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- EMDB-7065: Cryo-EM structure of human insulin degrading enzyme -

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Entry
Database: EMDB / ID: 7065
TitleCryo-EM structure of human insulin degrading enzyme
Map dataInsulin degrading enzyme in complex with insulinInsulin-degrading enzyme
SampleInsulin degrading enzymeInsulin-degrading enzyme:
Insulin-degrading enzyme
Function / homologyPeptidase M16, N-terminal / Peptidase M16, zinc-binding site / Peroxisomal protein import / Ub-specific processing proteases / Insulinase family, zinc-binding region signature. / Middle or third domain of peptidase_M16 / Peptidase M16 inactive domain / Insulinase (Peptidase family M16) / Peptidase M16, middle/third domain / Metalloenzyme, LuxS/M16 peptidase-like ...Peptidase M16, N-terminal / Peptidase M16, zinc-binding site / Peroxisomal protein import / Ub-specific processing proteases / Insulinase family, zinc-binding region signature. / Middle or third domain of peptidase_M16 / Peptidase M16 inactive domain / Insulinase (Peptidase family M16) / Peptidase M16, middle/third domain / Metalloenzyme, LuxS/M16 peptidase-like / Peptidase M16, C-terminal / 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 / virus receptor activity / metalloendopeptidase activity / protein homotetramerization / protein homooligomerization / ATPase activity / proteolysis / signaling receptor binding / cell surface / mitochondrion / protein homodimerization activity / zinc ion binding / extracellular space / ATP binding / identical protein binding / plasma membrane / nucleus / cytosol / cytoplasm / Insulin-degrading enzyme
Function and homology information
SourceHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / 6.5 Å resolution
AuthorsZhang Z / Liang WG / Bailey LJ / Tan YZ / Wei H / Kossiakoff AA / Carragher B / Potter SC / Tang WJ
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 ReportPDB-ID: 6b7y

SummaryFull reportAbout validation report
DateDeposition: Oct 5, 2017 / Header (metadata) release: Nov 8, 2017 / Map release: Nov 8, 2017 / Last update: Apr 11, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.014
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6b7y
  • Surface level: 0.014
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_7065.map.gz (map file in CCP4 format, 131073 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
320 pix
1.07 Å/pix.
= 343.36 Å
320 pix
1.07 Å/pix.
= 343.36 Å
320 pix
1.07 Å/pix.
= 343.36 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.073 Å
Density
Contour Level:0.014 (by author), 0.014 (movie #1):
Minimum - Maximum-0.1315158 - 0.08239051
Average (Standard dev.)0.00026647426 (0.003625321)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions320320320
Origin000
Limit319319319
Spacing320320320
CellA=B=C: 343.36 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0731.0731.073
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z343.360343.360343.360
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.1320.0820.000

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Supplemental data

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Mask #1

Fileemd_7065_msk_1.map ( map file in CCP4 format, 131073 KB )
Projections & Slices
AxesZYX
Projections
Slices (1/2)
Density Histograms
Data typeImage stored as Reals
Annotation detailsInsulin degrading enzyme in complex with insulin
Space group number1

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Mask #1~

Fileemd_7065_msk_1.map
Projections & Slices
AxesZYX
Projections
Slices (1/2)
Density Histograms

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Sample components

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Entire Insulin degrading enzyme

EntireName: Insulin degrading enzyme
Details: Cryo-EM structure of human Apo insulin degrading enzyme
Number of components: 2
MassExperimental: 100 kDa

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Component #1: protein, Insulin degrading enzyme

ProteinName: Insulin degrading enzymeInsulin-degrading enzyme
Details: Cryo-EM structure of human Apo insulin degrading enzyme
Recombinant expression: No
MassExperimental: 100 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli) / Strain: BL

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Component #2: protein, Insulin-degrading enzyme

ProteinName: Insulin-degrading enzyme / Recombinant expression: No
MassTheoretical: 111.866484 kDa
Source (engineered)Expression System: Homo sapiens (human)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.3 mg/ml / pH: 7.8
Support filmThe grids are homemade lacey gold nanowire grids
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 298 K / Humidity: 85 % / Details: The cryo grids were made using Spotiton

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Details: The image was collected at 20-50 degree tilt
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 6.8 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 22500 X (nominal), 46598 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 940.0 - 2200.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: K ( 70.0 - 70.0 K)
CameraDetector: GATAN K2 (4k x 4k)

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Image acquisition

Image acquisition #1Number of digital images: 509
Image acquisition #2Number of digital images: 620 / Sampling size: 5 microns

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 24425
3D reconstructionAlgorithm: FOURIER SPACE / Software: RELION / Resolution: 6.5 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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Atomic model buiding

Output model

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