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- EMDB-43878: Cas9d 20bp R-loop Complex -

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Basic information

Entry
Database: EMDB / ID: EMD-43878
TitleCas9d 20bp R-loop Complex
Map dataCas9d 20bp R-loop Complex
Sample
  • Complex: Cas9d ternary complex with a 20bp R-loop
    • Protein or peptide: HNH nuclease domain-containing protein
    • RNA: sgRNA
    • DNA: 5' Target Strand
    • DNA: Non-Target Strand
    • DNA: 3' Target Strand
KeywordsDNA/RNA CRISPR Cas9 Cas9d Nuclease sgRNA / DNA BINDING PROTEIN / RNA BINDING PROTEIN-DNA-RNA complex
Function / homology
Function and homology information


nucleic acid binding
Similarity search - Function
RRXRR domain / HNH endonuclease 5 / RRXRR protein / HNH endonuclease / : / HNH nucleases / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
HNH nuclease domain-containing protein
Similarity search - Component
Biological speciesDeltaproteobacteria (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.73 Å
AuthorsFregoso Ocampo R / Taylor DW / Bravo JPK
Funding support United States, 1 items
OrganizationGrant numberCountry
Welch FoundationF-1938 United States
CitationJournal: Nat Commun / Year: 2025
Title: DNA targeting by compact Cas9d and its resurrected ancestor.
Authors: Rodrigo Fregoso Ocampo / Jack P K Bravo / Tyler L Dangerfield / Isabel Nocedal / Samatar A Jirde / Lisa M Alexander / Nicole C Thomas / Anjali Das / Sarah Nielson / Kenneth A Johnson / ...Authors: Rodrigo Fregoso Ocampo / Jack P K Bravo / Tyler L Dangerfield / Isabel Nocedal / Samatar A Jirde / Lisa M Alexander / Nicole C Thomas / Anjali Das / Sarah Nielson / Kenneth A Johnson / Christopher T Brown / Cristina N Butterfield / Daniela S A Goltsman / David W Taylor /
Abstract: Type II CRISPR endonucleases are widely used programmable genome editing tools. Recently, CRISPR-Cas systems with highly compact nucleases have been discovered, including Cas9d (a type II-D nuclease). ...Type II CRISPR endonucleases are widely used programmable genome editing tools. Recently, CRISPR-Cas systems with highly compact nucleases have been discovered, including Cas9d (a type II-D nuclease). Here, we report the cryo-EM structures of a Cas9d nuclease (747 amino acids in length) in multiple functional states, revealing a stepwise process of DNA targeting involving a conformational switch in a REC2 domain insertion. Our structures provide insights into the intricately folded guide RNA which acts as a structural scaffold to anchor small, flexible protein domains for DNA recognition. The sgRNA can be truncated by up to ~25% yet still retain activity in vivo. Using ancestral sequence reconstruction, we generated compact nucleases capable of efficient genome editing in mammalian cells. Collectively, our results provide mechanistic insights into the evolution and DNA targeting of diverse type II CRISPR-Cas systems, providing a blueprint for future re-engineering of minimal RNA-guided DNA endonucleases.
History
DepositionFeb 29, 2024-
Header (metadata) releaseJan 15, 2025-
Map releaseJan 15, 2025-
UpdateJan 15, 2025-
Current statusJan 15, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43878.png

Downloads & links

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Map

FileDownload / File: emd_43878.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCas9d 20bp R-loop Complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 384 pix.
= 319.949 Å		0.83 Å/pix.
x 384 pix.
= 319.949 Å		0.83 Å/pix.
x 384 pix.
= 319.949 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8332 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.649
Minimum - Maximum-6.3181453 - 9.720691
Average (Standard dev.)-0.00028394145 (±0.112122186)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 319.9488 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Additional Map

Fileemd_43878_additional_1.map
AnnotationAdditional Map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map B

Fileemd_43878_half_map_1.map
AnnotationHalf Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map A

Fileemd_43878_half_map_2.map
AnnotationHalf Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cas9d ternary complex with a 20bp R-loop

EntireName: Cas9d ternary complex with a 20bp R-loop
Components
  • Complex: Cas9d ternary complex with a 20bp R-loop
    • Protein or peptide: HNH nuclease domain-containing protein
    • RNA: sgRNA
    • DNA: 5' Target Strand
    • DNA: Non-Target Strand
    • DNA: 3' Target Strand

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Supramolecule #1: Cas9d ternary complex with a 20bp R-loop

SupramoleculeName: Cas9d ternary complex with a 20bp R-loop / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Deltaproteobacteria (bacteria)
Molecular weightTheoretical: 171 KDa

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Macromolecule #1: HNH nuclease domain-containing protein

