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- PDB-6b7z: Cryo-EM structure of human insulin degrading enzyme in complex wi... -

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Basic information

Entry
Database: PDB / ID: 6b7z
TitleCryo-EM structure of human insulin degrading enzyme in complex with FAB H11 heavy chain and FAB H11 light chain
Components
  • FAB H11 heavy chain
  • FAB H11 light chain
  • Insulin-degrading enzyme
KeywordsHYDROLASE/IMMUNE SYSTEM / IDE / insulin degrading enzyme / amyloid beta / BIOSYNTHETIC PROTEIN / HYDROLASE-IMMUNE SYSTEM complex
Function / homologyPeptidase M16, C-terminal / Immunoglobulin-like domain superfamily / Immunoglobulin subtype / Immunoglobulin C1-set / Metalloenzyme, LuxS/M16 peptidase-like / Peptidase M16, N-terminal / Immunoglobulin/major histocompatibility complex, conserved site / Immunoglobulin V-set domain / Immunoglobulin-like fold / Peptidase M16, middle/third domain ...Peptidase M16, C-terminal / Immunoglobulin-like domain superfamily / Immunoglobulin subtype / Immunoglobulin C1-set / Metalloenzyme, LuxS/M16 peptidase-like / Peptidase M16, N-terminal / Immunoglobulin/major histocompatibility complex, conserved site / Immunoglobulin V-set domain / Immunoglobulin-like fold / Peptidase M16, middle/third domain / Peptidase M16, zinc-binding site / Insulinase (Peptidase family M16) / Immunoglobulin-like domain / Peptidase M16 inactive domain / Immunoglobulin C1-set domain / Immunoglobulin V-set domain / Middle or third domain of peptidase_M16 / Insulinase family, zinc-binding region signature. / Immunoglobulins and major histocompatibility complex proteins signature. / Ig-like domain profile. / Ub-specific processing proteases / Peroxisomal protein import / beta-endorphin binding / insulysin / bradykinin catabolic process / insulin catabolic process / ubiquitin recycling / insulin metabolic process / ubiquitin-dependent protein binding / hormone catabolic process / cytosolic proteasome complex / insulin binding / amyloid-beta clearance / determination of adult lifespan / peroxisomal matrix / positive regulation of protein oligomerization / protein heterooligomerization / amyloid-beta metabolic process / proteolysis involved in cellular protein catabolic process / protein targeting to peroxisome / positive regulation of protein catabolic process / peptide binding / negative regulation of proteolysis / insulin receptor signaling pathway / peroxisome / antigen binding / virus receptor activity / metalloendopeptidase activity / adaptive immune response / protein homotetramerization / protein homooligomerization / ATPase activity / proteolysis / signaling receptor binding / cell surface / mitochondrion / protein homodimerization activity / zinc ion binding / extracellular space / extracellular region / ATP binding / identical protein binding / plasma membrane / nucleus / cytosol / cytoplasm / Immunoglobulin gamma-1 heavy chain / Insulin-degrading enzyme / Uncharacterized protein
Function and homology information
Specimen sourceHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 6.5 Å resolution
AuthorsLiang, W.G. / Zhang, Z. / Bailey, L.J. / Kossiakoff, A.A. / Tan, Y.Z. / Wei, H. / Carragher, B. / Potter, S.C. / Tang, W.J.
CitationJournal: Elife / Year: 2018
Title: Ensemble cryoEM elucidates the mechanism of insulin capture and degradation by human insulin degrading enzyme.
Authors: Zhening Zhang / Wenguang G Liang / Lucas J Bailey / Yong Zi Tan / Hui Wei / Andrew Wang / Mara Farcasanu / Virgil A Woods / Lauren A McCord / David Lee / Weifeng Shang / Rebecca Deprez-Poulain / Benoit Deprez / David R Liu / Akiko Koide / Shohei Koide / Anthony A Kossiakoff / Sheng Li / Bridget Carragher / Clinton S Potter / Wei-Jen Tang
Abstract: Insulin degrading enzyme (IDE) plays key roles in degrading peptides vital in type two diabetes, Alzheimer's, inflammation, and other human diseases. However, the process through which IDE recognizes ...Insulin degrading enzyme (IDE) plays key roles in degrading peptides vital in type two diabetes, Alzheimer's, inflammation, and other human diseases. However, the process through which IDE recognizes peptides that tend to form amyloid fibrils remained unsolved. We used cryoEM to understand both the apo- and insulin-bound dimeric IDE states, revealing that IDE displays a large opening between the homologous ~55 kDa N- and C-terminal halves to allow selective substrate capture based on size and charge complementarity. We also used cryoEM, X-ray crystallography, SAXS, and HDX-MS to elucidate the molecular basis of how amyloidogenic peptides stabilize the disordered IDE catalytic cleft, thereby inducing selective degradation by substrate-assisted catalysis. Furthermore, our insulin-bound IDE structures explain how IDE processively degrades insulin by stochastically cutting either chain without breaking disulfide bonds. Together, our studies provide a mechanism for how IDE selectively degrades amyloidogenic peptides and offers structural insights for developing IDE-based therapies.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Oct 5, 2017 / Release: Jan 10, 2018
RevisionDateData content typeGroupCategoryItemProviderType
1.0Jan 10, 2018Structure modelrepositoryInitial release
1.1Jan 17, 2018Structure modelAuthor supporting evidencepdbx_audit_support_pdbx_audit_support.funding_organization
1.2Apr 11, 2018Structure modelData collection / Database referencescitation / citation_author_citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.name

