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- PDB-5iae: Caspase 3 V266F -

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Basic information

Entry
Database: PDB / ID: 5iae
TitleCaspase 3 V266F
Components
  • ACE-ASP-GLU-VAL-ASK
  • Caspase-3
KeywordsHydrolase/Hydrolase Inhibitor / Allostery / saturation mutagenesis / conformational selection / native ensemble / protein solvation / protein structure / protein dynamics / Hydrolase-Hydrolase Inhibitor complex
Function / homology
Function and homology information


Stimulation of the cell death response by PAK-2p34 / caspase-3 / phospholipase A2 activator activity / anterior neural tube closure / intrinsic apoptotic signaling pathway in response to osmotic stress / leukocyte apoptotic process / glial cell apoptotic process / positive regulation of pyroptotic inflammatory response / NADE modulates death signalling / luteolysis ...Stimulation of the cell death response by PAK-2p34 / caspase-3 / phospholipase A2 activator activity / anterior neural tube closure / intrinsic apoptotic signaling pathway in response to osmotic stress / leukocyte apoptotic process / glial cell apoptotic process / positive regulation of pyroptotic inflammatory response / NADE modulates death signalling / luteolysis / response to cobalt ion / : / cellular response to staurosporine / death-inducing signaling complex / cyclin-dependent protein serine/threonine kinase inhibitor activity / Apoptosis induced DNA fragmentation / Apoptotic cleavage of cell adhesion proteins / Caspase activation via Dependence Receptors in the absence of ligand / : / SMAC, XIAP-regulated apoptotic response / death receptor binding / axonal fasciculation / Activation of caspases through apoptosome-mediated cleavage / fibroblast apoptotic process / Signaling by Hippo / SMAC (DIABLO) binds to IAPs / SMAC(DIABLO)-mediated dissociation of IAP:caspase complexes / : / epithelial cell apoptotic process / negative regulation of cytokine production / platelet formation / Other interleukin signaling / execution phase of apoptosis / positive regulation of amyloid-beta formation / pyroptotic inflammatory response / negative regulation of B cell proliferation / T cell homeostasis / Apoptotic cleavage of cellular proteins / B cell homeostasis / negative regulation of activated T cell proliferation / neurotrophin TRK receptor signaling pathway / protein maturation / negative regulation of cell cycle / response to X-ray / Pyroptosis / cell fate commitment / response to amino acid / regulation of macroautophagy / Caspase-mediated cleavage of cytoskeletal proteins / response to tumor necrosis factor / response to glucose / response to UV / response to glucocorticoid / keratinocyte differentiation / striated muscle cell differentiation / enzyme activator activity / Degradation of the extracellular matrix / intrinsic apoptotic signaling pathway / erythrocyte differentiation / apoptotic signaling pathway / hippocampus development / response to nicotine / sensory perception of sound / regulation of protein stability / protein catabolic process / response to hydrogen peroxide / neuron differentiation / protein processing / response to wounding / positive regulation of neuron apoptotic process / response to estradiol / heart development / peptidase activity / protease binding / neuron apoptotic process / response to lipopolysaccharide / aspartic-type endopeptidase activity / learning or memory / response to hypoxia / response to xenobiotic stimulus / cysteine-type endopeptidase activity / neuronal cell body / DNA damage response / protein-containing complex binding / apoptotic process / proteolysis / nucleoplasm / nucleus / cytosol / cytoplasm
Similarity search - Function
Rossmann fold - #1460 / 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 ...Rossmann fold - #1460 / 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 / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Ac-Asp-Glu-Val-Asp-CMK / ACETATE ION / Caspase-3
Similarity search - Component
Biological speciesHomo sapiens (human)
unidentified (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / Resolution: 1.55 Å
AuthorsMaciag, J.J. / Mackenzie, S.H. / Tucker, M.B. / Schipper, J.L. / Swartz, P.D. / Clark, A.C.
CitationJournal: Proc.Natl.Acad.Sci.USA / Year: 2016
Title: Tunable allosteric library of caspase-3 identifies coupling between conserved water molecules and conformational selection.
Authors: Maciag, J.J. / Mackenzie, S.H. / Tucker, M.B. / Schipper, J.L. / Swartz, P. / Clark, A.C.
History
DepositionFeb 21, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 26, 2016Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Caspase-3
C: Caspase-3
D: ACE-ASP-GLU-VAL-ASK
E: ACE-ASP-GLU-VAL-ASK
hetero molecules


Theoretical massNumber of molelcules
Total (without water)65,1027
Polymers64,9724
Non-polymers1303
Water10,629590
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area7140 Å2
ΔGint-34 kcal/mol
Surface area19990 Å2
MethodPISA
Unit cell
Length a, b, c (Å)128.223, 68.765, 84.706
Angle α, β, γ (deg.)90.00, 131.34, 90.00
Int Tables number5
Space group name H-MC121

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Components

#1: Protein Caspase-3 / CASP-3 / Apopain / Cysteine protease CPP32 / CPP-32 / Protein Yama / SREBP cleavage activity 1 / SCA-1


