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- PDB-2fo5: Crystal structure of recombinant barley cysteine endoprotease B i... -

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Basic information

Entry
Database: PDB / ID: 2fo5
TitleCrystal structure of recombinant barley cysteine endoprotease B isoform 2 (EP-B2) in complex with leupeptin
Components
  • ACE-LEU-LEU-argininal (leupeptin)
  • Cysteine proteinase EP-B 2
KeywordsHYDROLASE/HYDROLASE INHIBITOR / EP-B2 / EPB2 / EPB / cysteine endoprotease / endopeptidase / leupeptin / HYDROLASE / HYDROLASE-HYDROLASE INHIBITOR COMPLEX
Function / homology
Function and homology information


cysteine-type peptidase activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases
Similarity search - Function
Cathepsin propeptide inhibitor domain (I29) / Cathepsin propeptide inhibitor domain (I29) / Cathepsin propeptide inhibitor domain (I29) / Papain-like cysteine endopeptidase / Cysteine peptidase, asparagine active site / Eukaryotic thiol (cysteine) proteases asparagine active site. / Cysteine peptidase, histidine active site / Eukaryotic thiol (cysteine) proteases histidine active site. / Peptidase C1A, papain C-terminal / Papain family cysteine protease ...Cathepsin propeptide inhibitor domain (I29) / Cathepsin propeptide inhibitor domain (I29) / Cathepsin propeptide inhibitor domain (I29) / Papain-like cysteine endopeptidase / Cysteine peptidase, asparagine active site / Eukaryotic thiol (cysteine) proteases asparagine active site. / Cysteine peptidase, histidine active site / Eukaryotic thiol (cysteine) proteases histidine active site. / Peptidase C1A, papain C-terminal / Papain family cysteine protease / Papain family cysteine protease / 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
LEUPEPTIN / : / Cysteine proteinase EP-B 2
Similarity search - Component
Biological speciesHordeum vulgare (barley)
Actinomycetes Streptomyces roseus MA 839-A1 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.2 Å
AuthorsBethune, M.T. / Strop, P. / Brunger, A.T. / Khosla, C.
CitationJournal: Chem.Biol. / Year: 2006
Title: Heterologous Expression, Purification, Refolding, and Structural-Functional Characterization of EP-B2, a Self-Activating Barley Cysteine Endoprotease.
Authors: Bethune, M.T. / Strop, P. / Tang, Y. / Sollid, L.M. / Khosla, C.
History
DepositionJan 12, 2006Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 18, 2006Provider: repository / Type: Initial release
Revision 1.1May 1, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Atomic model / Database references ...Atomic model / Database references / Derived calculations / Non-polymer description / Structure summary / Version format compliance
Revision 1.3Dec 12, 2012Group: Other
Revision 1.4Oct 18, 2017Group: Advisory / Refinement description / Category: pdbx_unobs_or_zero_occ_residues / software

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Cysteine proteinase EP-B 2
B: Cysteine proteinase EP-B 2
C: Cysteine proteinase EP-B 2
D: Cysteine proteinase EP-B 2
E: ACE-LEU-LEU-argininal (leupeptin)
F: ACE-LEU-LEU-argininal (leupeptin)
G: ACE-LEU-LEU-argininal (leupeptin)
H: ACE-LEU-LEU-argininal (leupeptin)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)113,62217
Polymers112,7578
Non-polymers8659
Water9,170509
1
A: Cysteine proteinase EP-B 2
E: ACE-LEU-LEU-argininal (leupeptin)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,4775
Polymers28,1892
Non-polymers2883
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1500 Å2
ΔGint-44 kcal/mol
Surface area9980 Å2
MethodPISA
2
B: Cysteine proteinase EP-B 2
F: ACE-LEU-LEU-argininal (leupeptin)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,3814
Polymers28,1892
Non-polymers1922
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1290 Å2
ΔGint-30 kcal/mol
Surface area9900 Å2
MethodPISA
3
C: Cysteine proteinase EP-B 2
G: ACE-LEU-LEU-argininal (leupeptin)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,3814
Polymers28,1892
Non-polymers1922
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1280 Å2
ΔGint-30 kcal/mol
Surface area9990 Å2
MethodPISA
4
D: Cysteine proteinase EP-B 2
H: ACE-LEU-LEU-argininal (leupeptin)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,3814
Polymers28,1892
Non-polymers1922
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1290 Å2
ΔGint-30 kcal/mol
Surface area9940 Å2
MethodPISA
Unit cell
Length a, b, c (Å)44.370, 51.730, 115.655
Angle α, β, γ (deg.)86.32, 86.46, 79.63
Int Tables number1
Space group name H-MP1

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Components

#1: Protein
Cysteine proteinase EP-B 2 /


Mass: 27759.693 Da / Num. of mol.: 4 / Fragment: Cystein proteinase EP-B2 domain, residues 133-356
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Hordeum vulgare (barley) / Gene: EPB2 / Plasmid: pMTB1 / Species (production host): Escherichia coli / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): BL21
References: UniProt: P25250, Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases
#2: Protein/peptide
ACE-LEU-LEU-argininal (leupeptin) /


