+Open data
-Basic information
Entry | Database: PDB / ID: 8r4i | |||||||||
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Title | Cryo-EM structure of human islet amyloid polypeptide (hIAPP) | |||||||||
Components | Islet amyloid polypeptide | |||||||||
Keywords | PROTEIN FIBRIL / Amyloid / 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 / bone resorption ...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 / 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 | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | ELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4.01 Å | |||||||||
Authors | Ooi, S.A. / Valli, D. / Maj, M. | |||||||||
Funding support | Sweden, 2items
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Citation | Journal: 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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8r4i.cif.gz | 48.8 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8r4i.ent.gz | 36.1 KB | Display | PDB format |
PDBx/mmJSON format | 8r4i.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8r4i_validation.pdf.gz | 1.2 MB | Display | wwPDB validaton report |
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Full document | 8r4i_full_validation.pdf.gz | 1.2 MB | Display | |
Data in XML | 8r4i_validation.xml.gz | 24.7 KB | Display | |
Data in CIF | 8r4i_validation.cif.gz | 34.3 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/r4/8r4i ftp://data.pdbj.org/pub/pdb/validation_reports/r4/8r4i | HTTPS FTP |
-Related structure data
Related structure data | 18887MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein/peptide | Mass: 3907.312 Da / Num. of mol.: 10 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: P10997 Has ligand of interest | N | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: FILAMENT / 3D reconstruction method: helical reconstruction |
-Sample preparation
Component | Name: Islet amyloid polypeptide / Type: CELL / Entity ID: all / Source: MULTIPLE SOURCES |
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Source (natural) | Organism: Homo sapiens (human) |
Source (recombinant) | Organism: synthetic construct (others) |
Buffer solution | pH: 7.4 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 1700 nm / Nominal defocus min: 500 nm |
Image recording | Electron dose: 39.926 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
-Processing
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION |
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Helical symmerty | Angular rotation/subunit: 178.25 ° / Axial rise/subunit: 2.46 Å / Axial symmetry: C2 |
3D reconstruction | Resolution: 4.01 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 10566 / Symmetry type: HELICAL |