Mass: 31951.283 Da / Num. of mol.: 2 / Mutation: V266F
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CASP3, CPP32 / Production host: Escherichia coli (E. coli) / References: UniProt: P42574, caspase-3
#2: Protein/peptide ACE-ASP-GLU-VAL-ASK


Type: Peptide-like / Class: Inhibitor / Mass: 534.946 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) unidentified (others) / References: Ac-Asp-Glu-Val-Asp-CMK
#3: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#4: Chemical ChemComp-ACT / ACETATE ION


Mass: 59.044 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H3O2
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 590 / 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: 2.16 Å3/Da / Density % sol: 43.05 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 8.5
Details: Caspase-3 variants were crystallized in the presence of Ac-DEVD-CMK. PROTEINS WERE DIALYZED IN A BUFFER OF 10 MM TRIS-HCL, PH 8.5, 1 MM DTT. THE PROTEIN WAS CONCENTRATED TO 10 MG/ML USING ...Details: Caspase-3 variants were crystallized in the presence of Ac-DEVD-CMK. PROTEINS WERE DIALYZED IN A BUFFER OF 10 MM TRIS-HCL, PH 8.5, 1 MM DTT. THE PROTEIN WAS CONCENTRATED TO 10 MG/ML USING AMICON ULTRAFREE CENTRIFUGAL FILTER DEVICES, AND INHIBITOR, AC-DEVD-CMK RECONSTITUTED IN DMSO, WAS THEN ADDED AT 5:1 WT:WT, INHIBITOR TO PEPTIDE. THE PROTEIN WAS DILUTED TO A CONCENTRATION OF 8 MG/ML BY ADDING 10 MM TRIS-HCL, PH 8.5, CONCENTRATED DTT AND CONCENTRATED NAN3 SO THAT THE FINAL BUFFER WAS 10 MM TRIS-HCL, PH 8.5, 10 MM DTT, 3 MM NAN3. 2 UL OF CONCENTRATED PROTEIN WAS MIXED 1:1 WITH WELL BUFFER THAT CONTAINED 100 MM SODIUM CITRATE, PH 5, 3 MM NAN3, 10 MM DTT AND 17% PEG 6000 W/V. SOLUTIONS WERE INCUBATED AT 18 DEG C USING THE HANGING DROP METHOD. CRYSTALS GREW WITHIN THREE DAYS FOR WILD- TYPE CASPASE-3 AND WITHIN TWO WEEKS FOR THE MUTANTS.

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 22-BM / Wavelength: 1 Å
DetectorType: MARMOSAIC 225 mm CCD / Detector: CCD / Date: Jun 24, 2010
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.545→23.975 Å / Num. obs: 80456 / % possible obs: 99.5 % / Redundancy: 3.7 % / Net I/σ(I): 1.34

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Processing

Software
NameVersionClassification
PHENIX(1.10.1_2155: ???)refinement
HKL-2000data reduction
HKL-2000data scaling
PHENIXphasing
RefinementResolution: 1.55→23.98 Å / SU ML: 0.12 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 17.22
Details: Authors state that there is a fundamental incompatibility in the naming of the inhibitor structure and the refinement program cannot be made to establish a bond that exists between the ...Details: Authors state that there is a fundamental incompatibility in the naming of the inhibitor structure and the refinement program cannot be made to establish a bond that exists between the inhibitor and the protein at the active site cysteine.
RfactorNum. reflection% reflection
Rfree0.182 2017 2.51 %
Rwork0.155 --
obs0.155 80456 99.5 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 1.55→23.98 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3969 0 6 590 4565
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0064397
X-RAY DIFFRACTIONf_angle_d0.8985958
X-RAY DIFFRACTIONf_dihedral_angle_d15.5652669
X-RAY DIFFRACTIONf_chiral_restr0.059632
X-RAY DIFFRACTIONf_plane_restr0.005782
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.5455-1.58410.24961340.20785217X-RAY DIFFRACTION93
1.5841-1.62690.20371430.19135624X-RAY DIFFRACTION100
1.6269-1.67480.20851440.1835554X-RAY DIFFRACTION100
1.6748-1.72890.23231450.17975653X-RAY DIFFRACTION100
1.7289-1.79060.18271450.17695596X-RAY DIFFRACTION100
1.7906-1.86230.18951450.16255619X-RAY DIFFRACTION100
1.8623-1.9470.19731420.165590X-RAY DIFFRACTION100
1.947-2.04960.1711440.14635593X-RAY DIFFRACTION100
2.0496-2.17790.19141440.14995638X-RAY DIFFRACTION100
2.1779-2.3460.19361440.14675666X-RAY DIFFRACTION100
2.346-2.58180.17881470.15435631X-RAY DIFFRACTION100
2.5818-2.95480.20841440.16385637X-RAY DIFFRACTION100
2.9548-3.72050.16261490.13815642X-RAY DIFFRACTION100
3.7205-23.9780.14791470.13995779X-RAY DIFFRACTION100

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