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- EMDB-18538: p97 in DNA origami cage -

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Basic information

Entry
Database: EMDB / ID: EMD-18538
Titlep97 in DNA origami cage
Map datap97 encapsulated within DNA origami cage with the N-terminal domains pointing outside the DNA compartment.
Sample
  • Complex: p97 in DNA origami cage
KeywordsDNA origami / p97 / DNA
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 16.0 Å
AuthorsManar E / Amelie HJ
Funding support Germany, 1 items
OrganizationGrant numberCountry
German Research Foundation (DFG) Germany
CitationJournal: Nat Nanotechnol / Year: 2024
Title: A modular DNA origami nanocompartment for engineering a cell-free, protein unfolding and degradation pathway.
Authors: J Huang / A Jaekel / J van den Boom / D Podlesainski / M Elnaggar / A Heuer-Jungemann / M Kaiser / H Meyer / B Saccà /
Abstract: Within the cell, chemical reactions are often confined and organized through a modular architecture. This facilitates the targeted localization of molecular species and their efficient translocation ...Within the cell, chemical reactions are often confined and organized through a modular architecture. This facilitates the targeted localization of molecular species and their efficient translocation to subsequent sites. Here we present a cell-free nanoscale model that exploits compartmentalization strategies to carry out regulated protein unfolding and degradation. Our synthetic model comprises two connected DNA origami nanocompartments (each measuring 25 nm × 41 nm × 53 nm): one containing the protein unfolding machine, p97, and the other housing the protease chymotrypsin. We achieve the unidirectional immobilization of p97 within the first compartment, establishing a gateway mechanism that controls substrate recruitment, translocation and processing within the second compartment. Our data show that, whereas spatial confinement increases the rate of the individual reactions by up to tenfold, the physical connection of the compartmentalized enzymes into a chimera efficiently couples the two reactions and reduces off-target proteolysis by almost sixfold. Hence, our modular approach may serve as a blueprint for engineering artificial nanofactories with reshaped catalytic performance and functionalities beyond those observed in natural systems.
History
DepositionSep 29, 2023-
Header (metadata) releaseJul 31, 2024-
Map releaseJul 31, 2024-
UpdateAug 7, 2024-
Current statusAug 7, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18538.png

Downloads & links

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Map

FileDownload / File: emd_18538.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationp97 encapsulated within DNA origami cage with the N-terminal domains pointing outside the DNA compartment.
Projections & slices

Image control

Size
Brightness
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AxesZ (Sec.)Y (Row.)X (Col.)
2.56 Å/pix.
x 200 pix.
= 512. Å		2.56 Å/pix.
x 200 pix.
= 512. Å		2.56 Å/pix.
x 200 pix.
= 512. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 2.56 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.016
Minimum - Maximum-0.038968924 - 0.15461798
Average (Standard dev.)0.002285061 (±0.020330833)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 512.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_18538_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

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Half map: #2

Fileemd_18538_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : p97 in DNA origami cage

EntireName: p97 in DNA origami cage
Components
  • Complex: p97 in DNA origami cage

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Supramolecule #1: p97 in DNA origami cage

SupramoleculeName: p97 in DNA origami cage / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)

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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

Concentration0.9 mg/mL
BufferpH: 8 / Component - Name: Tris Buffer / Details: 5 mM tris base, 1 mM Na2EDTA, 20 mM MgCl2
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 45 sec.
VitrificationCryogen name: NITROGEN / Chamber humidity: 95 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Number grids imaged: 4 / Number real images: 2468 / Average exposure time: 3.0 sec. / Average electron dose: 23.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 57194
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 16.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 14400
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
FSC plot (resolution estimation)

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