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- PDB-6hn4: Leucine-zippered human insulin receptor ectodomain with single bo... -

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Basic information

Entry
Database: PDB / ID: 6hn4
TitleLeucine-zippered human insulin receptor ectodomain with single bound insulin - "lower" membrane-proximal part
ComponentsInsulin receptor,Insulin receptor,General control protein GCN4
KeywordsSIGNALING PROTEIN / insulin / insulin receptor ectodomain / signal transdution
Function / homologyBasic-leucine zipper domain / Fibronectin type III / Protein kinase-like domain superfamily / Basic-leucine zipper (bZIP) domain signature. / Basic region leucine zipper / Protein tyrosine kinase / Receptor L domain / Furin-like cysteine rich region / Receptor L-domain superfamily / Fibronectin type III superfamily ...Basic-leucine zipper domain / Fibronectin type III / Protein kinase-like domain superfamily / Basic-leucine zipper (bZIP) domain signature. / Basic region leucine zipper / Protein tyrosine kinase / Receptor L domain / Furin-like cysteine rich region / Receptor L-domain superfamily / Fibronectin type III superfamily / Receptor L-domain / Tyrosine protein kinases specific active-site signature. / Tyrosine-protein kinase, catalytic domain / Protein kinase domain / Furin-like repeat / Tyrosine-protein kinase, active site / Protein kinase, ATP binding site / Tyrosine-protein kinase, insulin-like receptor / Growth factor receptor cysteine-rich domain superfamily / Immunoglobulin-like fold / Protein kinases ATP-binding region signature. / Serine-threonine/tyrosine-protein kinase, catalytic domain / Receptor tyrosine kinase class II signature. / PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling / Furin-like cysteine-rich domain / Insulin receptor recycling / Signaling by Insulin receptor / Insulin receptor signalling cascade / Protein kinase domain profile. / Tyrosine-protein kinase, receptor class II, conserved site / IRS activation / Signal attenuation / Fibronectin type-III domain profile. / Basic-leucine zipper (bZIP) domain profile. / regulation of female gonad development / positive regulation of meiotic cell cycle / exocrine pancreas development / positive regulation of developmental growth / male sex determination / insulin-like growth factor II binding / insulin receptor complex / insulin-like growth factor I binding / insulin-activated receptor activity / positive regulation of protein complex disassembly / positive regulation of transcription from RNA polymerase II promoter in response to nitrogen starvation / neuronal cell body membrane / positive regulation of cellular response to amino acid starvation / negative regulation of ribosomal protein gene transcription from RNA polymerase II promoter in response to nutrient levels / adrenal gland development / regulation of embryonic development / RNA polymerase II transcription regulator recruiting activity / dendritic spine maintenance / dendrite membrane / PTB domain binding / nitrogen catabolite activation of transcription from RNA polymerase II promoter / insulin binding / positive regulation of transcription initiation from RNA polymerase II promoter / epidermis development / amyloid-beta clearance / cellular amino acid biosynthetic process / positive regulation of respiratory burst / insulin receptor substrate binding / positive regulation of autophagy / heart morphogenesis / transformation of host cell by virus / phosphatidylinositol 3-kinase binding / positive regulation of glycogen biosynthetic process / positive regulation of glycolytic process / response to organic substance / cellular response to growth factor stimulus / neuron projection maintenance / insulin-like growth factor receptor binding / learning / positive regulation of mitotic nuclear division / activation of protein kinase activity / memory / positive regulation of RNA polymerase II transcriptional preinitiation complex assembly / RNA polymerase II transcription factor binding / caveola / activation of protein kinase B activity / positive regulation of glucose import / protein heterotetramerization / transmembrane receptor protein tyrosine kinase activity / receptor protein-tyrosine kinase / peptidyl-tyrosine autophosphorylation / male gonad development / protein self-association / insulin receptor signaling pathway / cellular response to insulin stimulus / peptidyl-tyrosine phosphorylation / glucose homeostasis / negative regulation of signal transduction / amyloid-beta binding / transcription factor complex / positive regulation of MAPK cascade / positive regulation of nitric oxide biosynthetic process / transmembrane signaling receptor activity / activation of MAPK activity / receptor complex / endosome membrane
Function and homology information
Specimen sourceHomo sapiens (human)
Saccharomyces cerevisiae (baker's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 4.2 Å resolution
AuthorsWeis, F. / Menting, J.G. / Margetts, M.B. / Chan, S.J. / Xu, Y. / Tennagels, N. / Wohlfart, P. / Langer, T. / Mueller, C.W. / Dreyer, M.K. / Lawrence, M.C.
CitationJournal: Nat Commun / Year: 2018
Title: The signalling conformation of the insulin receptor ectodomain.
Authors: Felix Weis / John G Menting / Mai B Margetts / Shu Jin Chan / Yibin Xu / Norbert Tennagels / Paulus Wohlfart / Thomas Langer / Christoph W Müller / Matthias K Dreyer / Michael C Lawrence
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Sep 14, 2018 / Release: Nov 21, 2018

