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- EMDB-32548: Cryo-EM structure of the AsCas12f1-sgRNAv1-dsDNA ternary complex -

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Basic information

Entry
Database: EMDB / ID: EMD-32548
TitleCryo-EM structure of the AsCas12f1-sgRNAv1-dsDNA ternary complex
Map data
Sample
  • Complex: AsCas12f1-sgRNAv1-dsDNA ternary complex
    • Complex: AsCas12f1
      • Protein or peptide: OrfB_Zn_ribbon domain-containing protein
    • Complex: sgRNA1
      • RNA: sgRNAv1
    • Complex: dsDNA
      • DNA: Target strand
      • DNA: Non-target strand
KeywordsCRISPR / targeting / RNA BINDING PROTEIN-RNA-DNA 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 speciesAcidibacillus sulfuroxidans (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.69 Å
AuthorsWu Z / Liu D
Funding support China, 3 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)21922705 China
National Natural Science Foundation of China (NSFC)91753127 China
National Natural Science Foundation of China (NSFC)2207783 China
CitationJournal: To Be Published
Title: Structure-directed functional evolution of the miniature CRISPR-AsCas12f1 system
Authors: Wu Z / Liu D / Pan D / Shen H / Ji Q
History
DepositionJan 7, 2022-
Header (metadata) releaseJan 18, 2023-
Map releaseJan 18, 2023-
UpdateJun 26, 2024-
Current statusJun 26, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_32548.png

Downloads & links

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Map

FileDownload / File: emd_32548.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 240 pix.
= 258.552 Å		1.08 Å/pix.
x 240 pix.
= 258.552 Å		1.08 Å/pix.
x 240 pix.
= 258.552 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0773 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.73
Minimum - Maximum-8.898664 - 13.899388999999999
Average (Standard dev.)-0.0006669837 (±0.24316305)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 258.552 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : AsCas12f1-sgRNAv1-dsDNA ternary complex

EntireName: AsCas12f1-sgRNAv1-dsDNA ternary complex
Components
  • Complex: AsCas12f1-sgRNAv1-dsDNA ternary complex
    • Complex: AsCas12f1
      • Protein or peptide: OrfB_Zn_ribbon domain-containing protein
    • Complex: sgRNA1
      • RNA: sgRNAv1
    • Complex: dsDNA
      • DNA: Target strand
      • DNA: Non-target strand

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Supramolecule #1: AsCas12f1-sgRNAv1-dsDNA ternary complex

SupramoleculeName: AsCas12f1-sgRNAv1-dsDNA ternary complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Acidibacillus sulfuroxidans (bacteria)

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Supramolecule #2: AsCas12f1

SupramoleculeName: AsCas12f1 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: synthetic construct (others)

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Supramolecule #3: sgRNA1

SupramoleculeName: sgRNA1 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: synthetic construct (others)

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Supramolecule #4: dsDNA

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

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Macromolecule #1: OrfB_Zn_ribbon domain-containing protein

MacromoleculeName: OrfB_Zn_ribbon domain-containing protein / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Acidibacillus sulfuroxidans (bacteria)
Molecular weightTheoretical: 48.719742 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MIKVYRYEIV KPLDLDWKEF GTILRQLQQE TRFALNKATQ LAWEWMGFSS DYKDNHGEYP KSKDILGYTN VHGYAYHTIK TKAYRLNSG NLSQTIKRAT DRFKAYQKEI LRGDMSIPSY KRDIPLDLIK ENISVNRMNH GDYIASLSLL SNPAKQEMNV K RKISVIII ...String:
MIKVYRYEIV KPLDLDWKEF GTILRQLQQE TRFALNKATQ LAWEWMGFSS DYKDNHGEYP KSKDILGYTN VHGYAYHTIK TKAYRLNSG NLSQTIKRAT DRFKAYQKEI LRGDMSIPSY KRDIPLDLIK ENISVNRMNH GDYIASLSLL SNPAKQEMNV K RKISVIII VRGAGKTIMD RILSGEYQVS ASQIIHDDRK NKWYLNISYD FEPQTRVLDL NKIMGIALGV AVAVYMAFQH TP ARYKLEG GEIENFRRQV ESRRISMLRQ GKYAGGARGG HGRDKRIKPI EQLRDKIANF RDTTNHRYSR YIVDMAIKEG CGT IQMEDL TNIRDIGSRF LQNWTYYDLQ QKIIYKAEEA GIKVIKIDPQ YTSQRCSECG NIDSGNRIGQ AIFKCRACGY EANA DYNAA RNIAIPNIDK IIAESIK

UniProtKB: CRISPR-associated endodeoxyribonuclease Cas12f1

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Macromolecule #2: sgRNAv1

MacromoleculeName: sgRNAv1 / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 61.643578 KDa
SequenceString:
AUUCGUCGGU UCAGCGACGA UAAGCCGAGA AGUGCCAAUA AAACUGUUAA GUGGUUUGGU AACGCUCGGU AAGGUAGCCA AAAGGCUGA AACUCCGUGC ACAAAGACCG CACGGACGCU UCACAUAUAG CUCAUAAACA AGGGUUUGCG AGCUAGCUUG U GGAGUGUG AACCUGGCGU UUUUCCAUAG GCU

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Macromolecule #3: Target strand

MacromoleculeName: Target strand / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 9.260985 KDa
SequenceString:
(DG)(DC)(DG)(DG)(DA)(DG)(DC)(DC)(DT)(DA) (DT)(DG)(DG)(DA)(DA)(DA)(DA)(DA)(DC)(DG) (DC)(DC)(DA)(DG)(DC)(DA)(DA)(DC)(DG) (DC)

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Macromolecule #4: Non-target strand

MacromoleculeName: Non-target strand / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 1.840227 KDa
SequenceString:
(DG)(DC)(DG)(DT)(DT)(DG)

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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: 7.5
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: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 1.5 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: 2.69 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 1770878
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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