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- PDB-5knz: Human Islet Amyloid Polypeptide Segment 19-SGNNFGAILSS-29 with Ea... -

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Basic information

Entry
Database: PDB / ID: 5knz
TitleHuman Islet Amyloid Polypeptide Segment 19-SGNNFGAILSS-29 with Early Onset S20G Mutation Determined by MicroED
ComponentshIAPP(residues 19-29)S20G
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 / 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 / electron crystallography / MOLECULAR REPLACEMENT / molecular replacement / cryo EM / Resolution: 1.9 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 / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / refine
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _refine.ls_d_res_high / _refine.ls_d_res_low

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Structure visualization

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Assembly

Deposited unit
A: hIAPP(residues 19-29)S20G


Theoretical massNumber of molelcules
Total (without water)1,0661
Polymers1,0661
Non-polymers00
Water181
1
A: hIAPP(residues 19-29)S20G

A: hIAPP(residues 19-29)S20G

A: hIAPP(residues 19-29)S20G

A: hIAPP(residues 19-29)S20G

A: hIAPP(residues 19-29)S20G

A: hIAPP(residues 19-29)S20G

A: hIAPP(residues 19-29)S20G

A: hIAPP(residues 19-29)S20G

A: hIAPP(residues 19-29)S20G

A: hIAPP(residues 19-29)S20G

A: hIAPP(residues 19-29)S20G

A: hIAPP(residues 19-29)S20G

A: hIAPP(residues 19-29)S20G

A: hIAPP(residues 19-29)S20G

A: hIAPP(residues 19-29)S20G

A: hIAPP(residues 19-29)S20G

A: hIAPP(residues 19-29)S20G

A: hIAPP(residues 19-29)S20G


Theoretical massNumber of molelcules
Total (without water)19,19018
Polymers19,19018
Non-polymers00
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
crystal symmetry operation4_155x-7/2,-y+1/2,-z1
crystal symmetry operation4_255x-5/2,-y+1/2,-z1
crystal symmetry operation4_355x-3/2,-y+1/2,-z1
crystal symmetry operation4_455x-1/2,-y+1/2,-z1
crystal symmetry operation4_555x+1/2,-y+1/2,-z1
crystal symmetry operation4_655x+3/2,-y+1/2,-z1
crystal symmetry operation4_755x+5/2,-y+1/2,-z1
crystal symmetry operation4_855x+7/2,-y+1/2,-z1
crystal symmetry operation4_955x+9/2,-y+1/2,-z1
Unit cell
Length a, b, c (Å)4.780, 18.600, 70.800
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
DetailsThe biological unit is an extended pair of beta sheets comprising peptides at positions X,Y,Z and 1/2+X,-1/2-Y,-Z repeated ad infinitum along the a crystal axis.

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Components

#1: Protein/peptide hIAPP(residues 19-29)S20G / Amylin / Diabetes-associated peptide / DAP / Insulinoma amyloid peptide


Mass: 1066.125 Da / Num. of mol.: 1 / Mutation: S20G / Source method: obtained synthetically / Details: islet amyloid / Source: (synth.) Homo sapiens (human) / References: UniProt: P10997
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1 / 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: 4.441 kDa/nm / Experimental value: YES
EM crystal formationInstrument: 1.5 mL Eppendorf tube / Atmosphere: Air, sealed chamber.
Details: 1 mM lyophilized peptide in PBS with 1% DMSO at room temperature under quiescent conditions. Crystals grew in a few hours and reached full size within 15 hours.
Temperature: 298 K / Time: 2 DAY
Buffer solutionpH: 7.4
Buffer component
IDConc.NameBuffer-ID
1phosphate-buffered saline1
21 %DMSO1
SpecimenConc.: 1.066 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: 295 K / Method: batch mode / pH: 7.4
Details: 1 mmol SGNNFGAILSS in 1 L phosphate-buffered saline with 1% DMSO incubated under quiescent conditions overnight

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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 shell
Resolution (Å)IDEM diffraction stats-IDFourier space coverage (%)MultiplicityNum. of structure factorsPhase residual (°)
4.25-221181.92.63590.01
3.01-4.242190.92.781100.01
2.45-33193.32.851110.01
2.13-2.444189.52.51530.01
1.9-2.125164.61.921150.01
EM diffraction statsDetails: Phasing statistics are not applicable. No imaging was used. The phases were obtained using molecular replacement.
Fourier space coverage: 83 % / High resolution: 1.9 Å / Num. of intensities measured: 1380 / Num. of structure factors: 548 / Phase error: 0.01 ° / Phase residual: 0.01 ° / Phase error rejection criteria: 0 / Rmerge: 0.106 / Rsym: 0.106
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 11, 2014
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: electron
Radiation wavelengthWavelength: 0.0251 Å / Relative weight: 1
ReflectionResolution: 1.9→35.4 Å / Num. obs: 548 / % possible obs: 82.9 % / Observed criterion σ(I): -3 / Biso Wilson estimate: 15.55 Å2 / CC1/2: 0.989 / Rmerge(I) obs: 0.106 / Rrim(I) all: 0.13 / Χ2: 0.931 / Net I/σ(I): 5.65 / Num. measured all: 1380
Reflection shell

Diffraction-ID: 1 / Rejects: _

Resolution (Å)Rmerge(I) obsMean I/σ(I) obsNum. measured obsNum. possibleNum. unique obsCC1/2Rrim(I) all% possible all
1.9-2.130.153.652211781150.9390.19164.6
2.13-2.450.1714.773821711530.9780.21189.5
2.45-3.010.1465.583161191110.9630.18293.3
3.01-4.250.1147.843061211100.9770.13890.9
4.25-35.40.0617.9215572590.9960.07381.9

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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: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 4.78 Å / B: 18.6 Å / C: 70.8 Å / Space group name: P212121 / Space group num: 19
CTF correctionType: NONE
3D reconstructionResolution: 1.9 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingB value: 14.512 / Protocol: OTHER / Space: RECIPROCAL / Target criteria: maximum likelihood
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.9→1.9 Å / SU ML: 0.13 / Cross valid method: FREE R-VALUE / σ(F): 1.49 / Phase error: 18.57
RfactorNum. reflection% reflection
Rfree0.2749 53 9.71 %
Rwork0.2275 --
obs0.2318 546 82.98 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 43.03 Å2 / Biso mean: 14.5116 Å2 / Biso min: 0.79 Å2
Refinement stepCycle: final / Resolution: 1.902→14.608 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms75 0 0 1 76
Biso mean---7.44 -
Num. residues----11
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.0175
ELECTRON CRYSTALLOGRAPHYf_angle_d1.233100
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.03711
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.00414
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d10.07924
LS refinement shellResolution: 1.9016→14.6087 Å / Total num. of bins used: 1
RfactorNum. reflection% reflection
Rfree0.2749 53 -
Rwork0.2275 493 -
all-546 -
obs--83 %

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