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- EMDB-43757: Cas9d Effector:sgRNA Binary Complex -

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Basic information

Entry
Database: EMDB / ID: EMD-43757
TitleCas9d Effector:sgRNA Binary Complex
Map data
Sample
  • Complex: Cas9d:sgRNA
    • Protein or peptide: HNH nuclease domain-containing protein
    • RNA: sgRNA
KeywordsCRISPR-Cas9 / Endonuclease / Binary Complex / Effector / sgRNA / IMMUNE SYSTEM-RNA complex
Biological speciesDeltaproteobacteria (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 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-
UpdateJun 4, 2025-
Current statusJun 4, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43757.png

Downloads & links

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Map

FileDownload / File: emd_43757.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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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

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8332 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.246
Minimum - Maximum-1.7545066 - 2.878404
Average (Standard dev.)-0.00021821843 (±0.04724821)
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_43757_additional_1.map
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Half map: #2

Fileemd_43757_half_map_1.map
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_43757_half_map_2.map
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Sample components

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Entire : Cas9d:sgRNA

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

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Supramolecule #1: Cas9d:sgRNA

SupramoleculeName: Cas9d:sgRNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Deltaproteobacteria (bacteria)
Molecular weightTheoretical: 137.34 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: 108.989773 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 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 KANSAEIEQS EKDNLKDAVL DKNRREEVKS FFRKHKTDEH IRKQVYDIAD NKLSGRTVF CKEHILSRGS ALHDRNPLSY KKGIITRRFM VTEIECGKED DVISETYREK LKEAFKRFDT KKGKCLTDKE AKEAGFCIKK NELVMSLKC SIKGTGPGQM IRINNNVFKT NVHNV(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)RKNPRLSK HFIE

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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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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: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 101411
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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