodeling hydrogen bond interactions results in relaxed specificity of Caspase-3.
Authors: Yao, L. / Swartz, P. / Hamilton, P.T. / Clark, A.C.
History
DepositionJul 29, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 27, 2021Provider: repository / Type: Initial release
Revision 1.1Mar 10, 2021Group: Database references / Category: citation / Item: _citation.journal_volume
Revision 1.2Oct 18, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Caspase 3, apoptosis-related cysteine protease a
C: Caspase 3, apoptosis-related cysteine protease a
B: Caspase 3, apoptosis-related cysteine protease a
D: Caspase 3, apoptosis-related cysteine protease a
F: ASP-GLU-VAL-ASP peptide


Theoretical massNumber of molelcules
Total (without water)63,5705
Polymers63,5705
Non-polymers00
Water1,964109
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area14220 Å2
ΔGint-92 kcal/mol
Surface area17650 Å2
MethodPISA
Unit cell
Length a, b, c (Å)56.305, 74.580, 133.413
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein Caspase 3, apoptosis-related cysteine protease a / / Caspase-3


Mass: 19512.988 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Danio rerio (zebrafish) / Gene: casp3a, casp3 / Production host: Escherichia coli (E. coli) / References: UniProt: Q98UI8, UniProt: B8JK21*PLUS
#2: Protein Caspase 3, apoptosis-related cysteine protease a / / Caspase-3


Mass: 12033.823 Da / Num. of mol.: 2 / Mutation: N213T
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Danio rerio (zebrafish) / Gene: casp3a, casp3 / Production host: Escherichia coli (E. coli) / References: UniProt: Q98UI8
#3: Protein/peptide ASP-GLU-VAL-ASP peptide


Mass: 476.435 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 109 / 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: 2.13 Å3/Da / Density % sol: 42.35 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop
Details: Protein was dialyzed in a buffer of 10 mM Tris-HCl, pH 8.5, 1 mM DTT, concentrated to 4 mg/mL, and inhibitor Ac-DEVD-CHO was added at a 5:1 (w/w) inhibitor/protein ratio. Crystals were ...Details: Protein was dialyzed in a buffer of 10 mM Tris-HCl, pH 8.5, 1 mM DTT, concentrated to 4 mg/mL, and inhibitor Ac-DEVD-CHO was added at a 5:1 (w/w) inhibitor/protein ratio. Crystals were obtained at 18 C by the hanging drop vapor diffusion method using a 4 uL drop that contained equal amounts of protein and reservoir solution. Each well contained a reservoir solution (500 uL) of 100 mM sodium citrate, pH 5.4, 23% PEG 6000, 10 mM DTT, and 3 mM NaN3. Flat, sheet-like crystals appeared within 14 days, and we used microseeding to obtain diffraction-quality crystals. In this case, crystal trays were set up as described above and incubated for 24 hours. The flat sheet crystals were collected and treated with seed beads using a kit from Hampton Research. A 4 uL drop containing flat sheet crystals was added to a tube containing seed beads. Reservoir solution (10 uL) was pipetted on the cover slide to remove all crystals from the coverslip, and the procedure was repeated five times. The resulting mixture of crystals and seed beads was vortexed for 30 seconds and cooled on ice for 10 seconds, and the procedure was repeated six times. Serial dilutions of the treated crystals were set up from 10-1 to 10-3, and 0.5 uL of the 10-2 dilution crystal seeds was added into the drops of the 24-hour crystal tray. Larger cube-shaped crystals appeared within 14 days. The crystals were collected and frozen in liquid nitrogen following the addition of 20% MPD plus the reservoir solution.

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Data collection

DiffractionMean temperature: 100 K / Ambient temp details: Constant temperature / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 22-BM / Wavelength: 1 Å
DetectorType: MARMOSAIC 300 mm CCD / Detector: CCD / Date: Aug 18, 2018
RadiationMonochromator: Insertion device / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.05→35.91 Å / Num. obs: 32428 / % possible obs: 90.51 % / Redundancy: 5.1 % / CC1/2: 0.843 / Net I/σ(I): 30.77
Reflection shellResolution: 2.07→2.11 Å / Num. unique obs: 91 / CC1/2: 0.069

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Processing

Software
NameVersionClassification
PHENIX1.18.2_3874refinement
PDB_EXTRACT3.25data extraction
HKL-2000data reduction
HKL-2000data scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5JFT

5jft


Resolution: 2.05→35.91 Å / SU ML: 0.31 / Cross valid method: THROUGHOUT / σ(F): 1.33 / Phase error: 28.06 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2533 2000 6.17 %
Rwork0.2018 30428 -
obs0.2051 32428 90.51 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 99.05 Å2 / Biso mean: 37.4882 Å2 / Biso min: 16.64 Å2
Refinement stepCycle: final / Resolution: 2.05→35.91 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3717 0 0 109 3826
Biso mean---38.92 -
Num. residues----476
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 14

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.05-2.110.37691270.34711932205982
2.11-2.160.31781420.28212153229591
2.16-2.230.32411410.26782155229691
2.23-2.30.46381410.31952153229491
2.3-2.380.2551410.23392127226890
2.38-2.480.29931400.24052142228290
2.48-2.590.3061390.23492114225389
2.59-2.720.30091400.23892129226990
2.72-2.890.30451390.23392109224888
2.9-3.120.25431380.21762104224287
3.12-3.430.26251380.19622112225089
3.43-3.930.20111470.15892231237891
3.93-4.950.17871590.14822418257798
4.95-35.910.2461680.18462549271799

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