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- EMDB-62146: Engineered IscB-Version 2 wRNA-Target DNA ternary complex(enIscB-... -

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Basic information

Entry
Database: EMDB / ID: EMD-62146
TitleEngineered IscB-Version 2 wRNA-Target DNA ternary complex(enIscB-v2 wRNA-target DNA)
Map data
Sample
  • Complex: EnIscB-wRNA-Target DNA
KeywordsIscB / RNA-guided nuclease / transposase / RNA
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / Resolution: 6.5 Å
AuthorsZhang S / Wang F / Hu C
Funding support Singapore, 1 items
OrganizationGrant numberCountry
Ministry of Education (MoE, Singapore)A-8001534 Singapore
CitationJournal: Cell Rep / Year: 2024
Title: Engineered IscB-ωRNA system with improved base editing efficiency for disease correction via single AAV delivery in mice.
Authors: Ruochen Guo / Xiaozhi Sun / Feizuo Wang / Dingyi Han / Qiaoxia Yang / Hua Gao / Zhifang Li / Zhuang Shao / Jinqi Shi / Rongrong Yang / Xiaona Huo / Junda Yan / Guoling Li / Qingquan Xiao / ...Authors: Ruochen Guo / Xiaozhi Sun / Feizuo Wang / Dingyi Han / Qiaoxia Yang / Hua Gao / Zhifang Li / Zhuang Shao / Jinqi Shi / Rongrong Yang / Xiaona Huo / Junda Yan / Guoling Li / Qingquan Xiao / Yuanhua Liu / Senfeng Zhang / Xinyu Liu / Yingsi Zhou / Leyun Wang / Chunyi Hu / Chunlong Xu /
Abstract: IscBs, as hypercompact ancestry proteins of Cas9 nuclease, are suitable for in vivo gene editing via single adeno-associated virus (AAV) delivery. Due to the low activity of natural IscBs in ...IscBs, as hypercompact ancestry proteins of Cas9 nuclease, are suitable for in vivo gene editing via single adeno-associated virus (AAV) delivery. Due to the low activity of natural IscBs in eukaryotic cells, recent studies have been focusing on improving OgeuIscB's gene editing efficiency via protein engineering. However, in vivo gene editing efficacy of IscBs for disease correction remained to be demonstrated. Here, we showed effective gene knockout and base editing in mouse embryos. To further improve IscB activity, we performed systematic engineering of IscB-associated ωRNA and identified a variant, ωRNA-v2, with enhanced gene editing efficiency. Furthermore, our study demonstrated the efficacy of an engineered IscB-ωRNA system for robust gene knockout and base editing in vivo. Single AAV delivery of IscB-derived cytosine and adenine base editors achieved disease correction in a mouse model of tyrosinemia. Therefore, our results indicated the great potential of miniature IscBs for developing single-AAV-based gene editing therapeutics.
History
DepositionOct 23, 2024-
Header (metadata) releaseApr 23, 2025-
Map releaseApr 23, 2025-
UpdateApr 23, 2025-
Current statusApr 23, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_62146.png

Downloads & links

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Map

FileDownload / File: emd_62146.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.67 Å/pix.
x 128 pix.
= 213.504 Å		1.67 Å/pix.
x 128 pix.
= 213.504 Å		1.67 Å/pix.
x 128 pix.
= 213.504 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.668 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.17
Minimum - Maximum-0.5185171 - 1.012431
Average (Standard dev.)0.0017289495 (±0.031639706)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 213.504 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_62146_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_62146_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : EnIscB-wRNA-Target DNA

EntireName: EnIscB-wRNA-Target DNA
Components
  • Complex: EnIscB-wRNA-Target DNA

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Supramolecule #1: EnIscB-wRNA-Target DNA

SupramoleculeName: EnIscB-wRNA-Target DNA / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli (E. coli) / Strain: BL21 DE3

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Experimental details

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Structure determination

Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 8 / Component - Concentration: 100.0 mM / Component - Formula: NaCl / Component - Name: Sodium Chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY

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Electron microscopy

MicroscopeTFS TALOS
TemperatureMax: 70.0 K
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 48.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 2.0 µm / Calibrated defocus min: 1.2 µm / Calibrated magnification: 67000 / Illumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Cs: 2.0 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 87000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 53280
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: OTHER

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