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- EMDB-47858: Tomogram of a minimized Enveloped Delivery Vehicle (miniEDV) -

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Basic information

Entry
Database: EMDB / ID: EMD-47858
TitleTomogram of a minimized Enveloped Delivery Vehicle (miniEDV)
Map data
Sample
  • Virus: Lentivirus derived Enveloped Delivery Vehicle
KeywordsEnveloped Delivery Vehicles / Gene editing / VIRUS LIKE PARTICLE
Biological speciesLentivirus derived Enveloped Delivery Vehicle
Methodelectron tomography / cryo EM
AuthorsPeukes J / Ngo W / Nogales E / Doudna J
Funding supportEuropean Union, Germany, Canada, United States, 5 items
OrganizationGrant numberCountry
European Molecular Biology Organization (EMBO)ALTF 1031-2021European Union
Alexander von Humboldt Foundation Germany
Natural Sciences and Engineering Research Council (NSERC, Canada)PDF-578176-2023 Canada
Howard Hughes Medical Institute (HHMI) United States
Other governmentDE-AC52-07NA27344
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: Mechanism-guided engineering of a minimal biological particle for genome editing.
Authors: Wayne Ngo / Julia Peukes / Alisha Baldwin / Zhiwei Wayne Xue / Sidney Hwang / Robert R Stickels / Zhi Lin / Ansuman T Satpathy / James A Wells / Randy Schekman / Eva Nogales / Jennifer A Doudna /
Abstract: The widespread application of genome editing to treat and cure disease requires the delivery of genome editors into the nucleus of target cells. Enveloped delivery vehicles (EDVs) are engineered ...The widespread application of genome editing to treat and cure disease requires the delivery of genome editors into the nucleus of target cells. Enveloped delivery vehicles (EDVs) are engineered virally derived particles capable of packaging and delivering CRISPR-Cas9 ribonucleoproteins (RNPs). However, the presence of lentiviral genome encapsulation and replication proteins in EDVs has obscured the underlying delivery mechanism and precluded particle optimization. Here, we show that Cas9 RNP nuclear delivery is independent of the native lentiviral capsid structure. Instead, EDV-mediated genome editing activity corresponds directly to the number of nuclear localization sequences on the Cas9 enzyme. EDV structural analysis using cryo-electron tomography and small molecule inhibitors guided the removal of ~80% of viral residues, creating a minimal EDV (miniEDV) that retains full RNP delivery capability. MiniEDVs are 25% smaller yet package equivalent amounts of Cas9 RNPs relative to the original EDVs and demonstrated increased editing in cell lines and therapeutically relevant primary human T cells. These results show that virally derived particles can be streamlined to create efficacious genome editing delivery vehicles with simpler production and manufacturing.
History
DepositionNov 12, 2024-
Header (metadata) releaseJan 15, 2025-
Map releaseJan 15, 2025-
UpdateJan 22, 2025-
Current statusJan 22, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47858.png

Downloads & links

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Map

FileDownload / File: emd_47858.map.gz / Format: CCP4 / Size: 1.6 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
6.68 Å/pix.
x 300 pix.
= 2004. Å		6.68 Å/pix.
x 1440 pix.
= 9619.2 Å		6.68 Å/pix.
x 1023 pix.
= 6833.64 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 6.68 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-0.121886596 - 0.19436517
Average (Standard dev.)0.007964415 (±0.013086674)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions14401023300
Spacing10231440300
CellA: 6833.6396 Å / B: 9619.2 Å / C: 2004.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Lentivirus derived Enveloped Delivery Vehicle

EntireName: Lentivirus derived Enveloped Delivery Vehicle
Components
  • Virus: Lentivirus derived Enveloped Delivery Vehicle

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Supramolecule #1: Lentivirus derived Enveloped Delivery Vehicle

SupramoleculeName: Lentivirus derived Enveloped Delivery Vehicle / type: virus / ID: 1 / Parent: 0
Details: Recombinantly expressed modified lentivirus derived virus like particles for the delivery of Cas9 proteins. This is the minimized version therefore termed minimized Enveloped Delivery Vehicle (miniEDV)
NCBI-ID: 11676
Sci species name: Lentivirus derived Enveloped Delivery Vehicle
Virus type: VIRUS-LIKE PARTICLE / Virus isolate: OTHER / Virus enveloped: Yes / Virus empty: No

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV
DetailsPurified lentivirus based virus like particles also referred to as minimized enveloped delivery vehicles (EDVs). This is the minimized version with cas9.
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: Aurion / Diameter: 10 nm

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsDetails: Tilt series were acquired using a dose-symmetric tilt scheme using the tilt series controller in serialEM.
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 2.92 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 4.5 µm / Nominal defocus min: 2.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionSoftware - Name: IMOD / Number images used: 41

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