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- EMDB-7041: Cryo-EM structure of human insulin degrading enzyme in complex wi... -

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Entry
Database: EMDB / ID: 7041
TitleCryo-EM structure of human insulin degrading enzyme in complex with insulin
Map dataThis is the sharpened map from the final, Insulin degrading enzyme in complex with insulin
SampleInsulin degrading enzyme/Insulin:
Insulin-degrading enzyme / Insulin
Function / homologyMiddle or third domain of peptidase_M16 / Insulin family signature. / Insulin-like / Insulin family / Peptidase M16, C-terminal / Insulin, conserved site / Peptidase M16, middle/third domain / Insulin-like superfamily / Insulin/IGF/Relaxin family / Insulinase (Peptidase family M16) ...Middle or third domain of peptidase_M16 / Insulin family signature. / Insulin-like / Insulin family / Peptidase M16, C-terminal / Insulin, conserved site / Peptidase M16, middle/third domain / Insulin-like superfamily / Insulin/IGF/Relaxin family / Insulinase (Peptidase family M16) / Peptidase M16 inactive domain / Insulin / Peptidase M16, zinc-binding site / Insulinase family, zinc-binding region signature. / Regulation of gene expression in beta cells / Metalloenzyme, LuxS/M16 peptidase-like / Insulin processing / Synthesis, secretion, and deacylation of Ghrelin / Regulation of insulin secretion / Ub-specific processing proteases / COPI-mediated anterograde transport / PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling / IRS activation / Signal attenuation / Insulin receptor signalling cascade / Signaling by Insulin receptor / Insulin receptor recycling / Peroxisomal protein import / Amyloid fiber formation / Peptidase M16, N-terminal / insulysin / beta-endorphin binding / bradykinin catabolic process / insulin catabolic process / insulin metabolic process / ubiquitin recycling / ubiquitin-dependent protein binding / hormone catabolic process / cytosolic proteasome complex / insulin binding / amyloid-beta clearance / alpha-beta T cell activation / negative regulation of glycogen catabolic process / negative regulation of fatty acid metabolic process / negative regulation of feeding behavior / negative regulation of NAD(P)H oxidase activity / determination of adult lifespan / regulation of cellular amino acid metabolic process / regulation of transmembrane transporter activity / positive regulation of respiratory burst / regulation of protein secretion / negative regulation of respiratory burst involved in inflammatory response / peroxisomal matrix / negative regulation of protein oligomerization / positive regulation of peptide hormone secretion / positive regulation of dendritic spine maintenance / negative regulation of oxidative stress-induced intrinsic apoptotic signaling pathway / positive regulation of lipid biosynthetic process / negative regulation of blood vessel diameter / negative regulation of gluconeogenesis / negative regulation of protein secretion / positive regulation of protein oligomerization / negative regulation of reactive oxygen species biosynthetic process / cognition / negative regulation of lipid catabolic process / positive regulation of cellular protein metabolic process / fatty acid homeostasis / regulation of protein localization to plasma membrane / positive regulation of glycolytic process / positive regulation of insulin receptor signaling pathway / positive regulation of glycogen biosynthetic process / positive regulation of nitric oxide mediated signal transduction / transport vesicle / endoplasmic reticulum-Golgi intermediate compartment membrane / regulation of synaptic plasticity / endosome lumen / positive regulation of brown fat cell differentiation / insulin-like growth factor receptor binding / neuron projection maintenance / positive regulation of protein autophosphorylation / amyloid-beta metabolic process / positive regulation of glucose import in response to insulin stimulus / protein heterooligomerization / positive regulation of cell differentiation / negative regulation of acute inflammatory response / regulation of protein localization / positive regulation of cytokine secretion / proteolysis involved in cellular protein catabolic process / positive regulation of mitotic nuclear division / protein targeting to peroxisome / positive regulation of protein catabolic process / positive regulation of long-term synaptic potentiation / peptide binding / positive regulation of glucose import / activation of protein kinase B activity / negative regulation of protein catabolic process / positive regulation of nitric-oxide synthase activity / negative regulation of proteolysis / insulin receptor binding / insulin receptor signaling pathway
Function and homology information
SourceHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / 3.7 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: 6b3q

SummaryFull report
PDB-ID: 6bfc

SummaryFull report
About validation report
DateDeposition: Sep 22, 2017 / Header (metadata) release: Nov 22, 2017 / Map release: Nov 22, 2017 / Last update: Jul 18, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.053
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.053
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6b3q
  • Surface level: 0.053
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: : PDB-6bfc
  • Surface level: 0.053
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_7041.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:0.053 (by author), 0.053 (movie #1):
Minimum - Maximum-0.23219995 - 0.18164448
Average (Standard dev.)0.00027707088 (0.0042306143)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions320320320
Origin0.0.0.
Limit319.319.319.
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-0.2320.1820.000

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Supplemental data

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Mask #1

Fileemd_7041_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/Insulin

EntireName: Insulin degrading enzyme/Insulin
Details: Cryo-EM structure of human insulin degrading enzyme in complex with insulin
Number of components: 3

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Component #1: protein, Insulin degrading enzyme/Insulin

ProteinName: Insulin degrading enzyme/Insulin
Details: Cryo-EM structure of human insulin degrading enzyme in complex with insulin
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 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 114.561562 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: protein, Insulin

ProteinName: Insulin / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 11.989862 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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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
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 298 K / Humidity: 85 %
Details: The cryo grids were made using Spotiton and homemade plunger.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 71.4 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 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 3085 / Sampling size: 5 microns

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 116122
3D reconstructionAlgorithm: FOURIER SPACE / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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Atomic model buiding

Modeling #1Refinement space: REAL / Overall bvalue: 92
Output model

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