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- PDB-8w2z: Cas9d 6bp R-loop Seed Complex -

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Basic information

Entry
Database: PDB / ID: 8w2z
TitleCas9d 6bp R-loop Seed Complex
Components
  • DNA Non-Target Strand
  • DNA Target Strand
  • HNH nuclease domain-containing protein
  • sgRNA
KeywordsIMMUNE SYSTEM/RNA/DNA / CRISPR-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
DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / HNH nuclease domain-containing protein
Similarity search - Component
Biological speciesDeltaproteobacteria (bacteria)
Escherichia phage Lambda (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.37 Å
AuthorsFregoso Ocampo, R. / Bravo, J.P.K. / Taylor, D.W.
Funding support United States, 1items
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, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 15, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: HNH nuclease domain-containing protein
B: sgRNA
C: DNA Target Strand
D: DNA Non-Target Strand


Theoretical massNumber of molelcules
Total (without water)158,5844
Polymers158,5844
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein HNH nuclease domain-containing protein


Mass: 86568.711 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Deltaproteobacteria (bacteria) / Gene: A2022_01700 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): NICO DE3 / References: UniProt: A0A1F8ZSN4
#2: RNA chain sgRNA


Mass: 50836.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Deltaproteobacteria (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): NICO DE3
#3: DNA chain DNA Target Strand


Mass: 16865.783 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia phage Lambda (virus)
#4: DNA chain DNA Non-Target Strand


Mass: 4313.830 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia phage Lambda (virus)
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Cas9d:sgRNA:DNA / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Source (natural)Organism: Deltaproteobacteria (bacteria) / Strain: NICO DE3
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: NICO DE3
Buffer solutionpH: 7.9
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 80 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.37 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 70093 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0058161
ELECTRON MICROSCOPYf_angle_d0.84511804
ELECTRON MICROSCOPYf_dihedral_angle_d22.0792919
ELECTRON MICROSCOPYf_chiral_restr0.0481440
ELECTRON MICROSCOPYf_plane_restr0.007887

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