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- PDB-3p7o: Rat Insulin Degrading Enzyme (Insulysin) E111F mutant with two bo... -

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Basic information

Entry
Database: PDB / ID: 3p7o
TitleRat Insulin Degrading Enzyme (Insulysin) E111F mutant with two bound peptides
Components
  • Insulin-degrading enzyme
  • active site bound peptide
  • distal site bound peptide
KeywordsHYDROLASE / metallopeptidase
Function / homology
Function and homology information


beta-endorphin binding / Peroxisomal protein import / Insulin receptor recycling / insulysin / Ub-specific processing proteases / ubiquitin recycling / insulin catabolic process / insulin metabolic process / amyloid-beta clearance by cellular catabolic process / hormone catabolic process ...beta-endorphin binding / Peroxisomal protein import / Insulin receptor recycling / insulysin / Ub-specific processing proteases / ubiquitin recycling / insulin catabolic process / insulin metabolic process / amyloid-beta clearance by cellular catabolic process / hormone catabolic process / bradykinin catabolic process / cytosolic proteasome complex / ubiquitin-modified protein reader activity / insulin binding / regulation of aerobic respiration / peptide catabolic process / insulin receptor recycling / amyloid-beta clearance / peptide hormone binding / peroxisomal matrix / amyloid-beta metabolic process / proteolysis involved in protein catabolic process / endosome lumen / peptide binding / negative regulation of proteolysis / protein catabolic process / metalloendopeptidase activity / antigen processing and presentation of endogenous peptide antigen via MHC class I / peroxisome / amyloid-beta binding / peptidase activity / basolateral plasma membrane / endopeptidase activity / response to oxidative stress / external side of plasma membrane / protein-containing complex binding / cell surface / ATP hydrolysis activity / protein homodimerization activity / mitochondrion / proteolysis / extracellular space / extracellular exosome / zinc ion binding / ATP binding / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Peptidase M16, middle/third domain / Middle or third domain of peptidase_M16 / Cytochrome Bc1 Complex; Chain A, domain 1 / Metalloenzyme, LuxS/M16 peptidase-like / Peptidase M16, zinc-binding site / Insulinase family, zinc-binding region signature. / Peptidase M16, C-terminal / Peptidase M16 inactive domain / Peptidase M16, N-terminal / Insulinase (Peptidase family M16) ...Peptidase M16, middle/third domain / Middle or third domain of peptidase_M16 / Cytochrome Bc1 Complex; Chain A, domain 1 / Metalloenzyme, LuxS/M16 peptidase-like / Peptidase M16, zinc-binding site / Insulinase family, zinc-binding region signature. / Peptidase M16, C-terminal / Peptidase M16 inactive domain / Peptidase M16, N-terminal / Insulinase (Peptidase family M16) / Metalloenzyme, LuxS/M16 peptidase-like / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
Insulin-degrading enzyme
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.1423 Å
AuthorsRodgers, D.W. / Noinaj, N.
CitationJournal: Plos One / Year: 2011
Title: Identification of the allosteric regulatory site of insulysin.
Authors: Noinaj, N. / Bhasin, S.K. / Song, E.S. / Scoggin, K.E. / Juliano, M.A. / Juliano, L. / Hersh, L.B. / Rodgers, D.W.
History
DepositionOct 12, 2010Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 27, 2011Provider: repository / Type: Initial release
Revision 1.1Nov 8, 2017Group: Advisory / Refinement description / Category: pdbx_unobs_or_zero_occ_atoms / software / Item: _software.name
Revision 1.2Feb 21, 2024Group: Advisory / Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_unobs_or_zero_occ_atoms / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Insulin-degrading enzyme
B: active site bound peptide
C: distal site bound peptide


Theoretical massNumber of molelcules
Total (without water)119,1953
Polymers119,1953
Non-polymers00
Water3,333185
1
A: Insulin-degrading enzyme
B: active site bound peptide
C: distal site bound peptide

A: Insulin-degrading enzyme
B: active site bound peptide
C: distal site bound peptide


Theoretical massNumber of molelcules
Total (without water)238,3916
Polymers238,3916
Non-polymers00
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_556-x,y,-z+11
2


