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Open data
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Basic information
Entry | Database: PDB / ID: 8wus | ||||||
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Title | SpCas9-MMLV RT-pegRNA-target DNA complex (termination) | ||||||
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![]() | RNA BINDING PROTEIN/RNA/DNA / CRISPR-Cas / RNA BINDING PROTEIN-RNA-DNA complex | ||||||
Function / homology | ![]() host cell late endosome membrane / maintenance of CRISPR repeat elements / virion assembly / 3'-5' exonuclease activity / DNA endonuclease activity / host multivesicular body / DNA integration / viral genome integration into host DNA / establishment of integrated proviral latency / RNA-directed DNA polymerase activity ...host cell late endosome membrane / maintenance of CRISPR repeat elements / virion assembly / 3'-5' exonuclease activity / DNA endonuclease activity / host multivesicular body / DNA integration / viral genome integration into host DNA / establishment of integrated proviral latency / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / viral nucleocapsid / DNA recombination / defense response to virus / structural constituent of virion / Hydrolases; Acting on ester bonds / aspartic-type endopeptidase activity / DNA-directed DNA polymerase activity / symbiont entry into host cell / host cell plasma membrane / proteolysis / DNA binding / RNA binding / zinc ion binding / metal ion binding / plasma membrane Similarity search - Function | ||||||
Biological species | ![]() ![]() ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å | ||||||
![]() | Shuto, Y. / Nakagawa, R. / Hoki, M. / Omura, S.N. / Hirano, H. / Itoh, Y. / Nureki, O. | ||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 535.1 KB | Display | ![]() |
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PDB format | ![]() | 415.4 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 37858MC ![]() 8wutC ![]() 8wuuC ![]() 8wuvC ![]() 8ygjC ![]() 39306 ![]() 39307 M: map data used to model this data C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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1 |
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Components
-Protein , 2 types, 2 molecules EA
#1: Protein | Mass: 55656.242 Da / Num. of mol.: 1 / Mutation: D200N, D209N, T306K, W313F, T330P, D335N Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Production host: ![]() ![]() References: UniProt: Q8UN00 |
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#2: Protein | Mass: 158457.578 Da / Num. of mol.: 1 / Mutation: H10A, H840A Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Gene: cas9 Production host: ![]() ![]() References: UniProt: Q99ZW2 |
-DNA chain , 3 types, 3 molecules CDF
#3: DNA chain | Mass: 15636.033 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() |
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#4: DNA chain | Mass: 5291.446 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() |
#6: DNA chain | Mass: 12315.929 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() |
-RNA chain , 1 types, 1 molecules B
#5: RNA chain | Mass: 37065.949 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() |
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-Details
Has ligand of interest | N |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: SpCas9-MMLV RT-pegRNA-target DNA complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT |
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Molecular weight | Experimental value: NO |
Source (natural) | Organism: ![]() |
Source (recombinant) | Organism: ![]() ![]() |
Buffer solution | pH: 8 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 297 K |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 1600 nm / Nominal defocus min: 800 nm |
Image recording | Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
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Processing
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
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3D reconstruction | Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 248187 / Symmetry type: POINT | ||||||||||||||||||||||||
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