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- EMDB-43760: Cas9d 6bp R-loop Seed Complex -

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Open data


ID or keywords:

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Basic information

Entry
Database: EMDB / ID: EMD-43760
TitleCas9d 6bp R-loop Seed Complex
Map data
Sample
  • Complex: Cas9d:sgRNA:DNA
    • Protein or peptide: HNH nuclease domain-containing protein
    • RNA: sgRNA
    • DNA: DNA Target Strand
    • DNA: DNA Non-Target Strand
KeywordsCRISPR-Cas9 / Endonuclease / Binary Complex / Effector / sgRNA / IMMUNE SYSTEM-RNA-DNA 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 (d-proteobacteria) / Escherichia phage Lambda (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.37 Å
AuthorsFregoso Ocampo R / Bravo JPK / Taylor DW
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 21, 2024-
Header (metadata) releaseJan 15, 2025-
Map releaseJan 15, 2025-
UpdateMay 28, 2025-
Current statusMay 28, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43760.png

Downloads & links

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Map

FileDownload / File: emd_43760.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 320 pix.
= 266.624 Å		0.83 Å/pix.
x 320 pix.
= 266.624 Å		0.83 Å/pix.
x 320 pix.
= 266.624 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: 0.275
Minimum - Maximum-1.741729 - 3.1508436
Average (Standard dev.)0.000056987836 (±0.052231174)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 266.624 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_43760_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_43760_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_43760_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 : Cas9d:sgRNA:DNA

EntireName: Cas9d:sgRNA:DNA
Components
  • Complex: Cas9d:sgRNA:DNA
    • Protein or peptide: HNH nuclease domain-containing protein
    • RNA: sgRNA
    • DNA: DNA Target Strand
    • DNA: DNA Non-Target Strand

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Supramolecule #1: Cas9d:sgRNA:DNA

SupramoleculeName: Cas9d:sgRNA:DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Deltaproteobacteria (d-proteobacteria) / Strain: NICO DE3

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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 (d-proteobacteria)
Molecular weightTheoretical: 86.568711 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
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 FPIESEKDNL KDAVLDKNRR EEVKSFFRKH KT DEHIRKQ VYDIADNKLS GRTVFCKEHI LEHTEHSKEE RKVFRLAPSL KTKIENVLAV IKDEILPKFT VNKVVMESNN FDI AAKTQG KKRLAKEEYG KGPREGKETR KEALLRETDG RCIYCGKSID ISNAHDDHIF PRKAGGLNIF ANLVACCAVC NENK KGRTP LESGISPKPE IIAFMKNDLK KKILEDARNI NTVDFNKYMS HASIGWRYMR DRLRESAGNK KLPIERQSGI YTAYF RRWW GFKKERGNTL HHALDAVILA SRKGYSDDGL VDMTLKPKYN KGGEFDPEKH LPEPIEFKMD KGSRGSALHD RNPLSY KKG IITRRFMVTE IECGKEDDVI SETYREKLKE AFKRFDTKKG KCLTDKEAKE AGFCIKKNEL VMSLKCSIKG TGPGQMI RI NNNVFKTNVH NVGVDVYLDE KGKKKAYERK NPRLSKHFIE PPPQPNGRVS FTLKRRDMVT VEGEDAIYRI KKLGTSPT I EAVVGSDGKT RTVSATKLTK ANSAE

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 (d-proteobacteria)
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: DNA Target Strand

MacromoleculeName: DNA Target Strand / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia phage Lambda (virus)
Molecular weightTheoretical: 16.865783 KDa
SequenceString: (DA)(DG)(DC)(DT)(DG)(DA)(DC)(DG)(DT)(DT) (DT)(DG)(DT)(DA)(DC)(DT)(DC)(DC)(DA)(DG) (DC)(DG)(DT)(DC)(DT)(DC)(DA)(DT)(DC) (DT)(DT)(DT)(DA)(DT)(DG)(DC)(DG)(DT)(DC) (DA) (DG)(DC)(DA)(DG)(DA)(DG) ...String:
(DA)(DG)(DC)(DT)(DG)(DA)(DC)(DG)(DT)(DT) (DT)(DG)(DT)(DA)(DC)(DT)(DC)(DC)(DA)(DG) (DC)(DG)(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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Macromolecule #4: DNA Non-Target Strand

MacromoleculeName: DNA Non-Target Strand / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia phage Lambda (virus)
Molecular weightTheoretical: 4.31383 KDa
SequenceString:
(DC)(DT)(DG)(DG)(DA)(DG)(DT)(DA)(DC)(DA) (DA)(DA)(DC)(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: 7.9
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

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.37 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 70093
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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