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Structure visualization

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  • Biological unit as author_defined_assembly
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  • Biological unit as author_defined_assembly
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  • Deposited structure unit
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  • Superimposition on EM map
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  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Insulin-degrading enzyme
B: Insulin-degrading enzyme
C: FAB H11 heavy chain
D: FAB H11 light chain
E: FAB H11 heavy chain
F: FAB H11 light chain


Theoretical massNumber of molelcules
Total (without water)316,0246
Polyers316,0246
Non-polymers00
Water0
1
A: Insulin-degrading enzyme
C: FAB H11 heavy chain
D: FAB H11 light chain


Theoretical massNumber of molelcules
Total (without water)158,0123
Polyers158,0123
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551
2
B: Insulin-degrading enzyme
E: FAB H11 heavy chain
F: FAB H11 light chain


Theoretical massNumber of molelcules
Total (without water)158,0123
Polyers158,0123
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 111866.484 Da / Num. of mol.: 2 / Fragment: residues 46-1011 / Source: (gene. exp.) Homo sapiens (human) / Gene: IDE / Production host: Escherichia coli (E. coli) / References: UniProt: P14735, insulysin
#2: Protein/peptide FAB H11 heavy chain


Mass: 23057.748 Da / Num. of mol.: 2
Source: (gene. exp.) Mus musculus (house mouse), (gene. exp.) Homo sapiens (human)
Production host: Escherichia coli (E. coli) / References: UniProt: P0DOX5
#3: Protein/peptide FAB H11 light chain


Mass: 23087.609 Da / Num. of mol.: 2
Source: (gene. exp.) Mus musculus (house mouse), (gene. exp.) Homo sapiens (human)
Production host: Escherichia coli (E. coli) / References: UniProt: Q6GMX0

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Insulin degrading enzymeInsulin-degrading enzyme / Type: COMPLEX
Details: Cryo-EM structure of human Apo insulin degrading enzyme
Entity ID: 1,2,3 / Source: RECOMBINANT
Molecular weightValue: 0.1 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL
Buffer solutionpH: 7.8
Buffer component
IDConc.NameFormulaBuffer ID
120 mmol/LHEPESC8H18N2O4S1
2300 mmol/LSodium chlorideNaCl1
320 mmol/LEDTAC10H16N2O81
SpecimenConc.: 0.3 mg/ml / Details: The sample was monodisperse / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: The grids are homemade lacey gold nanowire grids / Grid material: GOLD / Grid mesh size: 300 / Grid type: Homemade
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 85 % / Chamber temperature: 298 kelvins / Details: The cryo grids were made using Spotiton

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS / Details: The image was collected at 20-50 degree tilt
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 22500 / Calibrated magnification: 46598 / Nominal defocus max: 2200 nm / Nominal defocus min: 940 nm / Cs: 2.7 mm / C2 aperture diameter: 70 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 kelvins / Temperature (min): 70 kelvins / Residual tilt: 10 mradians
Image recording

Imaging ID: 1 / Average exposure time: 10 sec. / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 1

IDElectron doseNumber of real images
17.9509
26.8620
Image scansSampling size: 5 microns / Width: 3710 / Height: 3820 / Movie frames/image: 50 / Used frames/image: 1-50

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameVersionCategory
1DoG Pickerparticle selection
2Leginon3.3image acquisition
4GctfGctf-v0.50_sm_30_cu7.5_x86_64CTF correction
7CootCoot 0.8.9_premodel fitting
8UCSF ChimeraChimera 1.11.2model fitting
10PHENIXPhenix-1.12-2829model refinement
11RELION2.0initial Euler assignment
12RELION2.0final Euler assignment
13RELION2.0classification
14RELION2.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNumber of particles selected: 762283
3D reconstructionResolution: 6.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 16944 / Algorithm: FOURIER SPACE / Number of class averages: 1 / Symmetry type: POINT
Atomic model buildingOverall b value: 92 / Ref protocol: FLEXIBLE FIT / Ref space: REAL
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00822447
ELECTRON MICROSCOPYf_angle_d1.33930423
ELECTRON MICROSCOPYf_dihedral_angle_d8.77413510
ELECTRON MICROSCOPYf_chiral_restr0.0703329
ELECTRON MICROSCOPYf_plane_restr0.0083913

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