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- PDB-5ko0: Human Islet Amyloid Polypeptide Segment 15-FLVHSSNNFGA-25 Determi... -

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Basic information

Entry
Database: PDB / ID: 5ko0
TitleHuman Islet Amyloid Polypeptide Segment 15-FLVHSSNNFGA-25 Determined by MicroED
ComponentshIAPP(15-25)WT
KeywordsPROTEIN FIBRIL / Amyloid / islet amyloid polypeptide / Type II Diabetes / Toxic Spine / MicroED
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 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
THIOCYANATE ION / Islet amyloid polypeptide
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / MOLECULAR REPLACEMENT / molecular replacement / cryo EM / Resolution: 1.4 Å
AuthorsKrotee, P.A.L. / Rodriguez, J.A. / Sawaya, M.R. / Cascio, D. / Shi, D. / Nannenga, B.L. / Hattne, J. / Reyes, F.E. / Gonen, T. / Eisenberg, D.S.
Funding support United States, 2items
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, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 21, 2016Provider: repository / Type: Initial release
Revision 1.1Sep 13, 2017Group: Author supporting evidence / Data collection / Category: em_software / pdbx_audit_support
Item: _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.2Apr 25, 2018Group: Data collection / Category: diffrn_source / Item: _diffrn_source.source
Revision 1.3Jun 6, 2018Group: Data collection / Experimental preparation / Refinement description
Category: diffrn_radiation / exptl_crystal_grow / software
Item: _diffrn_radiation.pdbx_monochromatic_or_laue_m_l / _diffrn_radiation.pdbx_scattering_type ..._diffrn_radiation.pdbx_monochromatic_or_laue_m_l / _diffrn_radiation.pdbx_scattering_type / _exptl_crystal_grow.method / _software.classification
Revision 1.4Sep 4, 2019Group: Data collection / 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.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.5Nov 20, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.6Jun 30, 2021Group: Data collection / Category: diffrn_detector / Item: _diffrn_detector.detector
Revision 1.7Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: hIAPP(15-25)WT
B: hIAPP(15-25)WT
hetero molecules


Theoretical massNumber of molelcules
Total (without water)2,4453
Polymers2,3872
Non-polymers581
Water1086
1
A: hIAPP(15-25)WT
B: hIAPP(15-25)WT
hetero molecules

A: hIAPP(15-25)WT
B: hIAPP(15-25)WT
hetero molecules

A: hIAPP(15-25)WT
B: hIAPP(15-25)WT
hetero molecules

A: hIAPP(15-25)WT
B: hIAPP(15-25)WT
hetero molecules

A: hIAPP(15-25)WT
B: hIAPP(15-25)WT
hetero molecules

A: hIAPP(15-25)WT
B: hIAPP(15-25)WT
hetero molecules

A: hIAPP(15-25)WT
B: hIAPP(15-25)WT
hetero molecules

A: hIAPP(15-25)WT
B: hIAPP(15-25)WT
hetero molecules

A: hIAPP(15-25)WT
B: hIAPP(15-25)WT
hetero molecules


Theoretical massNumber of molelcules
Total (without water)22,00227
Polymers21,47918
Non-polymers5239
Water32418
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation1_155x-4,y,z1
crystal symmetry operation1_255x-3,y,z1
crystal symmetry operation1_355x-2,y,z1
crystal symmetry operation1_455x-1,y,z1
crystal symmetry operation1_655x+1,y,z1
crystal symmetry operation1_755x+2,y,z1
crystal symmetry operation1_855x+3,y,z1
crystal symmetry operation1_955x+4,y,z1
Unit cell
Length a, b, c (Å)11.680, 18.180, 19.930
Angle α, β, γ (deg.)62.840, 88.900, 87.600
Int Tables number1
Space group name H-MP1
DetailsThe biological unit is an extended beta sheet comprising peptides repeated ad infinitum along the a crystal axis.

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Components

#1: Protein/peptide hIAPP(15-25)WT / Amylin / Diabetes-associated peptide / DAP / Insulinoma amyloid peptide


Mass: 1193.289 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: islet amyloid / Source: (synth.) Homo sapiens (human) / References: UniProt: P10997
#2: Chemical ChemComp-SCN / THIOCYANATE ION


Mass: 58.082 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: CNS
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY
EM experimentAggregation state: 3D ARRAY / 3D reconstruction method: electron crystallography

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Sample preparation

ComponentName: Amyloid fiber / Type: COMPLEX / Entity ID: #1 / Source: MULTIPLE SOURCES
Molecular weightValue: 4.075 kDa/nm / Experimental value: YES
EM crystal formationInstrument: Hanging Drop Vapor Diffusion Tray / Atmosphere: Air, sealed chamber
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.
Temperature: 277 K / Time: 7 DAY
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
10.35 Msodium thiocyanateNaSCN1
235 %MPD1
SpecimenConc.: 20 mg/ml / Embedding 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
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop
Details: 2 uL 20 mg/mL FLVHSSNNFGA + 1 uL 35% MPD, 0.35 M sodium thiocyanate against a reservoir of 35% MPD, 0.35 M sodium thiocyanate. Crystals appeared within several hours.

