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- EMDB-40963: AP2 bound to MSP2N2 nanodisc with Tgn38 cargo peptide; focused re... -

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Basic information

Entry
Database: EMDB / ID: EMD-40963
TitleAP2 bound to MSP2N2 nanodisc with Tgn38 cargo peptide; focused refinement 1
Map dataSharpened map from cryoSPARC
Sample
  • Complex: AP2 bound to MSP2N2 nanodisc with Tgn38 cargo peptide
KeywordsClathrin-mediated endocytosis / peripheral membrane protein / ENDOCYTOSIS
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsBaker RW / Sarsam R / Cannon KS
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: J Struct Biol / Year: 2023
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
DepositionJun 2, 2023-
Header (metadata) releaseJul 12, 2023-
Map releaseJul 12, 2023-
UpdateJul 19, 2023-
Current statusJul 19, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40963.png

Downloads & links

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Map

FileDownload / File: emd_40963.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map from cryoSPARC
Voxel sizeX=Y=Z: 0.88 Å
Density
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-0.9086668 - 1.3861592
Average (Standard dev.)0.000044856297 (±0.020386629)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 337.91998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_40963_msk_1.map
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AxesZYX

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Additional map: Unsharpened map

Fileemd_40963_additional_1.map
AnnotationUnsharpened map
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Additional map: Sharpened map from deepEMhancer

Fileemd_40963_additional_2.map
AnnotationSharpened map from deepEMhancer
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Additional map: Locres map for coloring by local resolution

Fileemd_40963_additional_3.map
AnnotationLocres map for coloring by local resolution
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Additional map: Map filtered by local resolution

Fileemd_40963_additional_4.map
AnnotationMap filtered by local resolution
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Half map: Half map 2

Fileemd_40963_half_map_1.map
AnnotationHalf map 2
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Half map: Half map 1

Fileemd_40963_half_map_2.map
AnnotationHalf map 1
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Sample components

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Entire : AP2 bound to MSP2N2 nanodisc with Tgn38 cargo peptide

EntireName: AP2 bound to MSP2N2 nanodisc with Tgn38 cargo peptide
Components
  • Complex: AP2 bound to MSP2N2 nanodisc with Tgn38 cargo peptide

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Supramolecule #1: AP2 bound to MSP2N2 nanodisc with Tgn38 cargo peptide

SupramoleculeName: AP2 bound to MSP2N2 nanodisc with Tgn38 cargo peptide / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
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.
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 200 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.4 / Details: 20 mM HEPES pH 7.4, 100 mM NaCl
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Two applications of sample..
DetailsNanodiscs 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.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated magnification: 45000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 45000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 55.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2100000
Details: General model gmodel_phosnet_202005_N63_c17.h5 used for crYOLO picking.
Startup modelType of model: OTHER / Details: Ab initio model generation in cryoSPARC v3.2.
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2)
Details: Focused refinement using the Local Refine tool in cryoSPARC v3.2
Number images used: 268232
DetailsData collected in counting mode
FSC plot (resolution estimation)

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