MacromoleculeName: HNH nuclease domain-containing protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Deltaproteobacteria (bacteria)
Molecular weightTheoretical: 86.696844 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MERELVLGID YGGKYTGLAV VDRRHNQVLY ANRLKMRDDV AGILKDRRKQ RGIRRTAQTK KKRLRELKNY LKSIGYNEST ATFETVYSL AHKRGYDYAD MPEEKTSEEI EAMDVEERKQ WEKEKQEWEE TKRNSRHRKE VVKDVHKAMI EGRATEEQIK R VERIFNKQ ...String:
MERELVLGID YGGKYTGLAV VDRRHNQVLY ANRLKMRDDV AGILKDRRKQ RGIRRTAQTK KKRLRELKNY LKSIGYNEST ATFETVYSL AHKRGYDYAD MPEEKTSEEI EAMDVEERKQ WEKEKQEWEE TKRNSRHRKE VVKDVHKAMI EGRATEEQIK R VERIFNKQ YRPKRFNNRI LTKCKVEDCG VNTPLRKNVR DLLIENIVRF FPIEQSEKDN LKDAVLDKNR REEVKSFFRK HK TDEHIRK QVYDIADNKL SGRTVFCKEH ILEHTEHSKE ERKVFRLAPS LKTKIENVLA VIKDEILPKF TVNKVVMESN NFD IAAKTQ GKKRLAKEEY GKGPREGKET RKEALLRETD GRCIYCGKSI DISNAHDDHI FPRKAGGLNI FANLVACCAV CNEN KKGRT PLESGISPKP EIIAFMKNDL KKKILEDARN INTVDFNKYM SHASIGWRYM RDRLRESAGN KKLPIERQSG IYTAY FRRW WGFKKERGNT LHHALDAVIL ASRKGYSDDG LVDMTLKPKY NKGGEFDPEK HLPEPIEFKM DKGSRGSALH DRNPLS YKK GIITRRFMVT EIECGKEDDV ISETYREKLK EAFKRFDTKK GKCLTDKEAK EAGFCIKKNE LVMSLKCSIK GTGPGQM IR INNNVFKTNV HNVGVDVYLD EKGKKKAYER KNPRLSKHFI EPPPQPNGRV SFTLKRRDMV TVEGEDAIYR IKKLGTSP T IEAVVGSDGK TRTVSATKLT KANSAE

UniProtKB: HNH nuclease domain-containing protein

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Macromolecule #2: sgRNA

MacromoleculeName: sgRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Deltaproteobacteria (bacteria)
Molecular weightTheoretical: 50.836031 KDa
SequenceString:
GACGCAUAAA GAUGAGACGC GUUACAGUUA AGGCUCUGAA AAGAGCCUUA AUUGUAAAAC GCCUAUACAG UGAAGGGAUA UACGCUUGG GUUUGUCCAG CCUGAGCCUC UAUGCCAGAA AUGGCGCCUU CAUCGUGGGU UAGGACAUUU AAUUUUUUU

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Macromolecule #3: 5' Target Strand

MacromoleculeName: 5' Target Strand / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Deltaproteobacteria (bacteria)
Molecular weightTheoretical: 6.743352 KDa
SequenceString:
(DA)(DG)(DC)(DT)(DG)(DA)(DC)(DG)(DT)(DT) (DT)(DG)(DT)(DA)(DC)(DT)(DC)(DC)(DA)(DG) (DC)(DG)

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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: Deltaproteobacteria (bacteria)
Molecular weightTheoretical: 17.026959 KDa
SequenceString: (DA)(DG)(DC)(DA)(DG)(DA)(DA)(DA)(DT)(DC) (DT)(DC)(DT)(DG)(DC)(DT)(DG)(DA)(DC)(DG) (DC)(DA)(DT)(DA)(DA)(DA)(DG)(DA)(DT) (DG)(DA)(DG)(DA)(DC)(DG)(DC)(DT)(DG)(DG) (DA) (DG)(DT)(DA)(DC)(DA)(DA) ...String:
(DA)(DG)(DC)(DA)(DG)(DA)(DA)(DA)(DT)(DC) (DT)(DC)(DT)(DG)(DC)(DT)(DG)(DA)(DC)(DG) (DC)(DA)(DT)(DA)(DA)(DA)(DG)(DA)(DT) (DG)(DA)(DG)(DA)(DC)(DG)(DC)(DT)(DG)(DG) (DA) (DG)(DT)(DA)(DC)(DA)(DA)(DA)(DC) (DG)(DT)(DC)(DA)(DG)(DC)(DT)

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Macromolecule #5: 3' Target Strand

MacromoleculeName: 3' Target Strand / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Deltaproteobacteria (bacteria)
Molecular weightTheoretical: 10.077479 KDa
SequenceString:
(DT)(DC)(DT)(DC)(DA)(DT)(DC)(DT)(DT)(DT) (DA)(DT)(DG)(DC)(DG)(DT)(DC)(DA)(DG)(DC) (DA)(DG)(DA)(DG)(DA)(DT)(DT)(DT)(DC) (DT)(DG)(DC)(DT)

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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: 7.9
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: 80.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 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: 2.73 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 1164607
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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