+Open data
-Basic information
Entry | Database: PDB / ID: 8j1j | ||||||
---|---|---|---|---|---|---|---|
Title | Cryo-EM structure of the AsCas12f-YHAM-sgRNAS3-5v7-target DNA | ||||||
Components |
| ||||||
Keywords | RNA BINDING PROTEIN/DNA/RNA / CRISPR-Cas / RNA BINDING PROTEIN-DNA COMPLEX / RNA BINDING PROTEIN / RNA BINDING PROTEIN-DNA-RNA complex | ||||||
Function / homology | Function and homology information endonuclease activity / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding Similarity search - Function | ||||||
Biological species | Sulfoacidibacillus thermotolerans (bacteria) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.91 Å | ||||||
Authors | Hino, T. / Omura, N.S. / Nakagawa, R. / Togashi, T. / Takeda, N.S. / Hiramoto, T. / Tasaka, S. / Hirano, H. / Tokuyama, T. / Uosaki, H. ...Hino, T. / Omura, N.S. / Nakagawa, R. / Togashi, T. / Takeda, N.S. / Hiramoto, T. / Tasaka, S. / Hirano, H. / Tokuyama, T. / Uosaki, H. / Ishiguro, H. / Yamano, H. / Ozaki, Y. / Motooka, D. / Mori, H. / Kirita, Y. / Kise, Y. / Itoh, Y. / Matoba, S. / Aburatani, H. / Yachie, N. / Siksnys, V. / Ohmori, T. / Hoshino, A. / Nureki, O. | ||||||
Funding support | Japan, 1items
| ||||||
Citation | Journal: Cell / Year: 2023 Title: An AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis. Authors: Tomohiro Hino / Satoshi N Omura / Ryoya Nakagawa / Tomoki Togashi / Satoru N Takeda / Takafumi Hiramoto / Satoshi Tasaka / Hisato Hirano / Takeshi Tokuyama / Hideki Uosaki / Soh Ishiguro / ...Authors: Tomohiro Hino / Satoshi N Omura / Ryoya Nakagawa / Tomoki Togashi / Satoru N Takeda / Takafumi Hiramoto / Satoshi Tasaka / Hisato Hirano / Takeshi Tokuyama / Hideki Uosaki / Soh Ishiguro / Madina Kagieva / Hiroyuki Yamano / Yuki Ozaki / Daisuke Motooka / Hideto Mori / Yuhei Kirita / Yoshiaki Kise / Yuzuru Itoh / Satoaki Matoba / Hiroyuki Aburatani / Nozomu Yachie / Tautvydas Karvelis / Virginijus Siksnys / Tsukasa Ohmori / Atsushi Hoshino / Osamu Nureki / Abstract: SpCas9 and AsCas12a are widely utilized as genome-editing tools in human cells. However, their relatively large size poses a limitation for delivery by cargo-size-limited adeno-associated virus (AAV) ...SpCas9 and AsCas12a are widely utilized as genome-editing tools in human cells. However, their relatively large size poses a limitation for delivery by cargo-size-limited adeno-associated virus (AAV) vectors. The type V-F Cas12f from Acidibacillus sulfuroxidans is exceptionally compact (422 amino acids) and has been harnessed as a compact genome-editing tool. Here, we developed an approach, combining deep mutational scanning and structure-informed design, to successfully generate two AsCas12f activity-enhanced (enAsCas12f) variants. Remarkably, the enAsCas12f variants exhibited genome-editing activities in human cells comparable with those of SpCas9 and AsCas12a. The cryoelectron microscopy (cryo-EM) structures revealed that the mutations stabilize the dimer formation and reinforce interactions with nucleic acids to enhance their DNA cleavage activities. Moreover, enAsCas12f packaged with partner genes in an all-in-one AAV vector exhibited efficient knock-in/knock-out activities and transcriptional activation in mice. Taken together, enAsCas12f variants could offer a minimal genome-editing platform for in vivo gene therapy. | ||||||
History |
|
-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
---|
-Downloads & links
-Download
PDBx/mmCIF format | 8j1j.cif.gz | 259.3 KB | Display | PDBx/mmCIF format |
---|---|---|---|---|
PDB format | pdb8j1j.ent.gz | 197.5 KB | Display | PDB format |
PDBx/mmJSON format | 8j1j.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8j1j_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
---|---|---|---|---|
Full document | 8j1j_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | 8j1j_validation.xml.gz | 35 KB | Display | |
Data in CIF | 8j1j_validation.cif.gz | 53.9 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/j1/8j1j ftp://data.pdbj.org/pub/pdb/validation_reports/j1/8j1j | HTTPS FTP |
-Related structure data
Related structure data | 35926MC 8j12C 8j3rC M: map data used to model this data C: citing same article (ref.) |
---|---|
Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
|
---|---|
1 |
|
-Components
-DNA chain , 2 types, 2 molecules DE
#2: DNA chain | Mass: 11694.576 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Sulfoacidibacillus thermotolerans (bacteria) Production host: Escherichia coli (E. coli) |
---|---|
#3: DNA chain | Mass: 11689.535 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Sulfoacidibacillus thermotolerans (bacteria) Production host: Escherichia coli (E. coli) |
-Protein / RNA chain , 2 types, 3 molecules ABC
#1: Protein | Mass: 49966.105 Da / Num. of mol.: 2 / Mutation: F48Y, S188H, V233A, E317M Source method: isolated from a genetically manipulated source Source: (gene. exp.) Sulfoacidibacillus thermotolerans (bacteria) Gene: BM613_13600 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A2U3D0N8 #4: RNA chain | | Mass: 38288.805 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Sulfoacidibacillus thermotolerans (bacteria) Production host: Escherichia coli (E. coli) |
---|
-Non-polymers , 2 types, 4 molecules
#5: Chemical | #6: Chemical | |
---|
-Details
Has ligand of interest | N |
---|---|
Has protein modification | N |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
---|---|
EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: AsCas12f-YHAM-sgRNAS3-5v7-target DNA / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT |
---|---|
Molecular weight | Experimental value: NO |
Source (natural) | Organism: Sulfoacidibacillus thermotolerans (bacteria) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 7.6 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
---|---|
Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER |
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 BIOQUANTUM (6k x 4k) |
-Processing
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
3D reconstruction | Resolution: 2.91 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 146448 / Symmetry type: POINT | ||||||||||||||||||||||||
Refine LS restraints |
|