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- EMDB-0247: Leucine-zippered human insulin receptor ectodomain with single bo... -

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Basic information

Entry
Database: EMDB / ID: EMD-0247
TitleLeucine-zippered human insulin receptor ectodomain with single bound insulin - "upper" membrane-distal part
Map datahuman insulin receptor ectodomain with insulin bound - "upper" membrane-distal part
Sample
  • Complex: Leucine zippered human insulin receptor ectodomain (IR-A isoform, "deltabeta" mutant) in complex with insulin and two Fv 83-7 modules : "upper" membrane-distal part
    • Complex: insulin
      • Protein or peptide: Insulin
      • Protein or peptide: Insulin
    • Complex: Leucine zippered human insulin receptor ectodomain
      • Protein or peptide: Insulin receptor,General control protein GCN4
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homology
Function and homology information


regulation of female gonad development / positive regulation of meiotic cell cycle / protein localization to nuclear periphery / Activation of the AP-1 family of transcription factors / positive regulation of developmental growth / response to amino acid starvation / insulin-like growth factor II binding / mediator complex binding / negative regulation of ribosomal protein gene transcription by RNA polymerase II / positive regulation of cellular response to amino acid starvation ...regulation of female gonad development / positive regulation of meiotic cell cycle / protein localization to nuclear periphery / Activation of the AP-1 family of transcription factors / positive regulation of developmental growth / response to amino acid starvation / insulin-like growth factor II binding / mediator complex binding / negative regulation of ribosomal protein gene transcription by RNA polymerase II / positive regulation of cellular response to amino acid starvation / male sex determination / exocrine pancreas development / insulin receptor complex / insulin-like growth factor I binding / insulin receptor activity / nitrogen catabolite activation of transcription from RNA polymerase II promoter / positive regulation of protein-containing complex disassembly / cargo receptor activity / dendritic spine maintenance / insulin binding / PTB domain binding / negative regulation of NAD(P)H oxidase activity / neuronal cell body membrane / adrenal gland development / negative regulation of glycogen catabolic process / regulation of cellular amino acid metabolic process / Signaling by Insulin receptor / IRS activation / nitric oxide-cGMP-mediated signaling / negative regulation of fatty acid metabolic process / Insulin processing / negative regulation of feeding behavior / regulation of protein secretion / amyloid-beta clearance / positive regulation of peptide hormone secretion / activation of protein kinase activity / Regulation of gene expression in beta cells / positive regulation of respiratory burst / TFIID-class transcription factor complex binding / regulation of embryonic development / positive regulation of receptor internalization / transport across blood-brain barrier / positive regulation of dendritic spine maintenance / alpha-beta T cell activation / amino acid biosynthetic process / negative regulation of acute inflammatory response / negative regulation of respiratory burst involved in inflammatory response / insulin receptor substrate binding / negative regulation of protein secretion / positive regulation of transcription initiation by RNA polymerase II / positive regulation of RNA polymerase II transcription preinitiation complex assembly / fatty acid homeostasis / Synthesis, secretion, and deacylation of Ghrelin / epidermis development / positive regulation of glycogen biosynthetic process / positive regulation of lipid biosynthetic process / Signal attenuation / FOXO-mediated transcription of oxidative stress, metabolic and neuronal genes / negative regulation of gluconeogenesis / positive regulation of nitric oxide mediated signal transduction / regulation of protein localization to plasma membrane / COPI-mediated anterograde transport / phosphatidylinositol 3-kinase binding / negative regulation of lipid catabolic process / heart morphogenesis / negative regulation of oxidative stress-induced intrinsic apoptotic signaling pathway / negative regulation of reactive oxygen species biosynthetic process / positive regulation of insulin receptor signaling pathway / transport vesicle / positive regulation of protein autophosphorylation / dendrite membrane / Insulin receptor recycling / insulin-like growth factor receptor binding / cellular response to amino acid starvation / receptor-mediated endocytosis / NPAS4 regulates expression of target genes / positive regulation of protein metabolic process / neuron projection maintenance / endoplasmic reticulum-Golgi intermediate compartment membrane / positive regulation of brown fat cell differentiation / activation of protein kinase B activity / positive regulation of glycolytic process / Insulin receptor signalling cascade / positive regulation of mitotic nuclear division / learning / Regulation of insulin secretion / positive regulation of nitric-oxide synthase activity / positive regulation of long-term synaptic potentiation / caveola / endosome lumen / positive regulation of cytokine production / acute-phase response / positive regulation of protein secretion / regulation of transmembrane transporter activity / positive regulation of cell differentiation / positive regulation of glucose import / negative regulation of proteolysis / regulation of synaptic plasticity / positive regulation of MAP kinase activity / wound healing
Similarity search - Function
Insulin receptor, trans-membrane domain / Insulin receptor trans-membrane segment / Tyrosine-protein kinase, insulin-like receptor / Basic region leucine zipper / Basic-leucine zipper (bZIP) domain signature. / Basic-leucine zipper (bZIP) domain profile. / basic region leucin zipper / Basic-leucine zipper domain superfamily / Basic-leucine zipper domain / Tyrosine-protein kinase, receptor class II, conserved site ...Insulin receptor, trans-membrane domain / Insulin receptor trans-membrane segment / Tyrosine-protein kinase, insulin-like receptor / Basic region leucine zipper / Basic-leucine zipper (bZIP) domain signature. / Basic-leucine zipper (bZIP) domain profile. / basic region leucin zipper / Basic-leucine zipper domain superfamily / Basic-leucine zipper domain / Tyrosine-protein kinase, receptor class II, conserved site / Receptor tyrosine kinase class II signature. / Insulin / Insulin family / Insulin/IGF/Relaxin family / Insulin, conserved site / Insulin family signature. / Insulin-like / Insulin / insulin-like growth factor / relaxin family. / Insulin-like superfamily / Receptor L-domain / Furin-like cysteine-rich domain / Receptor L-domain superfamily / Furin-like cysteine rich region / Receptor L domain / Furin-like repeat / Furin-like repeats / Growth factor receptor cysteine-rich domain superfamily / Fibronectin type III domain / Fibronectin type 3 domain / Fibronectin type-III domain profile. / Fibronectin type III / Fibronectin type III superfamily / Tyrosine-protein kinase, catalytic domain / Tyrosine kinase, catalytic domain / Tyrosine protein kinases specific active-site signature. / Tyrosine-protein kinase, active site / Protein tyrosine and serine/threonine kinase / Serine-threonine/tyrosine-protein kinase, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Immunoglobulin-like fold / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Insulin / General control transcription factor GCN4 / Insulin receptor
Similarity search - Component
Biological speciesHomo sapiens (human) / Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsWeis F / Menting JG / Margetts MB / Chan SJ / Xu Y / Tennagels N / Wohlfart P / Langer T / Mueller CW / Dreyer MK / Lawrence MC
Funding support Australia, United States, 4 items
OrganizationGrant numberCountry
National Health and Medical Research Council (Australia)APP1099595 Australia
National Health and Medical Research Council (Australia)APP1128553 Australia
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)DK013914 United States
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)UC DRTC DK020595 United States
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 /
Abstract: Understanding the structural biology of the insulin receptor and how it signals is of key importance in the development of insulin analogs to treat diabetes. We report here a cryo-electron microscopy ...Understanding the structural biology of the insulin receptor and how it signals is of key importance in the development of insulin analogs to treat diabetes. We report here a cryo-electron microscopy structure of a single insulin bound to a physiologically relevant, high-affinity version of the receptor ectodomain, the latter generated through attachment of C-terminal leucine zipper elements to overcome the conformational flexibility associated with ectodomain truncation. The resolution of the cryo-electron microscopy maps is 3.2 Å in the insulin-binding region and 4.2 Å in the membrane-proximal region. The structure reveals how the membrane proximal domains of the receptor come together to effect signalling and how insulin's negative cooperativity of binding likely arises. Our structure further provides insight into the high affinity of certain super-mitogenic insulins. Together, these findings provide a new platform for insulin analog investigation and design.
History
DepositionSep 14, 2018-
Header (metadata) releaseNov 21, 2018-
Map releaseNov 21, 2018-
UpdateMar 30, 2022-
Current statusMar 30, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6hn5
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0247.png

