+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-35965 | |||||||||
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Title | Cryo-EM structure of the AsCas12f-HKRA-sgRNAS3-5v7-target DNA | |||||||||
Map data | ||||||||||
Sample |
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Keywords | CRISPR-Cas / RNA BINDING PROTEIN-DNA COMPLEX / RNA BINDING PROTEIN-DNA-RNA complex | |||||||||
Function / homology | : / Transposase IS605, OrfB, C-terminal / Putative transposase DNA-binding domain / endonuclease activity / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding / CRISPR-associated endodeoxyribonuclease Cas12f1 Function and homology information | |||||||||
Biological species | Sulfoacidibacillus thermotolerans (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.95 Å | |||||||||
Authors | Hino T / Omura NS / Nakagawa R / Togashi T / Takeda NS / Hiramoto T / Tasaka S / Hirano H / Tokuyama T / Uosaki H ...Hino T / Omura NS / Nakagawa R / Togashi T / Takeda NS / 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, 1 items
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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 |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_35965.map.gz | 8.2 MB | EMDB map data format | |
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Header (meta data) | emd-35965-v30.xml emd-35965.xml | 19.9 KB 19.9 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_35965_fsc.xml | 7.9 KB | Display | FSC data file |
Images | emd_35965.png | 122.8 KB | ||
Filedesc metadata | emd-35965.cif.gz | 6.3 KB | ||
Others | emd_35965_half_map_1.map.gz emd_35965_half_map_2.map.gz | 8.2 MB 8.2 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-35965 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-35965 | HTTPS FTP |
-Validation report
Summary document | emd_35965_validation.pdf.gz | 754 KB | Display | EMDB validaton report |
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Full document | emd_35965_full_validation.pdf.gz | 753.6 KB | Display | |
Data in XML | emd_35965_validation.xml.gz | 12.8 KB | Display | |
Data in CIF | emd_35965_validation.cif.gz | 16.8 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-35965 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-35965 | HTTPS FTP |
-Related structure data
Related structure data | 8j3rMC 8j12C 8j1jC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_35965.map.gz / Format: CCP4 / Size: 8.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.1 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: #1
File | emd_35965_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #2
File | emd_35965_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
+Entire : AsCas12f-HKRA-sgRNAS3-5v7-target DNA
+Supramolecule #1: AsCas12f-HKRA-sgRNAS3-5v7-target DNA
+Supramolecule #2: AsCas12f-HKRA
+Supramolecule #3: DNA
+Supramolecule #4: RNA
+Macromolecule #1: Transposase IS605 OrfB C-terminal domain-containing protein
+Macromolecule #2: DNA (37-MER)
+Macromolecule #3: DNA (38-MER)
+Macromolecule #4: RNA (118-MER)
+Macromolecule #5: ZINC ION
+Macromolecule #6: MAGNESIUM ION
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.6 |
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Vitrification | Cryogen name: ETHANE |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: OTHER / 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 |