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- PDB-8t1o: AP2 bound to MSP2N2 nanodisc with Tgn38 cargo peptide; composite map -
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Open data
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Basic information
Entry | Database: PDB / ID: 8t1o | ||||||
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Title | AP2 bound to MSP2N2 nanodisc with Tgn38 cargo peptide; composite map | ||||||
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![]() | ENDOCYTOSIS / Clathrin-mediated endocytosis / peripheral membrane protein | ||||||
Function / homology | ![]() Post-translational protein phosphorylation / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / Retrograde transport at the Trans-Golgi-Network / Formation of annular gap junctions / Gap junction degradation / LDL clearance / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / WNT5A-dependent internalization of FZD4 / Trafficking of GluR2-containing AMPA receptors / Retrograde neurotrophin signalling ...Post-translational protein phosphorylation / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / Retrograde transport at the Trans-Golgi-Network / Formation of annular gap junctions / Gap junction degradation / LDL clearance / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / WNT5A-dependent internalization of FZD4 / Trafficking of GluR2-containing AMPA receptors / Retrograde neurotrophin signalling / clathrin adaptor complex / clathrin coat / extrinsic component of presynaptic endocytic zone membrane / VLDLR internalisation and degradation / Golgi to endosome transport / Golgi Associated Vesicle Biogenesis / cardiac septum development / Recycling pathway of L1 / AP-2 adaptor complex / regulation of vesicle size / postsynaptic neurotransmitter receptor internalization / clathrin coat assembly / Cargo recognition for clathrin-mediated endocytosis / Cargo recognition for clathrin-mediated endocytosis / positive regulation of synaptic vesicle endocytosis / Clathrin-mediated endocytosis / clathrin adaptor activity / Clathrin-mediated endocytosis / vesicle budding from membrane / membrane coat / clathrin-dependent endocytosis / trans-Golgi network transport vesicle / MHC class II antigen presentation / coronary vasculature development / neurotransmitter receptor internalization / positive regulation of protein localization to membrane / signal sequence binding / aorta development / negative regulation of protein localization to plasma membrane / regulation of hematopoietic stem cell differentiation / ventricular septum development / low-density lipoprotein particle receptor binding / clathrin binding / positive regulation of endocytosis / positive regulation of receptor internalization / synaptic vesicle endocytosis / protein serine/threonine kinase binding / vesicle-mediated transport / Neutrophil degranulation / phosphatidylinositol binding / secretory granule / kidney development / intracellular protein transport / trans-Golgi network / cytoplasmic side of plasma membrane / kinase binding / disordered domain specific binding / synaptic vesicle / heart development / cytoplasmic vesicle / postsynapse / protein-containing complex assembly / transmembrane transporter binding / endosome / protein domain specific binding / intracellular membrane-bounded organelle / glutamatergic synapse / lipid binding / synapse / protein-containing complex binding / protein kinase binding / Golgi apparatus / mitochondrion / membrane / cytosol Similarity search - Function | ||||||
Biological species | ![]() ![]() ![]() ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å | ||||||
![]() | Baker, R.W. / Cannon, K.S. / Reta, S. | ||||||
Funding support | 1items
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![]() | ![]() Title: Lipid nanodiscs as a template for high-resolution cryo-EM structures of peripheral membrane proteins. Authors: Kevin S Cannon / Reta D Sarsam / Tanita Tedamrongwanish / Kevin Zhang / Richard W Baker / ![]() Abstract: Peripheral membrane proteins are ubiquitous throughout cell biology and are required for a variety of cellular processes such as signal transduction, membrane trafficking, and autophagy. Transient ...Peripheral membrane proteins are ubiquitous throughout cell biology and are required for a variety of cellular processes such as signal transduction, membrane trafficking, and autophagy. Transient binding to the membrane has a profound impact on protein function, serving to induce conformational changes and alter biochemical and biophysical parameters by increasing the local concentration of factors and restricting diffusion to two dimensions. Despite the centrality of the membrane in serving as a template for cell biology, there are few reported high-resolution structures of peripheral membrane proteins bound to the membrane. We analyzed the utility of lipid nanodiscs to serve as a template for cryo-EM analysis of peripheral membrane proteins. We tested a variety of nanodiscs and we report a 3.3 Å structure of the AP2 clathrin adaptor complex bound to a 17-nm nanodisc, with sufficient resolution to visualize a bound lipid head group. Our data demonstrate that lipid nanodiscs are amenable to high-resolution structure determination of peripheral membrane proteins and provide a framework for extending this analysis to other systems. | ||||||
History |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 349.9 KB | Display | ![]() |
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PDB format | ![]() | 278.8 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Summary document | ![]() | 1.3 MB | Display | ![]() |
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Full document | ![]() | 1.3 MB | Display | |
Data in XML | ![]() | 58.6 KB | Display | |
Data in CIF | ![]() | 85.7 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 40973MC C: citing same article ( M: map data used to model this data |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
-AP-2 complex subunit ... , 4 types, 4 molecules ABMS
#1: Protein | Mass: 70573.320 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
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#2: Protein | Mass: 66953.195 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
#3: Protein | Mass: 49726.641 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
#4: Protein | Mass: 17038.688 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
-Protein/peptide / Non-polymers , 2 types, 2 molecules P![](data/chem/img/PIO.gif)
![](data/chem/img/PIO.gif)
#5: Protein/peptide | Mass: 1723.009 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Details: Synthesized cargo peptide with an N-terminal oleic acid modification Source: (gene. exp.) ![]() ![]() ![]() ![]() |
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#6: Chemical | ChemComp-PIO / [( |
-Details
Has ligand of interest | Y |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: AP2 bound to MSP2N2 nanodisc with Tgn38 cargo peptide / Type: COMPLEX Details: Nanodiscs were assembled with a lipid mixture containing 75 mol% DOPC, 15 mol% DOPS, 10 mol% PIP2. Complex was formed by co-elution via gel filtration chromatography. Entity ID: #1-#5 / Source: MULTIPLE SOURCES | ||||||||||||
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Molecular weight | Value: 0.2 MDa / Experimental value: NO | ||||||||||||
Source (natural) |
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Source (recombinant) | Organism: ![]() ![]() | ||||||||||||
Buffer solution | pH: 7.4 / Details: 20 mM HEPES pH 7.4, 100 mM NaCl | ||||||||||||
Specimen | Conc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: Nanodiscs were assembled with a lipid mixture containing 75 mol% DOPC, 15 mol% DOPS, 10 mol% PIP2. Complex was formed by co-elution via gel filtration chromatography. | ||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277 K / Details: Two applications of sample. |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Talos Arctica / Image courtesy: FEI Company |
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Microscopy | Model: FEI TALOS ARCTICA |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 45000 X / Calibrated magnification: 45000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN |
Image recording | Electron dose: 55 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
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Processing
EM software |
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Image processing | Details: Data collected in counting mode | |||||||||||||||||||||||||
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||
Particle selection | Num. of particles selected: 2100000 Details: General model gmodel_phosnet_202005_N63_c17.h5 used for crYOLO picking. | |||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | |||||||||||||||||||||||||
3D reconstruction | Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 268232 Details: Composite map was made using the phenix.combine_focused_maps function. Three separate local refinements were combined to make the final map. Symmetry type: POINT | |||||||||||||||||||||||||
Refine LS restraints |
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