Downloads & links

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Map

FileDownload / File: emd_0247.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationhuman insulin receptor ectodomain with insulin bound - "upper" membrane-distal part
Voxel sizeX=Y=Z: 1.04 Å
Density
Contour LevelBy AUTHOR: 0.035 / Movie #1: 0.035
Minimum - Maximum-0.13270356 - 0.2162133
Average (Standard dev.)0.00019532986 (±0.0033116017)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 299.52 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.041.041.04
M x/y/z288288288
origin x/y/z0.0000.0000.000
length x/y/z299.520299.520299.520
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ240240240
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS288288288
D min/max/mean-0.1330.2160.000

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Supplemental data

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Sample components

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Entire : Leucine zippered human insulin receptor ectodomain (IR-A isoform,...

EntireName: Leucine zippered human insulin receptor ectodomain (IR-A isoform, "deltabeta" mutant) in complex with insulin and two Fv 83-7 modules : "upper" membrane-distal part
Components
  • Complex: Leucine zippered human insulin receptor ectodomain (IR-A isoform, "deltabeta" mutant) in complex with insulin and two Fv 83-7 modules : "upper" membrane-distal part
    • Complex: insulin
      • Protein or peptide: Insulin
      • Protein or peptide: Insulin
    • Complex: Leucine zippered human insulin receptor ectodomain
      • Protein or peptide: Insulin receptor,General control protein GCN4
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Leucine zippered human insulin receptor ectodomain (IR-A isoform,...