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Assembly

Deposited unit
E: Insulin receptor,Insulin receptor,General control protein GCN4
F: Insulin receptor,Insulin receptor,General control protein GCN4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)214,7848
Polyers213,4562
Non-polymers1,3276
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)4080
ΔGint (kcal/M)24
Surface area (Å2)37960
MethodPISA

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Components

#1: Protein/peptide Insulin receptor,Insulin receptor,General control protein GCN4 / IR / IR / Amino acid biosynthesis regulatory protein


Mass: 106728.211 Da / Num. of mol.: 2
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Gene: INSR, GCN4, AAS3, ARG9, YEL009C / Plasmid name: pEE14 / Strain: ATCC 204508 / S288c / Cell line (production host): Lec8 / Production host: Cricetulus griseus (Chinese hamster)
References: UniProt: P06213, UniProt: P03069, receptor protein-tyrosine kinase
#2: Chemical
ChemComp-NAG / N-ACETYL-D-GLUCOSAMINE


Mass: 221.208 Da / Num. of mol.: 6 / Formula: C8H15NO6 / N-Acetylglucosamine

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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: Leucine zippered human insulin receptor ectodomain (IR-A isoform, "deltabeta" mutant) in complex with insulin and two Fv 83-7 modules : "lower" membrane-proximal part
Type: COMPLEX
Details: Note: Attached to the leucine-zippered insulin receptor ectodomain are two Fv 83-7 modules. One of these is present within this map volume but it is very poorly ordered and thus left completely unmodelled. See the manuscript for further details.
Entity ID: 1 / Source: RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Cell: Lec8 / Organism: Cricetulus griseus (Chinese hamster) / Plasmid: pEE14
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer ID
110 mMHEPES-NaOH1
20.02 %sodium azide1
SpecimenConc.: 0.094 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283.15 kelvins

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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 FIELDBright-field microscopy / Nominal magnification: 130000 / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 microns / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 16 sec. / Electron dose: 1.85 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 1 / Number of real images: 2287
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 20 / Used frames/image: 1-20

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Processing

SoftwareName: PHENIX / Version: 1.13-2998_1692: / Classification: refinement
EM software
IDNameVersionCategory
1RELION2.1particle selection
2SerialEMimage acquisition
4GctfCTF correction
7UCSF Chimera1.11.2model fitting
8Coot0.8.7model fitting
10PHENIX1.13-2998-1692model refinement
11RELION2.1initial Euler assignment
12RELION2.1final Euler assignment
13RELION2.1classification
14RELION2.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNumber of particles selected: 747074
SymmetryPoint symmetry: C1
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 98481 / Algorithm: BACK PROJECTION / Number of class averages: 2 / Symmetry type: POINT
Atomic model buildingRef protocol: OTHER / Ref space: REAL / Target criteria: correlation coefficient
Atomic model buildingPDB-ID: 4ZXB

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