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- PDB-9udi: Cryo-EM structure of the RdCas12n-sgRNA-DNA ternary complex, Conf... -

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Basic information

Entry
Database: PDB / ID: 9udi
TitleCryo-EM structure of the RdCas12n-sgRNA-DNA ternary complex, Conformation 2
Components
  • DNA (40-MER)
  • DNA (5'-D(P*GP*CP*TP*GP*TP*GP*AP*GP*AP*AP*AP*CP*CP*G)-3')
  • Transposase
  • sgRNA
KeywordsDNA BINDING PROTEIN/DNA/RNA / CRISPR-Cas / Complex / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA-RNA complex
Function / homology
Function and homology information


transposition / DNA recombination / DNA binding / metal ion binding
Similarity search - Function
Transposase, putative, helix-turn-helix domain / Helix-turn-helix domain / Probable transposase, IS891/IS1136/IS1341 / Probable transposase / : / Transposase IS605, OrfB, C-terminal / Cas12f1-like, TNB domain
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / Transposase
Similarity search - Component
Biological speciesRothia dentocariosa (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.01 Å
AuthorsFu, W. / Ji, Q.
Funding support China, 3items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)21922705 China
National Natural Science Foundation of China (NSFC)2207783 China
National Natural Science Foundation of China (NSFC)91753127 China
CitationJournal: Nat Commun / Year: 2025
Title: Mechanisms and engineering of a miniature type V-N CRISPR-Cas12 effector enzyme.
Authors: Wenhan Fu / Jiacheng Ma / Zhipeng Wang / Na Tang / Deng Pan / Mengjiao Su / Zhaowei Wu / Jianhua Gan / Quanjiang Ji /
Abstract: Type V CRISPR-Cas12 systems are highly diverse in their functionality and molecular compositions, including miniature Cas12f1 and Cas12n genome editors that provide advantages for efficient in vivo ...Type V CRISPR-Cas12 systems are highly diverse in their functionality and molecular compositions, including miniature Cas12f1 and Cas12n genome editors that provide advantages for efficient in vivo therapeutic delivery due to their small size. In contrast to Cas12f1 nucleases that utilize a homodimer structure for DNA targeting and cleavage with a preference for T- or C-rich PAMs, Cas12n nucleases are likely monomeric proteins and uniquely recognize rare A-rich PAMs. However, the molecular mechanisms behind RNA-guided genome targeting and cleavage by Cas12n remain unclear. Here, we present the cryo-electron microscopy (cryo-EM) structure of Rothia dentocariosa Cas12n (RdCas12n) bound to a single guide RNA (sgRNA) and target DNA, illuminating the intricate molecular architecture of Cas12n and its sgRNA, as well as PAM recognition and nucleic-acid binding mechanisms. Through structural comparisons with other Cas12 nucleases and the ancestral precursor TnpB, we provide insights into the evolutionary significance of Cas12n in the progression from TnpB to various Cas12 nucleases. Additionally, we extensively modify the sgRNA and convert RdCas12n into an effective genome editor in human cells. Our findings enhance the understanding of the evolutionary mechanisms of type V CRISPR-Cas12 systems and offer a molecular foundation for engineering Cas12n genome editors.
History
DepositionApr 6, 2025Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Jun 4, 2025Provider: repository / Type: Initial release
Revision 1.0Jun 4, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jun 4, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jun 4, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 4, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 4, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jun 4, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Jun 4, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Jul 23, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Transposase
C: DNA (40-MER)
D: DNA (5'-D(P*GP*CP*TP*GP*TP*GP*AP*GP*AP*AP*AP*CP*CP*G)-3')
R: sgRNA


Theoretical massNumber of molelcules
Total (without water)147,1284
Polymers147,1284
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 Transposase / RdCas12n


Mass: 61318.246 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rothia dentocariosa (bacteria) / Gene: HXO56_11955 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A7D4LAR1
#2: DNA chain DNA (40-MER)


Mass: 12205.824 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (5'-D(P*GP*CP*TP*GP*TP*GP*AP*GP*AP*AP*AP*CP*CP*G)-3')


Mass: 4329.830 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: RNA chain sgRNA


Mass: 69274.062 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rothia dentocariosa (bacteria) / Production host: Escherichia coli (E. coli)
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: RdCas12n-sgRNA-DNA complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.144465 MDa / Experimental value: NO
Source (natural)Organism: Rothia dentocariosa (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5 / Details: 10mM MgCl2, 20mM Tris-HCl,500nM NaCl, 1mM DTT
Buffer component
IDConc.NameFormulaBuffer-ID
1500 mMsodium chlorideNaCl1
210 mMMagnesium chlorideMgCl21
320 mMTris (Hydroxymethyl) Aminomethane HydrochlorideTris-HCl1
41 mMDithiothreitolDTT1
SpecimenConc.: 0.277 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K

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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: 1800 nm / Nominal defocus min: 1200 nm
Image recordingAverage exposure time: 5.59 sec. / Electron dose: 60 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k) / Num. of grids imaged: 32 / Num. of real images: 5296
Image scansWidth: 4096 / Height: 4096

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.01 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 879820 / Symmetry type: POINT

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