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- PDB-8az0: IAPP S20G growth-phase fibril polymorph 2PF-L -

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Basic information

Entry
Database: PDB / ID: 8az0
TitleIAPP S20G growth-phase fibril polymorph 2PF-L
ComponentsIslet amyloid polypeptide
KeywordsPROTEIN FIBRIL / Amyloid / fibril / helical / cross-beta / Amylin / polymorph / islet / Diabetes
Function / homology
Function and homology information


amylin receptor 3 signaling pathway / amylin receptor 2 signaling pathway / amylin receptor 1 signaling pathway / amylin receptor signaling pathway / Calcitonin-like ligand receptors / negative regulation of amyloid fibril formation / negative regulation of bone resorption / eating behavior / positive regulation of cAMP/PKA signal transduction / negative regulation of osteoclast differentiation ...amylin receptor 3 signaling pathway / amylin receptor 2 signaling pathway / amylin receptor 1 signaling pathway / amylin receptor signaling pathway / Calcitonin-like ligand receptors / negative regulation of amyloid fibril formation / negative regulation of bone resorption / eating behavior / positive regulation of cAMP/PKA signal transduction / negative regulation of osteoclast differentiation / Regulation of gene expression in beta cells / bone resorption / negative regulation of protein-containing complex assembly / sensory perception of pain / positive regulation of calcium-mediated signaling / osteoclast differentiation / hormone activity / cell-cell signaling / amyloid-beta binding / G alpha (s) signalling events / positive regulation of MAPK cascade / positive regulation of apoptotic process / receptor ligand activity / Amyloid fiber formation / signaling receptor binding / neuronal cell body / lipid binding / apoptotic process / signal transduction / extracellular space / extracellular region / identical protein binding
Similarity search - Function
Islet amyloid polypeptide / Calcitonin-like / Calcitonin peptide-like / Calcitonin, conserved site / Calcitonin / CGRP / IAPP family signature. / calcitonin / Calcitonin/adrenomedullin / Calcitonin / CGRP / IAPP family
Similarity search - Domain/homology
Islet amyloid polypeptide
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsWilkinson, M. / Xu, Y. / Gallardo, R. / Radford, S.E. / Ranson, N.A.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Wellcome Trust United Kingdom
Medical Research Council (MRC, United Kingdom) United Kingdom
CitationJournal: Cell / Year: 2023
Title: Structural evolution of fibril polymorphs during amyloid assembly.
Authors: Martin Wilkinson / Yong Xu / Dev Thacker / Alexander I P Taylor / Declan G Fisher / Rodrigo U Gallardo / Sheena E Radford / Neil A Ranson /
Abstract: Cryoelectron microscopy (cryo-EM) has provided unprecedented insights into amyloid fibril structures, including those associated with disease. However, these structures represent the endpoints of ...Cryoelectron microscopy (cryo-EM) has provided unprecedented insights into amyloid fibril structures, including those associated with disease. However, these structures represent the endpoints of long assembly processes, and their relationship to fibrils formed early in assembly is unknown. Consequently, whether different fibril architectures, with potentially different pathological properties, form during assembly remains unknown. Here, we used cryo-EM to determine structures of amyloid fibrils at different times during in vitro fibrillation of a disease-related variant of human islet amyloid polypeptide (IAPP-S20G). Strikingly, the fibrils formed in the lag, growth, and plateau phases have different structures, with new forms appearing and others disappearing as fibrillation proceeds. A time course with wild-type hIAPP also shows fibrils changing with time, suggesting that this is a general property of IAPP amyloid assembly. The observation of transiently populated fibril structures has implications for understanding amyloid assembly mechanisms with potential new insights into amyloid progression in disease.
History
DepositionSep 5, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 10, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 6, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Islet amyloid polypeptide
B: Islet amyloid polypeptide
C: Islet amyloid polypeptide
D: Islet amyloid polypeptide
E: Islet amyloid polypeptide
F: Islet amyloid polypeptide
G: Islet amyloid polypeptide
H: Islet amyloid polypeptide
I: Islet amyloid polypeptide
J: Islet amyloid polypeptide


Theoretical massNumber of molelcules
Total (without water)38,77310
Polymers38,77310
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, Negative stain EM, microscopy, AFM
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein/peptide
Islet amyloid polypeptide / Amylin / Diabetes-associated peptide / DAP / Insulinoma amyloid peptide


Mass: 3877.286 Da / Num. of mol.: 10 / Mutation: S20G / Source method: obtained synthetically / Details: Synthesised with C-terminal amidation / Source: (synth.) Homo sapiens (human) / References: UniProt: P10997
Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: IAPP S20G growth-phase fibril polymorph 2PF-L / Type: COMPLEX
Details: In vitro fibril growth for 6 weeks at room temperature
Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: synthetic construct (others)
Buffer solutionpH: 6.8
Buffer componentConc.: 20 mM / Name: ammonium acetate / Formula: NH3CH3CO2
SpecimenConc.: 0.12 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Fibrillation conditions: 30 uM monomeric IAPP-S20G, quiescent at room temp for 6 weeks
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: EMS Lacey Carbon
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277 K / Details: 6s blot

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 96000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1300 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 5 sec. / Electron dose: 39 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2350
Details: 1204 raw EER frames were collected per image and combined into 30 fractions for processing

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFINDCTF correction
7Cootmodel fitting
9RELION4initial Euler assignment
10RELION4final Euler assignment
11RELION4classification
12RELION43D reconstruction
13PHENIX1.17model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 178.77 ° / Axial rise/subunit: 2.39 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 842792
Details: Manually picked a subset of images to train a model for automatic fibril segment picking in crYOLO
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 12934 / Symmetry type: HELICAL
Atomic model buildingB value: 68 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: Correlation coefficient

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