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- EMDB-47745: Subtomogram average of capsid (CA) from immature Enveloped Delive... -

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Basic information

Entry
Database: EMDB / ID: EMD-47745
TitleSubtomogram average of capsid (CA) from immature Enveloped Delivery Vehicles (EDVs) containing Cas9
Map data
Sample
  • Virus: lentivirus derived envelope delivery vehicle
    • Complex: capsid (immature Gag)
KeywordsCas9 delivery / Gene editing / EDV / VIRUS LIKE PARTICLE
Biological specieslentivirus derived Envelope Delivery Vehicle (EDV) / lentivirus derived envelope delivery vehicle
Methodsubtomogram averaging / cryo EM / Resolution: 9.5 Å
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 7, 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_47745.png

Downloads & links

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Map

FileDownload / File: emd_47745.map.gz / Format: CCP4 / Size: 6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.34 Å/pix.
x 116 pix.
= 387.44 Å		3.34 Å/pix.
x 116 pix.
= 387.44 Å		3.34 Å/pix.
x 116 pix.
= 387.44 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.34 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.22
Minimum - Maximum-0.5966451 - 0.64140147
Average (Standard dev.)0.0002600451 (±0.070908524)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions116116116
Spacing116116116
CellA=B=C: 387.44 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_47745_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_47745_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : lentivirus derived envelope delivery vehicle

EntireName: lentivirus derived envelope delivery vehicle
Components
  • Virus: lentivirus derived envelope delivery vehicle
    • Complex: capsid (immature Gag)

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Supramolecule #1: lentivirus derived envelope delivery vehicle

SupramoleculeName: lentivirus derived envelope delivery vehicle / type: virus / ID: 1 / Parent: 0 / NCBI-ID: 11676
Sci species name: lentivirus derived envelope delivery vehicle
Virus type: VIRUS-LIKE PARTICLE / Virus isolate: OTHER / Virus enveloped: Yes / Virus empty: No

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Supramolecule #2: capsid (immature Gag)

SupramoleculeName: capsid (immature Gag) / type: complex / ID: 2 / Parent: 1
Details: The capsid map was generated by subtomogram averaging of the capsid protein density from immature Gag in tomograms of Cas9 containing EDVs that were generated by expressing constructs ...Details: The capsid map was generated by subtomogram averaging of the capsid protein density from immature Gag in tomograms of Cas9 containing EDVs that were generated by expressing constructs derived from lentiviral vectors.
Source (natural)Organism: lentivirus derived Envelope Delivery Vehicle (EDV)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
GridModel: Quantifoil R2/2 / Support film - Material: CARBON
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV
DetailsPurified virus like particles also referred to as Enveloped Delivery Vehicles (EDVs) containing Cas9.

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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 reconstructionResolution.type: BY AUTHOR / Resolution: 9.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: subTOM / Number subtomograms used: 42366
ExtractionNumber tomograms: 38 / Number images used: 465070
Final angle assignmentType: OTHER
Details: The first 2 euler angles were assigned based on the orientation relative to the membrane. The final in plane rotation was randomized.

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