  • Idetical with deposited unit
  • defined by software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1700 Å2
ΔGint-9 kcal/mol
Surface area38390 Å2
MethodPISA
Unit cell
Length a, b, c (Å)115.52, 70.91, 114.36
Angle α, β, γ (deg.)90.00, 92.97, 90.00
Int Tables number5
Space group name H-MC121
Components on special symmetry positions
IDModelComponents
11A-1176-

HOH

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Components

#1: Protein Insulin-degrading enzyme / / Insulin protease / Insulinase / Insulysin


Mass: 117882.883 Da / Num. of mol.: 1 / Mutation: E111F
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Ide / Production host: Escherichia coli (E. coli) / References: UniProt: P35559, insulysin
#2: Protein/peptide active site bound peptide


Mass: 698.854 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Production host: Escherichia coli (E. coli)
#3: Protein/peptide distal site bound peptide


Mass: 613.749 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Production host: Escherichia coli (E. coli)
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 185 / Source method: isolated from a natural source / Formula: H2O
Sequence detailsPEPTIDES B AND C ARE PART OF A CLEAVED EXPRESSION TAG CONSISTING OF THE SEQUENCE (ACE)-SYYHHHHHHDYDIPTTENLYFQ

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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: 1.96 Å3/Da / Density % sol: 37.31 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: 100 mM sodium citrate pH 6.5, 100 mM ammonium acetate, 20% PEG 4000, 8 mg/ml protein, VAPOR DIFFUSION, HANGING DROP, temperature 298K

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Data collection

DiffractionMean temperature: 110 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 22-ID / Wavelength: 1 Å
DetectorType: MARMOSAIC 300 mm CCD / Detector: CCD / Date: Aug 21, 2008
RadiationMonochromator: Si(111) sagitally focused / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.14→50 Å / Num. all: 50790 / Num. obs: 47233 / % possible obs: 98.7 % / Observed criterion σ(F): -0.81 / Observed criterion σ(I): -0.25 / Redundancy: 2.8 % / Rmerge(I) obs: 0.088 / Net I/σ(I): 14.96
Reflection shellResolution: 2.14→2.24 Å / % possible obs: 99.6 % / Redundancy: 2 % / Rmerge(I) obs: 0.41 / Mean I/σ(I) obs: 3.98 / % possible all: 99.6

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Processing

Software
NameVersionClassification
MAR345data collection
PHASERphasing
PHENIX(phenix.refine: 1.6.1_357)refinement
HKL-2000data reduction
HKL-2000data scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.1423→28.84 Å / SU ML: 0.36 / σ(F): 1.44 / Phase error: 32.39 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.2846 4748 10.05 %random
Rwork0.2077 ---
all0.2213 50931 --
obs0.2154 47228 92.73 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL / Bsol: 32.178 Å2 / ksol: 0.295 e/Å3
Displacement parameters
Baniso -1Baniso -2Baniso -3
1-0.4169 Å20 Å20.7604 Å2
2--0.7948 Å2-0 Å2
3----1.2117 Å2
Refinement stepCycle: LAST / Resolution: 2.1423→28.84 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7909 0 0 185 8094
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0098105
X-RAY DIFFRACTIONf_angle_d1.15810964
X-RAY DIFFRACTIONf_dihedral_angle_d16.3873052
X-RAY DIFFRACTIONf_chiral_restr0.0781179
X-RAY DIFFRACTIONf_plane_restr0.0051422
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.1423-2.21880.40923730.32963601X-RAY DIFFRACTION79
2.2188-2.30760.39084400.29763985X-RAY DIFFRACTION88
2.3076-2.41260.34044520.27544147X-RAY DIFFRACTION90
2.4126-2.53970.35794680.26354094X-RAY DIFFRACTION91
2.5397-2.69870.32924970.24184239X-RAY DIFFRACTION93
2.6987-2.90690.31224990.22924269X-RAY DIFFRACTION94
2.9069-3.19910.28334820.22094440X-RAY DIFFRACTION97
3.1991-3.66120.28065030.19234492X-RAY DIFFRACTION98
3.6612-4.60970.24235110.1584540X-RAY DIFFRACTION99
4.6097-28.84250.23415230.17444673X-RAY DIFFRACTION99

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