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- PDB-6jr3: Crystal structure of insulin hexamer fitted into cryo EM density ... -

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Basic information

Entry
Database: PDB / ID: 6jr3
TitleCrystal structure of insulin hexamer fitted into cryo EM density map where each dimer was kept as rigid body
Components
  • Insulin A chain
  • Insulin B chain
KeywordsHORMONE / Insulin fibrillation / Natural polyphenols / Anti-Amyloid activity / Insulin hexamer / Bioavailability
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 / negative regulation of fatty acid metabolic process / nitric oxide-cGMP-mediated signaling / 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 / negative regulation of fatty acid metabolic process / nitric oxide-cGMP-mediated signaling / 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 / regulation of protein localization to plasma membrane / positive regulation of nitric oxide mediated signal transduction / COPI-mediated anterograde transport / negative regulation of lipid catabolic process / 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 / insulin-like growth factor receptor binding / Insulin receptor recycling / positive regulation of protein metabolic process / NPAS4 regulates expression of target genes / endoplasmic reticulum-Golgi intermediate compartment membrane / neuron projection maintenance / 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 / regulation of transmembrane transporter activity / positive regulation of protein secretion / positive regulation of cell differentiation / positive regulation of glucose import / negative regulation of proteolysis / regulation of synaptic plasticity / wound healing / insulin receptor binding / cognition / negative regulation of protein catabolic process / positive regulation of neuron projection development / hormone activity / vasodilation / Golgi lumen / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 14.5 Å
AuthorsSengupta, J. / Pathak, B.K. / Bhakta, S.
Funding support India, 3items
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, 2019Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 22, 2020Provider: repository / Type: Initial release
Revision 1.1Apr 29, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jul 8, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Mar 27, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Insulin A chain
B: Insulin B chain
C: Insulin A chain
D: Insulin B chain
H: Insulin B chain
G: Insulin A chain
F: Insulin B chain
E: Insulin A chain
I: Insulin A chain
J: Insulin B chain
K: Insulin A chain
L: Insulin B chain


Theoretical massNumber of molelcules
Total (without water)34,90612
Polymers34,90612
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: isothermal titration calorimetry, assay for oligomerization
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein/peptide
Insulin A chain / Small chain / Coordinate model: Cα atoms only


Mass: 2383.698 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: INS / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P01308
#2: Protein/peptide
Insulin B chain / Large chain / Coordinate model: Cα atoms only


Mass: 3433.953 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: INS / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P01308

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Insulin oligomer and Resveratrol complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 10 e/Å2 / Film or detector model: FEI EAGLE (4k x 4k)
Image scansWidth: 4096 / Height: 4096

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Processing

EM softwareName: SPIDER / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 14.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 11000 / Symmetry type: POINT
Atomic model buildingPDB-ID: 1EV6
Accession code: 1EV6 / Source name: PDB / Type: experimental model

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