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- PDB-8j3r: Cryo-EM structure of the AsCas12f-HKRA-sgRNAS3-5v7-target DNA -

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Basic information

Entry
Database: PDB / ID: 8j3r
TitleCryo-EM structure of the AsCas12f-HKRA-sgRNAS3-5v7-target DNA
Components
  • DNA (37-MER)
  • DNA (38-MER)
  • RNA (118-MER)
  • Transposase IS605 OrfB C-terminal domain-containing protein
KeywordsRNA BINDING PROTEIN/DNA/RNA / CRISPR-Cas / RNA BINDING PROTEIN-DNA COMPLEX / 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
: / Transposase IS605, OrfB, C-terminal / Cas12f1-like, TNB domain
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / CRISPR-associated endodeoxyribonuclease Cas12f1
Similarity search - Component
Biological speciesSulfoacidibacillus thermotolerans (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.95 Å
AuthorsHino, 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
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED) Japan
CitationJournal: 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
DepositionApr 18, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 27, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 9, 2024Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Transposase IS605 OrfB C-terminal domain-containing protein
D: DNA (37-MER)
E: DNA (38-MER)
C: RNA (118-MER)
B: Transposase IS605 OrfB C-terminal domain-containing protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)161,4699
Polymers161,3315
Non-polymers1384
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA chain , 2 types, 2 molecules DE

#2: DNA chain DNA (37-MER)


Mass: 11388.354 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 DNA (38-MER)


Mass: 11698.549 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)

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Protein / RNA chain , 2 types, 3 molecules ABC

#1: Protein Transposase IS605 OrfB C-terminal domain-containing protein


Mass: 49978.141 Da / Num. of mol.: 2 / Mutation: I123H, D195K, D208R, V232A
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 RNA (118-MER)


Mass: 38287.820 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)

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Non-polymers , 2 types, 4 molecules

#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestN
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1AsCas12f-HKRA-sgRNAS3-5v7-target DNACOMPLEX#1-#40RECOMBINANT
2AsCas12f-HKRACOMPLEX#11RECOMBINANT
3DNACOMPLEX#2-#31RECOMBINANT
4RNACOMPLEX#41RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Sulfoacidibacillus thermotolerans (bacteria)1765684
32Sulfoacidibacillus thermotolerans (bacteria)1765684
43Sulfoacidibacillus thermotolerans (bacteria)1765684
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli (E. coli)562
32Escherichia coli (E. coli)562
43Escherichia coli (E. coli)562
Buffer solutionpH: 7.6
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1600 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.95 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 155198 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0029997
ELECTRON MICROSCOPYf_angle_d0.45614143
ELECTRON MICROSCOPYf_dihedral_angle_d16.9672521
ELECTRON MICROSCOPYf_chiral_restr0.0361628
ELECTRON MICROSCOPYf_plane_restr0.0031293

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