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- PDB-9lgi: Cryo-EM structure of a type II-D CRISPR-Cas9 in complex with sing... -

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Basic information

Entry
Database: PDB / ID: 9lgi
TitleCryo-EM structure of a type II-D CRISPR-Cas9 in complex with single-guided RNA and double-stranded DNA
Components
  • (DNA (41-MER)) x 2
  • HNH nuclease domain-containing protein
  • sgRNA (156-MER)
KeywordsHYDROLASE / Complex / CRISPR
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 speciesNitrospirae bacterium RBG_13_39_12 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.86 Å
AuthorsWang, K. / Wang, Y.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Nat Commun / Year: 2025
Title: Structural insights into Type II-D Cas9 and its robust cleavage activity.
Authors: Kangkang Wang / Jiuyu Wang / Xiaoqi Yang / Wei Sun / Gang Sheng / Yanli Wang /
Abstract: Type II-D Cas9 proteins (Cas9d) are more compact than typical Type II-A/B/C Cas9s. Here, we demonstrate that NsCas9d from Nitrospirae bacterium RBG_13_39_12 derived from a metagenomic assembly ...Type II-D Cas9 proteins (Cas9d) are more compact than typical Type II-A/B/C Cas9s. Here, we demonstrate that NsCas9d from Nitrospirae bacterium RBG_13_39_12 derived from a metagenomic assembly exhibits robust dsDNA cleavage activity comparable to SpCas9 in vitro. Unlike typical Cas9 enzymes that generate blunt ends, NsCas9d produces 3-nucleotide staggered overhangs. Our high-resolution cryo-EM structure of the NsCas9d-sgRNA-dsDNA complex in its catalytic state reveals the target and non-target DNA strands positioned within the HNH and RuvC catalytic pockets, respectively. NsCas9d recognizes the 5'-NRG-3' protospacer adjacent motif (PAM), with 5'-NGG-3' showing the highest cleavage efficiency. Its sgRNA structure, resembling the 5' end of IscB ωRNA, along with structural features shared with other Cas9 variants, suggests that Cas9d are hypothesized to resemble evolutionary intermediates between other Cas9 sub-types and IscB. These findings deepen our understanding of Cas9 evolution and mechanisms, highlighting NsCas9d as a promising genome-editing tool due to its compact size, DNA cleavage pattern, and efficient PAM recognition.
History
DepositionJan 10, 2025Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Aug 20, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: HNH nuclease domain-containing protein
B: sgRNA (156-MER)
C: DNA (41-MER)
D: DNA (41-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)163,8316
Polymers163,7004
Non-polymers1312
Water1086
1


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

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Components

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DNA chain , 2 types, 2 molecules CD

#3: DNA chain DNA (41-MER)


Mass: 12489.061 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (41-MER)


Mass: 12560.096 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein / RNA chain , 2 types, 2 molecules AB

#1: Protein HNH nuclease domain-containing protein / CRISPR-associated endonuclease Cas9/Csn1


Mass: 88439.391 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Nitrospirae bacterium RBG_13_39_12 (bacteria)
Gene: A2Y97_00035 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A1G1G089
#2: RNA chain sgRNA (156-MER)


Mass: 50211.645 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Nitrospirae bacterium RBG_13_39_12 (bacteria)
Production host: Escherichia coli (E. coli)

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Non-polymers , 2 types, 8 molecules

#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
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: Ternary complex of NsCas9-sgRNA-dsDNA / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Source (natural)Organism: Nitrospirae bacterium RBG_13_39_12 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1400 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: NONE
3D reconstructionResolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 215731 / Symmetry type: POINT

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