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- EMDB-30299: Cryo-EM structure of the Cas12f1-sgRNA-target DNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-30299
TitleCryo-EM structure of the Cas12f1-sgRNA-target DNA complex
Map data
Sample
  • Complex: Cryo-EM structure of the Cas12f1-sgRNA-target DNA complex
    • Complex: Cas12f1
      • Protein or peptide: CRISPR-associated protein Cas14a.1
    • Complex: target DNA
      • DNA: DNA (40-mer)
      • DNA: DNA (40-mer)
    • Organelle or cellular component: sgRNA
      • RNA: sgRNA
  • Ligand: ZINC ION
KeywordsCas12f / Cas14 / sgRNA / target DNA / CRISPR / RNA BINDING PROTEIN-RNA-DNA complex
Function / homologyTransposase IS605, OrfB, C-terminal / Putative transposase DNA-binding domain / transposition / DNA recombination / DNA binding / CRISPR-associated protein Cas14a.1
Function and homology information
Biological speciesuncultured archaeon (environmental samples) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsTakeda NS / Nakagawa R
CitationJournal: Mol Cell / Year: 2021
Title: Structure of the miniature type V-F CRISPR-Cas effector enzyme.
Authors: Satoru N Takeda / Ryoya Nakagawa / Sae Okazaki / Hisato Hirano / Kan Kobayashi / Tsukasa Kusakizako / Tomohiro Nishizawa / Keitaro Yamashita / Hiroshi Nishimasu / Osamu Nureki /
Abstract: RNA-guided DNA endonucleases derived from CRISPR-Cas adaptive immune systems are widely used as powerful genome-engineering tools. Among the diverse CRISPR-Cas nucleases, the type V-F Cas12f (also ...RNA-guided DNA endonucleases derived from CRISPR-Cas adaptive immune systems are widely used as powerful genome-engineering tools. Among the diverse CRISPR-Cas nucleases, the type V-F Cas12f (also known as Cas14) proteins are exceptionally compact and associate with a guide RNA to cleave single- and double-stranded DNA targets. Here, we report the cryo-electron microscopy structure of Cas12f1 (also known as Cas14a) in complex with a guide RNA and its target DNA. Unexpectedly, the structure revealed that two Cas12f1 molecules assemble with the single guide RNA to recognize the double-stranded DNA target. Each Cas12f1 protomer adopts a different conformation and plays distinct roles in nucleic acid recognition and DNA cleavage, thereby explaining how the miniature Cas12f1 enzyme achieves RNA-guided DNA cleavage as an "asymmetric homodimer." Our findings augment the mechanistic understanding of diverse CRISPR-Cas nucleases and provide a framework for the development of compact genome-engineering tools critical for therapeutic genome editing.
History
DepositionMay 26, 2020-
Header (metadata) releaseDec 23, 2020-
Map releaseDec 23, 2020-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7c7l
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30299.png

Downloads & links

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Map

FileDownload / File: emd_30299.map.gz / Format: CCP4 / Size: 12.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.05133 Å
Density
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.025
Minimum - Maximum-0.123241425 - 0.21197207
Average (Standard dev.)0.0009314228 (±0.008136102)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions150150150
Spacing150150150
CellA=B=C: 157.6995 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.05132666666671.05132666666671.0513266666667
M x/y/z150150150
origin x/y/z0.0000.0000.000
length x/y/z157.699157.699157.699
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS150150150
D min/max/mean-0.1230.2120.001

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Supplemental data

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Mask #1

Fileemd_30299_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_30299_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_30299_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 : Cryo-EM structure of the Cas12f1-sgRNA-target DNA complex

EntireName: Cryo-EM structure of the Cas12f1-sgRNA-target DNA complex
Components
  • Complex: Cryo-EM structure of the Cas12f1-sgRNA-target DNA complex
    • Complex: Cas12f1
      • Protein or peptide: CRISPR-associated protein Cas14a.1
    • Complex: target DNA
      • DNA: DNA (40-mer)
      • DNA: DNA (40-mer)
    • Organelle or cellular component: sgRNA
      • RNA: sgRNA
  • Ligand: ZINC ION

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Supramolecule #1: Cryo-EM structure of the Cas12f1-sgRNA-target DNA complex

