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- EMDB-18887: Cryo-EM structure of human islet amyloid polypeptide (hIAPP) -

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Basic information

Entry
Database: EMDB / ID: EMD-18887
TitleCryo-EM structure of human islet amyloid polypeptide (hIAPP)
Map data
Sample
  • Cell: Islet amyloid polypeptideAmylin
    • Protein or peptide: Islet amyloid polypeptideAmylin
KeywordsAmyloid / fibril / PROTEIN FIBRIL
Function / homology
Function and homology information


: / amylin receptor signaling pathway / Calcitonin-like ligand receptors / negative regulation of amyloid fibril formation / negative regulation of bone resorption / eating behavior / negative regulation of osteoclast differentiation / positive regulation of protein kinase A signaling / Regulation of gene expression in beta cells / negative regulation of protein-containing complex assembly ...: / amylin receptor signaling pathway / Calcitonin-like ligand receptors / negative regulation of amyloid fibril formation / negative regulation of bone resorption / eating behavior / negative regulation of osteoclast differentiation / positive regulation of protein kinase A signaling / Regulation of gene expression in beta cells / negative regulation of protein-containing complex assembly / positive regulation of cAMP-mediated signaling / positive regulation of calcium-mediated signaling / bone resorption / sensory perception of pain / osteoclast differentiation / hormone activity / cell-cell signaling / amyloid-beta binding / G alpha (s) signalling events / positive regulation of MAPK cascade / receptor ligand activity / positive regulation of apoptotic process / Amyloid fiber formation / signaling receptor binding / 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)
Methodhelical reconstruction / cryo EM / Resolution: 4.01 Å
AuthorsOoi SA / Valli D / Maj M
Funding support Sweden, 2 items
OrganizationGrant numberCountry
Swedish Research Council2020-05403 Sweden
Other privateSwedish Society for Medical Research (S20-0156) Sweden
CitationJournal: Biophys J / Year: 2024
Title: Improving cryo-EM grids for amyloid fibrils using interface-active solutions and spectator proteins.
Authors: Dylan Valli / Saik Ann Ooi / Giorgio Scattolini / Himanshu Chaudhary / Alesia A Tietze / Michał Maj /
Abstract: Preparation of cryoelectron microscopy (cryo-EM) grids for imaging of amyloid fibrils is notoriously challenging. The human islet amyloid polypeptide (hIAPP) serves as a notable example, as the ...Preparation of cryoelectron microscopy (cryo-EM) grids for imaging of amyloid fibrils is notoriously challenging. The human islet amyloid polypeptide (hIAPP) serves as a notable example, as the majority of reported structures have relied on the use of nonphysiological pH buffers, N-terminal tags, and seeding. This highlights the need for more efficient, reproducible methodologies that can elucidate amyloid fibril structures formed under diverse conditions. In this work, we demonstrate that the distribution of fibrils on cryo-EM grids is predominantly determined by the solution composition, which is critical for the stability of thin vitreous ice films. We discover that, among physiological pH buffers, HEPES uniquely enhances the distribution of fibrils on cryo-EM grids and improves the stability of ice layers. This improvement is attributed to direct interactions between HEPES molecules and hIAPP, effectively minimizing the tendency of hIAPP to form dense clusters in solutions and preventing ice nucleation. Furthermore, we provide additional support for the idea that denatured protein monolayers forming at the interface are also capable of eliciting a surfactant-like effect, leading to improved particle coverage. This phenomenon is illustrated by the addition of nonamyloidogenic rat IAPP (rIAPP) to a solution of preaggregated hIAPP just before the freezing process. The resultant grids, supplemented with this "spectator protein", exhibit notably enhanced coverage and improved ice quality. Unlike conventional surfactants, rIAPP is additionally capable of disentangling the dense clusters formed by hIAPP. By applying the proposed strategies, we have resolved the structure of the dominant hIAPP polymorph, formed in vitro at pH 7.4, to a final resolution of 4 Å. The advances in grid preparation presented in this work hold significant promise for enabling structural determination of amyloid proteins which are particularly resistant to conventional grid preparation techniques.
History
DepositionNov 13, 2023-
Header (metadata) releaseMar 6, 2024-
Map releaseMar 6, 2024-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18887.png

Downloads & links

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Map

FileDownload / File: emd_18887.map.gz / Format: CCP4 / Size: 19.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 50 pix.
= 41.4 Å		0.83 Å/pix.
x 320 pix.
= 264.96 Å		0.83 Å/pix.
x 318 pix.
= 263.304 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 0.828 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.008
Minimum - Maximum-0.024138808 - 0.036960907
Average (Standard dev.)0.000033502285 (±0.002071296)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin02180
Dimensions32031850
Spacing31832050
CellA: 263.30402 Å / B: 264.96 Å / C: 41.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_18887_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_18887_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Islet amyloid polypeptide

EntireName: Islet amyloid polypeptideAmylin
Components
  • Cell: Islet amyloid polypeptideAmylin
    • Protein or peptide: Islet amyloid polypeptideAmylin

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Supramolecule #1: Islet amyloid polypeptide

SupramoleculeName: Islet amyloid polypeptide / type: cell / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Islet amyloid polypeptide

MacromoleculeName: Islet amyloid polypeptide / type: protein_or_peptide / ID: 1 / Number of copies: 10 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 3.907312 KDa
SequenceString:
KCNTATCATQ RLANFLVHSS NNFGAILSST NVGSNTY(NH2)

UniProtKB: Islet amyloid polypeptide

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.4
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: 1.7 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 39.926 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Helical parameters - Δz: 2.46 Å
Applied symmetry - Helical parameters - Δ&Phi: 178.25 °
Applied symmetry - Helical parameters - Axial symmetry: C2 (2 fold cyclic)
Resolution.type: BY AUTHOR / Resolution: 4.01 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 10566
FSC plot (resolution estimation)

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