SupramoleculeName: Leucine zippered human insulin receptor ectodomain (IR-A isoform, "deltabeta" mutant) in complex with insulin and two Fv 83-7 modules : "upper" membrane-distal part
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
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 ...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.

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Supramolecule #2: insulin

SupramoleculeName: insulin / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)

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Supramolecule #3: Leucine zippered human insulin receptor ectodomain

SupramoleculeName: Leucine zippered human insulin receptor ectodomain / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: synthetic construct (others)

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Macromolecule #1: Insulin

MacromoleculeName: Insulin / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 2.383698 KDa
SequenceString:
GIVEQCCTSI CSLYQLENYC N

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Macromolecule #2: Insulin

MacromoleculeName: Insulin / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 3.433953 KDa
SequenceString:
FVNQHLCGSH LVEALYLVCG ERGFFYTPKT

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Macromolecule #3: Insulin receptor,General control protein GCN4

MacromoleculeName: Insulin receptor,General control protein GCN4 / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO / EC number: receptor protein-tyrosine kinase
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 106.728211 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: HLYPGEVCPG MDIRNNLTRL HELENCSVIE GHLQILLMFK TRPEDFRDLS FPKLIMITDY LLLFRVYGLE SLKDLFPNLT VIRGSRLFF NYALVIFEMV HLKELGLYNL MNITRGSVRI EKNNELCYLA TIDWSRILDS VEDNHIVLNK DDNEECGDIC P GTAKGKTN ...String:
HLYPGEVCPG MDIRNNLTRL HELENCSVIE GHLQILLMFK TRPEDFRDLS FPKLIMITDY LLLFRVYGLE SLKDLFPNLT VIRGSRLFF NYALVIFEMV HLKELGLYNL MNITRGSVRI EKNNELCYLA TIDWSRILDS VEDNHIVLNK DDNEECGDIC P GTAKGKTN CPATVINGQF VERCWTHSHC QKVCPTICKS HGCTAEGLCC HSECLGNCSQ PDDPTKCVAC RNFYLDGRCV ET CPPPYYH FQDWRCVNFS FCQDLHHKCK NSRRQGCHQY VIHNNKCIPE CPSGYTMNSS NLLCTPCLGP CPKVCHLLEG EKT IDSVTS AQELRGCTVI NGSLIINIRG GNNLAAELEA NLGLIEEISG YLKIRRSYAL VSLSFFRKLR LIRGETLEIG NYSF YALDN QNLRQLWDWS KHNLTTTQGK LFFHYNPKLC LSEIHKMEEV SGTKGRQERN DIALKTNGDK ASCENELLKF SYIRT SFDK ILLRWEPYWP PDFRDLLGFM LFYKEAPYQN VTEFDGQDAC GSNSWTVVDI DPPLRSNDPK SQNHPGWLMR GLKPWT QYA IFVKTLVTFS DERRTYGAKS DIIYVQTDAT NPSVPLDPIS VSNSSSQIIL KWKPPSDPNG NITHYLVFWE RQAEDSE LF ELDYCLKGLK LPSRTWSPPF ESEDSQKHNQ SEYEDSAGEC CSCPKTDSQI LKELEESSFR KTFEDYLHNV VFVPRPSR K RRSLGDVGNA GNNEEHRPFE KVVNKESLVI SGLRHFTGYR IELQACNQDT PEERCSVAAY VSARTMPEAK ADDIVGPVT HEIFENNVVH LMWQEPKEPN GLIVLYEVSY RRYGDEELHL CVSRKHFALE RGCRLRGLSP GNYSVRIRAT SLAGNGSWTE PTYFYVTDY LDVPSNIARM KQLEDKVEEL LSKNYHLENE VARLKKLVGE R

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Macromolecule #5: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 5 / Number of copies: 9 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.094 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
10.0 mMHEPES-NaOH
0.02 %sodium azide
GridModel: C-flat-2/2 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Details: PELCO easiGlow factory settings
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-20 / Number grids imaged: 1 / Number real images: 2287 / Average exposure time: 16.0 sec. / Average electron dose: 1.85 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 747074
CTF correctionSoftware - Name: Gctf
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4zxb


Details: symmetrised domains L1, CR and L2 bound to Fv 83-7
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.1)
Final 3D classificationNumber classes: 8 / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.1)
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 213867

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Atomic model buiding 1

Initial modelPDB ID:

4zxb

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation coefficient
Output model

PDB-6hn5:
Leucine-zippered human insulin receptor ectodomain with single bound insulin - "upper" membrane-distal part

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