SupramoleculeName: Cryo-EM structure of the Cas12f1-sgRNA-target DNA complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: uncultured archaeon (environmental samples)

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Supramolecule #2: Cas12f1

SupramoleculeName: Cas12f1 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1

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Supramolecule #4: target DNA

SupramoleculeName: target DNA / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3-#4

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Supramolecule #3: sgRNA

SupramoleculeName: sgRNA / type: organelle_or_cellular_component / ID: 3 / Parent: 1 / Macromolecule list: #2

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Macromolecule #1: CRISPR-associated protein Cas14a.1

MacromoleculeName: CRISPR-associated protein Cas14a.1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: uncultured archaeon (environmental samples)
Molecular weightTheoretical: 62.748598 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGHHHHHHGS MAKNTITKTL KLRIVRPYNS AEVEKIVADE KNNREKIALE KNKDKVKEAC SKHLKVAAYC TTQVERNACL FCKARKLDD KFYQKLRGQF PDAVFWQEIS EIFRQLQKQA AEIYNQSLIE LYYEIFIKGK GIANASSVEH YLSDVCYTRA A ELFKNAAI ...String:
MGHHHHHHGS MAKNTITKTL KLRIVRPYNS AEVEKIVADE KNNREKIALE KNKDKVKEAC SKHLKVAAYC TTQVERNACL FCKARKLDD KFYQKLRGQF PDAVFWQEIS EIFRQLQKQA AEIYNQSLIE LYYEIFIKGK GIANASSVEH YLSDVCYTRA A ELFKNAAI ASGLRSKIKS NFRLKELKNM KSGLPTTKSD NFPIPLVKQK GGQYTGFEIS NHNSDFIIKI PFGRWQVKKE ID KYRPWEK FDFEQVQKSP KPISLLLSTQ RRKRNKGWSK DEGTEAEIKK VMNGDYQTSY IEVKRGSKIG EKSAWMLNLS IDV PKIDKG VDPSIIGGIA VGVKSPLVCA INNAFSRYSI SDNDLFHFNK KMFARRRILL KKNRHKRAGH GAKNKLKPIT ILTE KSERF RKKLIERWAC EIADFFIKNK VGTVQMENLE SMKRKEDSYF NIRLRGFWPY AEMQNKIEFK LKQYGIEIRK VAPNN TSKT CSKCGHLNNY FNFEYRKKNK FPHFKCEKCN FKENADYNAA LNISNPKLKS TKEEP

UniProtKB: CRISPR-associated protein Cas14a.1

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Macromolecule #2: sgRNA

MacromoleculeName: sgRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: uncultured archaeon (environmental samples)
Molecular weightTheoretical: 58.133543 KDa
SequenceString:
UUCACUGAUA AAGUGGAGAA CCGCUUCACC AAAAGCUGUC CCUUAGGGGA UUAGAACUUG AGUGAAGGUG GGCUGCUUGC AUCAGCCUA AUGUCGAGAA GUGCUUUCUU CGGAAAGUAA CCCUCGAAAC AAAUUCAUUU GAAAGAAUGA AGGAAUGCAA C GGAAAUUA GGUGCGCUUG GC

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Macromolecule #3: DNA (40-mer)

MacromoleculeName: DNA (40-mer) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 12.297954 KDa
SequenceString:
(DG)(DA)(DA)(DT)(DG)(DG)(DT)(DT)(DG)(DC) (DC)(DA)(DA)(DG)(DC)(DG)(DC)(DA)(DC)(DC) (DT)(DA)(DA)(DT)(DT)(DT)(DC)(DC)(DC) (DA)(DA)(DA)(DT)(DT)(DA)(DG)(DA)(DA)(DA) (DA)

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Macromolecule #4: DNA (40-mer)

MacromoleculeName: DNA (40-mer) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 12.323922 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DC)(DT)(DA)(DA)(DT)(DT) (DT)(DG)(DG)(DG)(DA)(DA)(DA)(DT)(DT)(DA) (DG)(DG)(DT)(DG)(DC)(DG)(DC)(DT)(DT) (DG)(DG)(DC)(DA)(DA)(DC)(DC)(DA)(DT)(DT) (DC)

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 3 / Formula: ZN
Molecular weightTheoretical: 65.409 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: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 48.7 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 87253
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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