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- EMDB-40305: Cryo-EM structure of insulin amyloid-like fibril that is composed... -

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Entry
Database: EMDB / ID: EMD-40305
TitleCryo-EM structure of insulin amyloid-like fibril that is composed of two antiparallel protofilaments
Map dataCryo-EM structure of insulin amyloid-like fibril that is composed of two antiparallel protofilaments.
Sample
  • Complex: Cryo-EM structure of insulin amyloid-like fibril that is composed of two antiparallel protofilaments
    • Protein or peptide: Insulin B chain
    • Protein or peptide: Insulin A chain
Keywordsamyloid-like fibril / SIGNALING PROTEIN
Function / homology
Function and homology information


negative regulation of NAD(P)H oxidase activity / 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 ...negative regulation of NAD(P)H oxidase activity / 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 / positive regulation of peptide hormone secretion / Regulation of gene expression in beta cells / positive regulation of respiratory burst / positive regulation of dendritic spine maintenance / alpha-beta T cell activation / negative regulation of acute inflammatory response / negative regulation of respiratory burst involved in inflammatory response / negative regulation of protein secretion / fatty acid homeostasis / Synthesis, secretion, and deacylation of Ghrelin / 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 / negative regulation of lipid catabolic process / negative regulation of oxidative stress-induced intrinsic apoptotic signaling pathway / positive regulation of insulin receptor signaling pathway / negative regulation of reactive oxygen species biosynthetic process / transport vesicle / positive regulation of protein autophosphorylation / Insulin receptor recycling / insulin-like growth factor receptor binding / 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 / Regulation of insulin secretion / positive regulation of nitric-oxide synthase activity / positive regulation of long-term synaptic potentiation / 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 / wound healing / insulin receptor binding / negative regulation of protein catabolic process / positive regulation of neuron projection development / hormone activity / cognition / Golgi lumen / vasodilation / positive regulation of protein localization to nucleus / glucose metabolic process / regulation of protein localization / glucose homeostasis / cell-cell signaling / insulin receptor signaling pathway / positive regulation of NF-kappaB transcription factor activity / PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling / positive regulation of cell growth / secretory granule lumen / protease binding / positive regulation of MAPK cascade / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / positive regulation of cell migration / G protein-coupled receptor signaling pathway / Amyloid fiber formation / endoplasmic reticulum lumen / Golgi membrane / negative regulation of gene expression / positive regulation of cell population proliferation / positive regulation of gene expression / regulation of DNA-templated transcription / extracellular space / extracellular region / identical protein binding
Similarity search - Function
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
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsWang LW / Hall C / Uchikawa E / Chen DL / Choi E / Zhang XW / Bai XC
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM130289 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM136976 United States
CitationJournal: Sci Adv / Year: 2023
Title: Structural basis of insulin fibrillation.
Authors: Liwei Wang / Catherine E Hall / Emiko Uchikawa / Dailu Chen / Eunhee Choi / Xuewu Zhang / Xiao-Chen Bai /
Abstract: Insulin is a hormone responsible for maintaining normal glucose levels by activating insulin receptor (IR) and is the primary treatment for diabetes. However, insulin is prone to unfolding and ...Insulin is a hormone responsible for maintaining normal glucose levels by activating insulin receptor (IR) and is the primary treatment for diabetes. However, insulin is prone to unfolding and forming cross-β fibers. Fibrillation complicates insulin storage and therapeutic application. Molecular details of insulin fibrillation remain unclear, hindering efforts to prevent fibrillation process. Here, we characterized insulin fibrils using cryo-electron microscopy (cryo-EM), showing multiple forms that contain one or more of the protofilaments containing both the A and B chains of insulin linked by disulfide bonds. We solved the cryo-EM structure of one of the fibril forms composed of two protofilaments at 3.2-Å resolution, which reveals both the β sheet conformation of the protofilament and the packing interaction between them that underlie the fibrillation. On the basis of this structure, we designed several insulin mutants that display reduced fibrillation while maintaining native IR signaling activity. These designed insulin analogs may be developed into more effective therapeutics for type 1 diabetes.
History
DepositionApr 3, 2023-
Header (metadata) releaseAug 30, 2023-
Map releaseAug 30, 2023-
UpdateSep 27, 2023-
Current statusSep 27, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40305.png

Downloads & links

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Map

FileDownload / File: emd_40305.map.gz / Format: CCP4 / Size: 33.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of insulin amyloid-like fibril that is composed of two antiparallel protofilaments.
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.017
Minimum - Maximum-0.04264341 - 0.073963016
Average (Standard dev.)0.00015444584 (±0.0022375959)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions206206206
Spacing206206206
CellA=B=C: 170.98 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Cryo-EM structure of insulin amyloid-like fibril that is...

Fileemd_40305_half_map_1.map
AnnotationCryo-EM structure of insulin amyloid-like fibril that is composed of two antiparallel protofilaments. Half map 1.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM structure of insulin amyloid-like fibril that is...

Fileemd_40305_half_map_2.map
AnnotationCryo-EM structure of insulin amyloid-like fibril that is composed of two antiparallel protofilaments. Half map 2.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cryo-EM structure of insulin amyloid-like fibril that is composed...

EntireName: Cryo-EM structure of insulin amyloid-like fibril that is composed of two antiparallel protofilaments
Components
  • Complex: Cryo-EM structure of insulin amyloid-like fibril that is composed of two antiparallel protofilaments
    • Protein or peptide: Insulin B chain
    • Protein or peptide: Insulin A chain

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Supramolecule #1: Cryo-EM structure of insulin amyloid-like fibril that is composed...

SupramoleculeName: Cryo-EM structure of insulin amyloid-like fibril that is composed of two antiparallel protofilaments
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Insulin B chain

MacromoleculeName: Insulin B chain / type: protein_or_peptide / ID: 1 / Number of copies: 16 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 3.433953 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
FVNQHLCGSH LVEALYLVCG ERGFFYTPKT

UniProtKB: Insulin

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Macromolecule #2: Insulin A chain

MacromoleculeName: Insulin A chain / type: protein_or_peptide / ID: 2 / Number of copies: 16 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 2.383698 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GIVEQCCTSI CSLYQLENYC N

UniProtKB: Insulin

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

Concentration1 mg/mL
BufferpH: 2
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.6 µm
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Average exposure time: 8.0 sec. / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 551345
Startup modelType of model: OTHER / Details: Generated from RELION
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 56537
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8sbd:
Cryo-EM structure of insulin amyloid-like fibril that is composed of two antiparallel protofilaments

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