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- EMDB-9878: Cryo EM density map of Resveratrol-stabilized bioactive insulin o... -

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Basic information

Entry
Database: EMDB / ID: EMD-9878
TitleCryo EM density map of Resveratrol-stabilized bioactive insulin oligomer
Map dataResveratrol-stabilized oligomeric form of insulin which is fully biologically active
Sample
  • Complex: Insulin oligomer and Resveratrol complex
    • Protein or peptide: Insulin A chain
    • Protein or peptide: Insulin B chain
KeywordsInsulin fibrillation / Natural polyphenols / Anti-Amyloid activity / Insulin hexamer / Bioavailability / HORMONE
Function / homology
Function and homology information


negative regulation of NAD(P)H oxidase activity / negative regulation of glycogen catabolic process / positive regulation of nitric oxide mediated signal transduction / negative regulation of fatty acid metabolic process / Signaling by Insulin receptor / negative regulation of feeding behavior / IRS activation / Insulin processing / regulation of protein secretion / positive regulation of peptide hormone secretion ...negative regulation of NAD(P)H oxidase activity / negative regulation of glycogen catabolic process / positive regulation of nitric oxide mediated signal transduction / negative regulation of fatty acid metabolic process / Signaling by Insulin receptor / negative regulation of feeding behavior / IRS activation / Insulin processing / regulation of protein secretion / positive regulation of peptide hormone secretion / positive regulation of respiratory burst / Regulation of gene expression in beta cells / negative regulation of acute inflammatory response / positive regulation of protein autophosphorylation / alpha-beta T cell activation / positive regulation of dendritic spine maintenance / Synthesis, secretion, and deacylation of Ghrelin / negative regulation of respiratory burst involved in inflammatory response / negative regulation of protein secretion / positive regulation of glycogen biosynthetic process / negative regulation of gluconeogenesis / Signal attenuation / fatty acid homeostasis / FOXO-mediated transcription of oxidative stress, metabolic and neuronal genes / positive regulation of insulin receptor signaling pathway / negative regulation of lipid catabolic process / regulation of protein localization to plasma membrane / positive regulation of lipid biosynthetic process / negative regulation of oxidative stress-induced intrinsic apoptotic signaling pathway / activation of protein kinase B activity / COPI-mediated anterograde transport / transport vesicle / nitric oxide-cGMP-mediated signaling / negative regulation of reactive oxygen species biosynthetic process / Insulin receptor recycling / insulin-like growth factor receptor binding / positive regulation of brown fat cell differentiation / NPAS4 regulates expression of target genes / neuron projection maintenance / endoplasmic reticulum-Golgi intermediate compartment membrane / positive regulation of nitric-oxide synthase activity / positive regulation of mitotic nuclear division / Insulin receptor signalling cascade / regulation of transmembrane transporter activity / positive regulation of glycolytic process / positive regulation of long-term synaptic potentiation / endosome lumen / positive regulation of cytokine production / acute-phase response / positive regulation of protein secretion / positive regulation of D-glucose import / positive regulation of cell differentiation / Regulation of insulin secretion / insulin receptor binding / wound healing / negative regulation of protein catabolic process / hormone activity / regulation of synaptic plasticity / positive regulation of neuron projection development / positive regulation of protein localization to nucleus / Golgi lumen / cognition / glucose metabolic process / vasodilation / insulin receptor signaling pathway / glucose homeostasis / cell-cell signaling / regulation of protein localization / PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling / protease binding / positive regulation of cell growth / secretory granule lumen / positive regulation of canonical NF-kappaB signal transduction / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / positive regulation of MAPK cascade / 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-like / Insulin/IGF/Relaxin family / Insulin / insulin-like growth factor / relaxin family. / Insulin, conserved site / Insulin family signature. / Insulin-like superfamily
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 14.5 Å
AuthorsSengupta J / Pathak BK
Funding support India, 3 items
OrganizationGrant numberCountry
Department of Biotechnology (India) India
Department of Science & Technology (India) India
Council of Scientific & Industrial Research India
CitationJournal: J Comput Aided Mol Des / Year: 2020
Title: Resveratrol as a nontoxic excipient stabilizes insulin in a bioactive hexameric form.
Authors: Bani Kumar Pathak / Debajyoti Das / Sayan Bhakta / Partha Chakrabarti / Jayati Sengupta /
Abstract: Insulin aggregation is the leading cause of considerable reduction in the amount of active drug molecules in liquid formulations manufactured for diabetes management. Phenolic compounds, such as ...Insulin aggregation is the leading cause of considerable reduction in the amount of active drug molecules in liquid formulations manufactured for diabetes management. Phenolic compounds, such as phenol and m-cresol, are routinely used to stabilize insulin in a hexameric form during its commercial preparation. However, long term usage of commercial insulin results in various adverse secondary responses, for which toxicity of the phenolic excipients is primarily responsible. In this study we aimed to find out a nontoxic insulin stabilizer. To that end, we have selected resveratrol, a natural polyphenol, as a prospective nontoxic insulin stabilizer because of its structural similarity with commercially used phenolic compounds. Atomic force microscopy visualization of resveratrol-treated human insulin revealed that resveratrol has a unique ability to arrest hINS in a soluble oligomeric form having discrete spherical morphology. Most importantly, resveratrol-treated insulin is nontoxic for HepG2 cells and it effectively maintains low blood glucose in a mouse model. Cryo-electron microscopy revealed 3D morphology of resveratrol-stabilized insulin that strikingly resembles crystal structures of insulin hexamer formulated with m-cresol. Significantly, we found that, in a condition inductive to amyloid fibrillation at physiological pH, resveratrol is capable of stabilizing insulin more efficiently than m-cresol. Thus, this study describes resveratrol as an effective nontoxic natural molecule that can be used for stabilizing insulin in a bioactive oligomeric form during its commercial formulation.
History
DepositionApr 2, 2019-
Header (metadata) releaseApr 22, 2020-
Map releaseApr 22, 2020-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6jr3
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9878.png