Type: Oligopeptide / Class: Enzyme inhibitor / Mass: 429.578 Da / Num. of mol.: 4 / Source method: obtained synthetically / Details: Chemically synthesized
Source: (synth.) Actinomycetes Streptomyces roseus MA 839-A1 (bacteria)
References: NOR: NOR00487, LEUPEPTIN
#3: Chemical
ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: SO4
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 509 / Source method: isolated from a natural source / Formula: H2O
Compound detailsTHE LEUPEPTIN IS COVALENTLY CONNECTED TO ACTIVE_SITE CYS 28 OF THE ENZYME TO FORM A HEMITHIOACETAL.
Sequence detailsTHERE IS A DISCREPANCY IN THE NORINE AND PDB NUMBERING, AS NORINE COUNTS ACE AND LEU TOGETHER AS ...THERE IS A DISCREPANCY IN THE NORINE AND PDB NUMBERING, AS NORINE COUNTS ACE AND LEU TOGETHER AS ONE RESIDUE. SO THE DBREF WILL REPORT 4 PDB RESIDUES MATCHING NORINE 3 RESIDUE

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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: 2.2 Å3/Da / Density % sol: 44 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 7
Details: 1.4 M LiSO4 0.1 M HEPES, pH 7.0, VAPOR DIFFUSION, HANGING DROP, temperature 298K

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Data collection

DiffractionMean temperature: 113 K
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 8.3.1 / Wavelength: 1.115869 Å
DetectorType: ADSC QUANTUM 4 / Detector: CCD / Date: Oct 22, 2005
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.115869 Å / Relative weight: 1
ReflectionResolution: 2.15→50 Å / Num. obs: 48898 / % possible obs: 89.9 % / Redundancy: 3.3 % / Rmerge(I) obs: 0.059 / Χ2: 1.181 / Net I/σ(I): 11.7
Reflection shell
Resolution (Å)% possible obs (%)Redundancy (%)Rmerge(I) obsNum. unique obsΧ2Diffraction-ID
2.15-2.2359.52.80.28432311.1371
2.23-2.3282.32.80.22444901.1861
2.32-2.4290.13.10.19748721.1761
2.42-2.5592.93.40.1750691.2011
2.55-2.71923.40.12749941.1961
2.71-2.9293.73.40.0950921.2171
2.92-3.2195.23.50.06252161.2321
3.21-3.6897.73.60.04353171.2331
3.68-4.6397.93.70.03252981.1071
4.63-5097.73.70.0353191.1131

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Phasing

Phasing MR
Highest resolutionLowest resolution
Rotation2.5 Å14.96 Å
Translation2.5 Å14.96 Å

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Processing

Software
NameVersionClassificationNB
DENZOdata reduction
SCALEPACKdata scaling
PHASERphasing
CNSrefinement
PDB_EXTRACT1.701data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.2→15 Å / FOM work R set: 0.843 / σ(F): 0 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.233 3395 6.7 %random
Rwork0.201 ---
obs-46985 92 %-
Solvent computationBsol: 44.175 Å2
Displacement parametersBiso mean: 26.942 Å2
Baniso -1Baniso -2Baniso -3
1--10.935 Å22.045 Å2-3.243 Å2
2--2.964 Å2-2.316 Å2
3---7.971 Å2
Refinement stepCycle: LAST / Resolution: 2.2→15 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6740 0 45 509 7294
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_angle_deg1.4
X-RAY DIFFRACTIONc_bond_d0.007
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 50

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection obs
2.2-2.210.267400.251615655
2.21-2.230.277480.262619667
2.23-2.250.321560.265657713
2.25-2.260.306430.285698741
2.26-2.280.295560.286827883
2.28-2.30.298590.244825884
2.3-2.310.273680.215844912
2.31-2.330.188420.19851893
2.33-2.350.298530.23873926
2.35-2.370.269650.218852917
2.37-2.390.226620.221848910
2.39-2.410.277830.216840923
2.41-2.430.291810.236843924
2.43-2.450.282810.228888969
2.45-2.480.29910.231869960
2.48-2.50.305650.246843908
2.5-2.530.242640.223872936
2.53-2.550.246680.214908976
2.55-2.580.272660.219883949
2.58-2.610.222630.211846909
2.61-2.640.215670.199898965
2.64-2.670.323640.227849913
2.67-2.70.275660.241907973
2.7-2.730.28710.226871942
2.73-2.770.255600.222891951
2.77-2.810.309780.212875953
2.81-2.850.257680.213893961
2.85-2.890.263660.221898964
2.89-2.930.296650.23884949
2.93-2.980.29750.221880955
2.98-3.030.225750.202892967
3.03-3.090.288740.222917991
3.09-3.150.25790.203890969
3.15-3.210.238760.208904980
3.21-3.280.233700.214917987
3.28-3.350.214740.204924998
3.35-3.440.222770.1999501027
3.44-3.530.198690.186888957
3.53-3.630.216690.1699611030
3.63-3.750.196840.1799211005
3.75-3.880.169670.169922989
3.88-4.030.172560.1689531009
4.03-4.210.189650.152913978
4.21-4.430.156770.159491026
4.43-4.70.174860.149897983
4.7-5.050.19780.155920998
5.05-5.530.197660.1819541020
5.53-6.280.288810.2239311012
6.28-7.730.191650.2239371002
7.73-150.266730.234903976
Xplor file
Refine-IDSerial noParam file
X-RAY DIFFRACTION1CNS_TOPPAR:protein_rep.param
X-RAY DIFFRACTION2CNS_TOPPAR:carbohydrate.param
X-RAY DIFFRACTION3CNS_TOPPAR:water_rep.param
X-RAY DIFFRACTION4CNS_TOPPAR:ion.param
X-RAY DIFFRACTION5ligand.par

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