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- EMDB-8273: Human Islet Amyloid Polypeptide Segment 15-FLVHSSNNFGA-25 Determi... -

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Basic information

Entry
Database: EMDB / ID: EMD-8273
TitleHuman Islet Amyloid Polypeptide Segment 15-FLVHSSNNFGA-25 Determined by MicroED
Map dataSigma-A-weighted 2Fo-Fc map
Sample
  • Complex: Amyloid fiber
    • Protein or peptide: hIAPP(15-25)WT
  • Ligand: THIOCYANATE ION
  • Ligand: water
KeywordsAmyloid / islet amyloid polypeptide / Type II Diabetes / Toxic Spine / MicroED / 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 / positive regulation of protein kinase A signaling / negative regulation of osteoclast differentiation / Regulation of gene expression in beta cells / negative regulation of protein-containing complex assembly / bone resorption ...amylin receptor signaling pathway / Calcitonin-like ligand receptors / negative regulation of amyloid fibril formation / negative regulation of bone resorption / eating behavior / positive regulation of protein kinase A signaling / negative regulation of osteoclast differentiation / Regulation of gene expression in beta cells / negative regulation of protein-containing complex assembly / bone resorption / 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 / 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)
Methodelectron crystallography / cryo EM / Resolution: 1.4 Å
AuthorsKrotee PAL / Rodriguez JA
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute on Aging (NIH/NIA)R01 AG029430 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Elife / Year: 2017
Title: Atomic structures of fibrillar segments of hIAPP suggest tightly mated β-sheets are important for cytotoxicity.
Authors: Pascal Krotee / Jose A Rodriguez / Michael R Sawaya / Duilio Cascio / Francis E Reyes / Dan Shi / Johan Hattne / Brent L Nannenga / Marie E Oskarsson / Stephan Philipp / Sarah Griner / Lin ...Authors: Pascal Krotee / Jose A Rodriguez / Michael R Sawaya / Duilio Cascio / Francis E Reyes / Dan Shi / Johan Hattne / Brent L Nannenga / Marie E Oskarsson / Stephan Philipp / Sarah Griner / Lin Jiang / Charles G Glabe / Gunilla T Westermark / Tamir Gonen / David S Eisenberg /
Abstract: hIAPP fibrils are associated with Type-II Diabetes, but the link of hIAPP structure to islet cell death remains elusive. Here we observe that hIAPP fibrils are cytotoxic to cultured pancreatic β- ...hIAPP fibrils are associated with Type-II Diabetes, but the link of hIAPP structure to islet cell death remains elusive. Here we observe that hIAPP fibrils are cytotoxic to cultured pancreatic β-cells, leading us to determine the structure and cytotoxicity of protein segments composing the amyloid spine of hIAPP. Using the cryoEM method MicroED, we discover that one segment, 19-29 S20G, forms pairs of β-sheets mated by a dry interface that share structural features with and are similarly cytotoxic to full-length hIAPP fibrils. In contrast, a second segment, 15-25 WT, forms non-toxic labile β-sheets. These segments possess different structures and cytotoxic effects, however, both can seed full-length hIAPP, and cause hIAPP to take on the cytotoxic and structural features of that segment. These results suggest that protein segment structures represent polymorphs of their parent protein and that segment 19-29 S20G may serve as a model for the toxic spine of hIAPP.
History
DepositionJun 28, 2016-
Header (metadata) releaseOct 5, 2016-
Map releaseDec 21, 2016-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5ko0
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8273.png

