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- EMDB-35965: Cryo-EM structure of the AsCas12f-HKRA-sgRNAS3-5v7-target DNA -

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Basic information

Entry
Database: EMDB / ID: EMD-35965
TitleCryo-EM structure of the AsCas12f-HKRA-sgRNAS3-5v7-target DNA
Map data
Sample
  • Complex: AsCas12f-HKRA-sgRNAS3-5v7-target DNA
    • Complex: AsCas12f-HKRA
      • Protein or peptide: Transposase IS605 OrfB C-terminal domain-containing protein
    • Complex: DNA
      • DNA: DNA (37-MER)
      • DNA: DNA (38-MER)
    • Complex: RNA
      • RNA: RNA (118-MER)
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
KeywordsCRISPR-Cas / RNA BINDING PROTEIN-DNA COMPLEX / RNA BINDING PROTEIN-DNA-RNA complex
Function / homology: / Transposase IS605, OrfB, C-terminal / Cas12f1-like, TNB domain / endonuclease activity / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding / CRISPR-associated endodeoxyribonuclease Cas12f1
Function and homology information
Biological speciesSulfoacidibacillus thermotolerans (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.95 Å
AuthorsHino 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
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, 2023-
Header (metadata) releaseSep 27, 2023-
Map releaseSep 27, 2023-
UpdateOct 9, 2024-
Current statusOct 9, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_35965.png

Downloads & links

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Map

FileDownload / File: emd_35965.map.gz / Format: CCP4 / Size: 8.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 132 pix.
= 145.2 Å		1.1 Å/pix.
x 132 pix.
= 145.2 Å		1.1 Å/pix.
x 132 pix.
= 145.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-0.0068531483 - 1.8033289
Average (Standard dev.)0.009842393 (±0.07597584)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin545454
Dimensions132132132
Spacing132132132
CellA=B=C: 145.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_35965_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_35965_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : AsCas12f-HKRA-sgRNAS3-5v7-target DNA

EntireName: AsCas12f-HKRA-sgRNAS3-5v7-target DNA
Components
  • Complex: AsCas12f-HKRA-sgRNAS3-5v7-target DNA
    • Complex: AsCas12f-HKRA
      • Protein or peptide: Transposase IS605 OrfB C-terminal domain-containing protein
    • Complex: DNA
      • DNA: DNA (37-MER)
      • DNA: DNA (38-MER)
    • Complex: RNA
      • RNA: RNA (118-MER)
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION

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Supramolecule #1: AsCas12f-HKRA-sgRNAS3-5v7-target DNA

SupramoleculeName: AsCas12f-HKRA-sgRNAS3-5v7-target DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Sulfoacidibacillus thermotolerans (bacteria)

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Supramolecule #2: AsCas12f-HKRA

SupramoleculeName: AsCas12f-HKRA / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Sulfoacidibacillus thermotolerans (bacteria)

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Sulfoacidibacillus thermotolerans (bacteria)

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Supramolecule #4: RNA

SupramoleculeName: RNA / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #4

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

MacromoleculeName: Transposase IS605 OrfB C-terminal domain-containing protein
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Sulfoacidibacillus thermotolerans (bacteria)
Molecular weightTheoretical: 49.978141 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGHHHHHHGS MIKVYRYEIV KPLDLDWKEF GTILRQLQQE TRFALNKATQ LAWEWMGFSS DYKDNHGEYP KSKDILGYTN VHGYAYHTI KTKAYRLNSG NLSQTIKRAT DRFKAYQKEI LRGDMSIPSY KRDHPLDLIK ENISVNRMNH GDYIASLSLL S NPAKQEMN ...String:
MGHHHHHHGS MIKVYRYEIV KPLDLDWKEF GTILRQLQQE TRFALNKATQ LAWEWMGFSS DYKDNHGEYP KSKDILGYTN VHGYAYHTI KTKAYRLNSG NLSQTIKRAT DRFKAYQKEI LRGDMSIPSY KRDHPLDLIK ENISVNRMNH GDYIASLSLL S NPAKQEMN VKRKISVIII VRGAGKTIMD RILSGEYQVS ASQIIHKDRK NKWYLNISYR FEPQTRVLDL NKIMGIDLGV AV AAYMAFQ HTPARYKLEG GEIENFRRQV ESRRISMLRQ GKYAGGARGG HGRDKRIKPI EQLRDKIANF RDTTNHRYSR YIV DMAIKE GCGTIQMEDL TNIRDIGSRF LQNWTYYDLQ QKIIYKAEEA GIKVIKIDPQ YTSQRCSECG NIDSGNRIGQ AIFK CRACG YEANADYNAA RNIAIPNIDK IIAESIK

UniProtKB: CRISPR-associated endodeoxyribonuclease Cas12f1

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Macromolecule #2: DNA (37-MER)

MacromoleculeName: DNA (37-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Sulfoacidibacillus thermotolerans (bacteria)
Molecular weightTheoretical: 11.388354 KDa
SequenceString:
(DG)(DA)(DA)(DT)(DG)(DG)(DT)(DT)(DC)(DA) (DG)(DC)(DG)(DC)(DG)(DC)(DC)(DT)(DA)(DA) (DT)(DT)(DT)(DC)(DC)(DT)(DA)(DA)(DT) (DT)(DT)(DA)(DG)(DA)(DA)(DA)(DA)

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Macromolecule #3: DNA (38-MER)

MacromoleculeName: DNA (38-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Sulfoacidibacillus thermotolerans (bacteria)
Molecular weightTheoretical: 11.698549 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DC)(DT)(DA)(DA)(DA)(DT) (DT)(DA)(DG)(DG)(DA)(DA)(DA)(DT)(DT)(DA) (DG)(DG)(DT)(DG)(DC)(DG)(DC)(DT)(DT) (DG)(DA)(DA)(DC)(DC)(DA)(DT)(DT)(DC)

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Macromolecule #4: RNA (118-MER)

MacromoleculeName: RNA (118-MER) / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Sulfoacidibacillus thermotolerans (bacteria)
Molecular weightTheoretical: 38.28782 KDa
SequenceString:
GGAUUCGUCG GUUCAGCGAC GAUAAGCCGA GAAGUGCCAA UAAAACUGUU AAGUGGUUUG GUAACGCUCG GUAAGGUCCG AAAGGAGAA CCACUGAACG GAAAUUAGGC GCGCUUGGC

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 3 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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: 7.6
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination 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

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.95 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 155198
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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