Downloads & links

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Map

FileDownload / File: emd_9878.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationResveratrol-stabilized oligomeric form of insulin which is fully biologically active
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.89 Å/pix.
x 128 pix.
= 241.92 Å		1.89 Å/pix.
x 128 pix.
= 241.92 Å		1.89 Å/pix.
x 128 pix.
= 241.92 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.89 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.12147528 - 0.2776239
Average (Standard dev.)-0.000007990561 (±0.008368772)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 241.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.891.891.89
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z241.920241.920241.920
α/β/γ90.00090.00090.000
start NX/NY/NZ-200-200-200
NX/NY/NZ401401401
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.1210.278-0.000

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Supplemental data

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Sample components

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Entire : Insulin oligomer and Resveratrol complex

EntireName: Insulin oligomer and Resveratrol complex
Components
  • Complex: Insulin oligomer and Resveratrol complex
    • Protein or peptide: Insulin A chain
    • Protein or peptide: Insulin B chain

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Supramolecule #1: Insulin oligomer and Resveratrol complex

SupramoleculeName: Insulin oligomer and Resveratrol complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Insulin A chain

MacromoleculeName: Insulin A chain / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 2.383698 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString:
GIVEQCCTSI CSLYQLENYC N

UniProtKB: Insulin

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Macromolecule #2: Insulin B chain

MacromoleculeName: Insulin B chain / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 3.433953 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString:
FVNQHLCGSH LVEALYLVCG ERGFFYTPKT

UniProtKB: Insulin

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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

BufferpH: 7.4
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI POLARA 300
Image recordingFilm or detector model: FEI EAGLE (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Average electron dose: 10.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 14.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: SPIDER / Number images used: 11000
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

1ev6


Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-6jr3:
Crystal structure of insulin hexamer fitted into cryo EM density map where each dimer was kept as rigid body

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