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Data collection

MicroscopyModel: FEI TECNAI 20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN
Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Temperature (max): 100 K / Temperature (min): 100 K
Image recordingAverage exposure time: 2 sec. / Electron dose: 0.01 e/Å2 / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Num. of diffraction images: 879 / Num. of grids imaged: 2
Details: The detector was operated in rolling shutter mode with 2x2 pixel binning.
Image scansSampling size: 15.6 µm / Width: 4096 / Height: 4096
EM diffractionCamera length: 1840 mm
EM diffraction shellResolution: 1.4→22 Å / Fourier space coverage: 75 % / Multiplicity: 4.1 / Num. of structure factors: 2180 / Phase residual: 0.01 °
EM diffraction statsDetails: Phasing statistics are not applicable. No imaging was used. The phases were obtained using molecular replacement.
Fourier space coverage: 75 % / High resolution: 1.4 Å / Num. of intensities measured: 9014 / Num. of structure factors: 2180 / Phase error: 0.01 ° / Phase residual: 0.01 ° / Phase error rejection criteria: 0 / Rmerge: 0.193 / Rsym: 0.193
DiffractionMean temperature: 100 K
Diffraction sourceSource: ELECTRON MICROSCOPE / Type: TECNAI F20 TEM / Wavelength: 0.0251 Å
DetectorType: TVIPS F416 CMOS CAMERA / Detector: CMOS / Date: Nov 4, 2014
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: electron
Radiation wavelengthWavelength: 0.0251 Å / Relative weight: 1
ReflectionResolution: 1.4→17.733 Å / Num. obs: 2180 / % possible obs: 75 % / Observed criterion σ(I): -3 / Biso Wilson estimate: 7.76 Å2 / CC1/2: 0.985 / Rmerge(I) obs: 0.193 / Rrim(I) all: 0.215 / Χ2: 0.847 / Net I/σ(I): 4.33 / Num. measured all: 9014
Reflection shell

Diffraction-ID: 1 / Rejects: _

Resolution (Å)Rmerge(I) obsMean I/σ(I) obsNum. measured obsNum. possibleNum. unique obsRrim(I) all% possible allCC1/2
1.4-1.440.5051.1153238840.64835.3
1.44-1.470.5591.22401891190.7163
1.47-1.520.4091.723442171440.51266.40.644
1.52-1.560.2791.923891801400.32977.80.956
1.56-1.620.4152.325862071660.47280.20.841
1.62-1.670.3892.566151881520.44280.90.896
1.67-1.730.462.544961521240.52181.60.842
1.73-1.810.3213.236341851510.36481.60.916
1.81-1.890.2833.956521681380.31582.10.961
1.89-1.980.274.776721601320.30282.50.909
1.98-2.090.2025.835931431210.22184.60.933
2.09-2.210.2256.956461491230.24682.60.976
2.21-2.360.1846.045851341150.20285.80.979
2.36-2.550.2616.1505125990.2979.20.9
2.55-2.80.2426.47458109870.26779.80.951
2.8-3.130.2176.95407100830.245830.979
3.13-3.610.1589.2336991730.17580.20.977
3.61-4.420.13310.1933781610.14675.30.983
4.42-6.260.1299.9323759450.1476.30.997
6.26-17.7330.148.39632230.15871.90.984

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO

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Processing

Software
NameVersionClassificationNB
XSCALEdata scaling
PHASER2.5.6phasing
PHENIXrefinement
PDB_EXTRACT3.2data extraction
EM software
IDNameVersionCategory
1EM-Menuimage acquisition
5XDSdiffraction indexing
6Coot0.8.2model fitting
8Phaser2.5.6molecular replacement
13PHENIX1.9model refinement
EM 3D crystal entity∠α: 62.8 ° / ∠β: 88.9 ° / ∠γ: 87.6 ° / A: 11.68 Å / B: 18.18 Å / C: 19.93 Å / Space group name: P1 / Space group num: 1
CTF correctionType: NONE
3D reconstructionResolution: 1.4 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingB value: 9.03 / Protocol: OTHER / Space: RECIPROCAL / Target criteria: maximum likelihood
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.4→9.749 Å / SU ML: 0.21 / Cross valid method: NONE / σ(F): 2.01 / Phase error: 29.83
RfactorNum. reflection% reflection
Rfree0.259 218 10.01 %
Rwork0.2247 --
obs0.2287 2177 75.56 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 21.98 Å2 / Biso mean: 9.0302 Å2 / Biso min: 1.1 Å2
Refinement stepCycle: final / Resolution: 1.399→9.749 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms170 0 3 6 179
Biso mean--17.67 11.86 -
Num. residues----22
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.008176
ELECTRON CRYSTALLOGRAPHYf_angle_d1.216235
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.04724
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.00432
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d12.1752
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.4-1.76050.3155990.2506897ELECTRON CRYSTALLOGRAPHY69
1.7605-9.7490.24011190.21521062ELECTRON CRYSTALLOGRAPHY82

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