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- EMDB-39315: SpCas9-MMLV RT-pegRNA-target DNA complex (elongation 16-nt) focus... -

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Basic information

Entry
Database: EMDB / ID: EMD-39315
TitleSpCas9-MMLV RT-pegRNA-target DNA complex (elongation 16-nt) focused map
Map data
Sample
  • Complex: SpCas9-MMLV RT-pegRNA-target DNA complex
KeywordsCRISPR-Cas / RNA BINDING PROTEIN-RNA-DNA complex / RNA BINDING PROTEIN
Biological speciesStreptococcus pyogenes (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.06 Å
AuthorsShuto Y / Nakagawa R / Mizuki H / Satoshi NO / Hisato H / Yuzuru I / Nureki O
Funding support Japan, 1 items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)JP23ama121012 Japan
CitationJournal: Nature / Year: 2024
Title: Structural basis for pegRNA-guided reverse transcription by a prime editor.
Authors: Yutaro Shuto / Ryoya Nakagawa / Shiyou Zhu / Mizuki Hoki / Satoshi N Omura / Hisato Hirano / Yuzuru Itoh / Feng Zhang / Osamu Nureki /
Abstract: The prime editor system composed of Streptococcus pyogenes Cas9 nickase (nSpCas9) and engineered Moloney murine leukaemia virus reverse transcriptase (M-MLV RT) collaborates with a prime editing ...The prime editor system composed of Streptococcus pyogenes Cas9 nickase (nSpCas9) and engineered Moloney murine leukaemia virus reverse transcriptase (M-MLV RT) collaborates with a prime editing guide RNA (pegRNA) to facilitate a wide variety of precise genome edits in living cells. However, owing to a lack of structural information, the molecular mechanism of pegRNA-guided reverse transcription by the prime editor remains poorly understood. Here we present cryo-electron microscopy structures of the SpCas9-M-MLV RTΔRNaseH-pegRNA-target DNA complex in multiple states. The termination structure, along with our functional analysis, reveals that M-MLV RT extends reverse transcription beyond the expected site, resulting in scaffold-derived incorporations that cause undesired edits at the target loci. Furthermore, structural comparisons among the pre-initiation, initiation and elongation states show that M-MLV RT remains in a consistent position relative to SpCas9 during reverse transcription, whereas the pegRNA-synthesized DNA heteroduplex builds up along the surface of SpCas9. On the basis of our structural insights, we rationally engineered pegRNA variants and prime-editor variants in which M-MLV RT is fused within SpCas9. Collectively, our findings provide structural insights into the stepwise mechanism of prime editing, and will pave the way for the development of a versatile prime editing toolbox.
History
DepositionFeb 29, 2024-
Header (metadata) releaseJan 15, 2025-
Map releaseJan 15, 2025-
UpdateJan 22, 2025-
Current statusJan 22, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_39315.png

Downloads & links

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Map

FileDownload / File: emd_39315.map.gz / Format: CCP4 / Size: 30.5 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.)
1.3 Å/pix.
x 200 pix.
= 259.38 Å		1.3 Å/pix.
x 200 pix.
= 259.38 Å		1.3 Å/pix.
x 200 pix.
= 259.38 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.2969 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.07
Minimum - Maximum-0.36652532 - 0.4095255
Average (Standard dev.)-0.0046074605 (±0.010776783)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin282828
Dimensions200200200
Spacing200200200
CellA=B=C: 259.38 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_39315_msk_1.map
Projections & Slices
AxesZYX

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Half map: #1

Fileemd_39315_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_39315_half_map_2.map
Projections & Slices
AxesZYX

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Sample components

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Entire : SpCas9-MMLV RT-pegRNA-target DNA complex

EntireName: SpCas9-MMLV RT-pegRNA-target DNA complex
Components
  • Complex: SpCas9-MMLV RT-pegRNA-target DNA complex

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Supramolecule #1: SpCas9-MMLV RT-pegRNA-target DNA complex

SupramoleculeName: SpCas9-MMLV RT-pegRNA-target DNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Streptococcus pyogenes (bacteria)

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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

BufferpH: 8
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 297 K

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.06 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 133710
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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