Downloads & links

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Map

FileDownload / File: emd_8273.map.gz / Format: CCP4 / Size: 179.7 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSigma-A-weighted 2Fo-Fc map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesY (Sec.)X (Row.)Z (Col.)
0.46 Å/pix.
x 40 pix.
= 20.02 Å		0.45 Å/pix.
x 26 pix.
= 17.96 Å		0.45 Å/pix.
x 44 pix.
= 11.778 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX: 0.449 Å / Y: 0.455 Å / Z: 0.453 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.14 / Movie #1: 0.3
Minimum - Maximum-0.53375727 - 1.3294561
Average (Standard dev.)0.000000000259126 (±0.22236347)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZXY
Origin-2600
Dimensions264440
Spacing404426
CellA: 17.96 Å / B: 20.02 Å / C: 11.778 Å
α: 62.843 ° / β: 88.9 ° / γ: 87.603 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.4490.4550.453
M x/y/z404426
origin x/y/z0.0000.0000.000
length x/y/z17.96020.02011.778
α/β/γ62.84388.90087.603
start NX/NY/NZ-2600
NX/NY/NZ264044
MAP C/R/S312
start NC/NR/NS0-260
NC/NR/NS442640
D min/max/mean-0.5341.3290.000

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Supplemental data

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Sample components

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Entire : Amyloid fiber

EntireName: Amyloid fiber
Components
  • Complex: Amyloid fiber
    • Protein or peptide: hIAPP(15-25)WT
  • Ligand: THIOCYANATE ION
  • Ligand: water

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Supramolecule #1: Amyloid fiber

SupramoleculeName: Amyloid fiber / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Molecular weightTheoretical: 4.075 kDa/nm

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Macromolecule #1: hIAPP(15-25)WT

MacromoleculeName: hIAPP(15-25)WT / type: protein_or_peptide / ID: 1 / Details: islet amyloid / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 1.193289 KDa
SequenceString:
FLVHSSNNFG A

UniProtKB: Islet amyloid polypeptide

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Macromolecule #2: THIOCYANATE ION

MacromoleculeName: THIOCYANATE ION / type: ligand / ID: 2 / Number of copies: 1 / Formula: SCN
Molecular weightTheoretical: 58.082 Da
Chemical component information

ChemComp-SCN:
THIOCYANATE ION

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 6 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron crystallography
Aggregation state3D array

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Sample preparation

Concentration20 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
0.35 MNaSCNsodium thiocyanate
35.0 %MPD
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 30 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE
Crystal formationInstrument: Hanging Drop Vapor Diffusion Tray / Atmosphere: Air, sealed chamber / Temperature: 277.0 K / Time: 7.0 DAY
Details: 20 mg/mL Lyophilized peptide in ice-cold water. Crystals were grown using the hanging drop vapor diffusion method at 4 degrees C in 0.35 M sodium thiocyanate and 35% MPD.

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Electron microscopy

MicroscopeFEI TECNAI 20
TemperatureMin: 100.0 K / Max: 100.0 K
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 2 / Number diffraction images: 879 / Average exposure time: 2.0 sec. / Average electron dose: 0.01 e/Å2
Details: The detector was operated in rolling shutter mode with 2x2 pixel binning.
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Camera length: 1840 mm
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 1.4 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES
Startup modelType of model: PDB ENTRY / Details: 5.0E+61
Molecular replacementSoftware - Name: Phaser (ver. 2.5.6)
Crystallography statisticsNumber intensities measured: 9014 / Number structure factors: 2180 / Fourier space coverage: 75 / R sym: 0.193 / R merge: 0.193 / Overall phase error: 0.01 / Overall phase residual: 0.01 / Phase error rejection criteria: 0 / High resolution: 1.4 Å
Details: Phasing statistics are not applicable. No imaging was used. The phases were obtained using molecular replacement.
Shell - Shell ID: 1 / Shell - High resolution: 1.4 Å / Shell - Low resolution: 22.0 Å / Shell - Number structure factors: 2180 / Shell - Phase residual: 0.01 / Shell - Fourier space coverage: 75 / Shell - Multiplicity: 4.1

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: OTHER / Overall B value: 9.03 / Target criteria: maximum likelihood
Output model

PDB-5ko0:
Human Islet Amyloid Polypeptide Segment 15-FLVHSSNNFGA-25 Determined by MicroED

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