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- PDB-6b3q: Cryo-EM structure of human insulin degrading enzyme in complex wi... -

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Entry
Database: PDB / ID: 6b3q
TitleCryo-EM structure of human insulin degrading enzyme in complex with insulin
DescriptorInsulin-degrading enzyme (E.C.3.4.24.56), Insulin
KeywordsHYDROLASE/HORMONE / IDE / insulin degrading enzyme / amyloid beta / HYDROLASE-HORMONE complex
Specimen sourceHomo sapiens / human
MethodElectron microscopy (3.7 Å resolution / Particle / Single particle)
AuthorsLiang, W.G. / Zhang, Z. / Bailey, L.J. / Kossiakoff, A.A. / Tan, Y.Z. / Wei, H. / Carragher, B. / Potter, S.C. / Tang, W.J.
CitationTo Be Published

To Be Published Search PubMed
Ensemble cryo-EM elucidates the mechanism of insulin capture and degradation by human insulin degrading enzyme
Zhang, Z. / Liang, G.W. / Bailey, J.L. / Tan, Y.Z. / Wei, H. / McCord, A.L. / Woods, A. / Farcasanu, M. / Wang, A. / Lee, D. / Shang, W. / Meredith, C.S. / Deprez-Poulain, R. / Deprez, B. / Liu, D. / Koide, S. / Lissiakoff, A.A. / Li, S. / Carraghter, B. / Potter, S.C. / Tang, W.J.

Validation Report
SummaryFull reportAbout validation report
DateDeposition: Sep 22, 2017 / Release: Nov 22, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0Nov 22, 2017Structure modelrepositoryInitial release
1.1Dec 6, 2017Structure modelAuthor supporting evidencepdbx_audit_support_pdbx_audit_support.funding_organization

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Structure visualization

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Assembly

Deposited unit
A: Insulin-degrading enzyme
B: Insulin-degrading enzyme
a: Insulin
b: Insulin


Theoretical massNumber of molelcules
Total (without water)253,1034
Polyers253,1034
Non-polymers00
Water0
#1
A: Insulin-degrading enzyme
a: Insulin


Theoretical massNumber of molelcules
Total (without water)126,5512
Polyers126,5512
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551
#2
B: Insulin-degrading enzyme
b: Insulin


Theoretical massNumber of molelcules
Total (without water)126,5512
Polyers126,5512
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Polypeptide(L)Insulin-degrading enzyme / Abeta-degrading protease / Insulin protease / Insulinase / Insulysin


Mass: 114561.562 Da / Num. of mol.: 2 / Fragment: residues 42-1019 / Source: (gene. exp.) Homo sapiens / human / References: UniProt: P14735, EC: 3.4.24.56
#2: Polypeptide(L)Insulin


Mass: 11989.862 Da / Num. of mol.: 2 / Source: (gene. exp.) Homo sapiens / human / References: UniProt: P01308

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE

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Sample preparation

ComponentName: Insulin degrading enzyme/Insulin / Type: COMPLEX
Details: Cryo-EM structure of human insulin degrading enzyme in complex with insulin
Entity ID: 1,2 / Source: RECOMBINANT
Molecular weightValue: 0.1 deg. / Units: MEGADALTONS / Experimental value: YES
Source (natural)Organism: Homo sapiens
Source (recombinant)Organism: Escherichia coli / Strain: BL
Buffer solutionpH: 7.8
Buffer component
IDConc.UnitsNameFormulaBuffer ID
120mmol/LHEPESC8H18N2O4S1
2300mmol/LSodium chlorideNaCl1
320mmol/LEDTAC10H16N2O81
SpecimenConc.: 0.3 mg/ml / Details: The sample was monodisperse / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 / Grid type: homemade nanowire grid
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 85 % / Chamber temperature: 298 kelvins
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
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / 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 recordingAverage exposure time: 10 sec. / Electron dose: 71.4 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 3 / Number of real images: 3085
Image scansSampling size: 5 microns / Dimension width: 3710 / Dimension height: 3838 / Movie frames/image: 50 / Used frames/image: 1-50

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameVersionCategoryImage processing IDImaging IDFitting ID
1DoG PickerPARTICLE SELECTION1
2Leginon3.3IMAGE ACQUISITION1
4GctfGctf-v0.50_sm_30_cu7.5_x86_64CTF CORRECTION1
7CootCoot 0.8.9_preMODEL FITTING1
8ChimeraChimera 1.11.2MODEL FITTING1
10RELION2.0INITIAL EULER ASSIGNMENT1
11RELION2.0FINAL EULER ASSIGNMENT1
12RELION2.0CLASSIFICATION1
13RELION2.1RECONSTRUCTION1
20PHENIXPhenix-1.12-2829MODEL REFINEMENT1
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNumber of particles selected: 762283
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 116122 / 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_d0.00516115
ELECTRON MICROSCOPYf_angle_d1.04121795
ELECTRON MICROSCOPYf_dihedral_angle_d9.0219745
ELECTRON MICROSCOPYf_chiral_restr0.0592352
ELECTRON MICROSCOPYf_plane_restr0.0082815

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