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- EMDB-61051: Cryo-EM structure of the RdCas12n-sgRNA-DNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-61051
TitleCryo-EM structure of the RdCas12n-sgRNA-DNA complex
Map data
Sample
  • Complex: RdCas12n-sgRNA-DNA complex
    • Protein or peptide: Transposase
    • DNA: DNA (40-MER)
    • DNA: DNA (5'-D(P*GP*CP*TP*GP*TP*GP*AP*GP*AP*AP*AP*CP*CP*G)-3')
    • RNA: sgRNA
KeywordsCRISPR-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
Biological speciesRothia dentocariosa (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.95 Å
AuthorsFu W / Ji Q
Funding support China, 3 items
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
DepositionAug 2, 2024-
Header (metadata) releaseJun 4, 2025-
Map releaseJun 4, 2025-
UpdateJul 23, 2025-
Current statusJul 23, 2025Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_61051.png

Downloads & links

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Map

FileDownload / File: emd_61051.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.96 Å/pix.
x 384 pix.
= 368.64 Å		0.96 Å/pix.
x 384 pix.
= 368.64 Å		0.96 Å/pix.
x 384 pix.
= 368.64 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.96 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-6.8850236 - 8.364497
Average (Standard dev.)-0.0000964759 (±0.06752649)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 368.63998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_61051_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_61051_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_61051_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 : RdCas12n-sgRNA-DNA complex

EntireName: RdCas12n-sgRNA-DNA complex
Components
  • Complex: RdCas12n-sgRNA-DNA complex
    • Protein or peptide: Transposase
    • DNA: DNA (40-MER)
    • DNA: DNA (5'-D(P*GP*CP*TP*GP*TP*GP*AP*GP*AP*AP*AP*CP*CP*G)-3')
    • RNA: sgRNA

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Supramolecule #1: RdCas12n-sgRNA-DNA complex

SupramoleculeName: RdCas12n-sgRNA-DNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Rothia dentocariosa (bacteria)
Molecular weightTheoretical: 144.465 KDa

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Macromolecule #1: Transposase

MacromoleculeName: Transposase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Rothia dentocariosa (bacteria)
Molecular weightTheoretical: 61.318246 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MATTDKKDEE NLRAYKFRLD PNQAQTTALY QAVGAARYTY NMLTAYNLEV NRLRDDYWKR RHDEDISDAD IKKELNALAK EDKRYKQLN YGAFGTQYLT PEKKRHEQAE HRIENGEDPS VVWNQETERS ANPWLHTANQ RVLVSGLQNA SDAWDNFWAS R TGKRAGRL ...String:
MATTDKKDEE NLRAYKFRLD PNQAQTTALY QAVGAARYTY NMLTAYNLEV NRLRDDYWKR RHDEDISDAD IKKELNALAK EDKRYKQLN YGAFGTQYLT PEKKRHEQAE HRIENGEDPS VVWNQETERS ANPWLHTANQ RVLVSGLQNA SDAWDNFWAS R TGKRAGRL VGTPRFKKKG VSRDSFTVPA PEKMGAYGTA YLRGEPAYKQ GRRKITDYRH VRLSYLGTIR TFNSTKPLVK AV VAGAKIR SYTVSRNADR WYVSFLVKFS EPIRRSATKR ARAAGSVGVD LGVKYLASLS DSEAPQRFPN LKFVEGLPSL ENP RWSEAS SRRLHKLQRA LARSQKGSNR RSRLVKQIAR LHHMTALRRE SNLHQLTKKL ATEYTLVGFE DLNVSGMTAS AKGT VENPG KNVAQKSGLN RVVLDAAFGV FRNQLEYKAV WYGSAFEKVD RYFASSQTCS ECGRKAKTKL TLRDRVFDCA YCGNM MDRD LNAAVNICRE AQRLFDEKLA SEDRESLNGR GSRGALRGAE TVEASRPPAS HRRGSP

UniProtKB: Transposase

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Macromolecule #2: DNA (40-MER)

MacromoleculeName: DNA (40-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 12.205824 KDa
SequenceString:
(DG)(DG)(DC)(DA)(DG)(DT)(DG)(DT)(DT)(DT) (DT)(DC)(DA)(DC)(DT)(DT)(DT)(DC)(DA)(DC) (DC)(DT)(DG)(DA)(DA)(DC)(DC)(DG)(DG) (DT)(DT)(DT)(DC)(DT)(DC)(DA)(DC)(DA)(DG) (DC)

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Macromolecule #3: DNA (5'-D(P*GP*CP*TP*GP*TP*GP*AP*GP*AP*AP*AP*CP*CP*G)-3')

MacromoleculeName: DNA (5'-D(P*GP*CP*TP*GP*TP*GP*AP*GP*AP*AP*AP*CP*CP*G)-3')
type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 4.32983 KDa
SequenceString:
(DG)(DC)(DT)(DG)(DT)(DG)(DA)(DG)(DA)(DA) (DA)(DC)(DC)(DG)

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Macromolecule #4: sgRNA

MacromoleculeName: sgRNA / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Rothia dentocariosa (bacteria)
Molecular weightTheoretical: 69.274062 KDa
SequenceString: CUUUUUGACG AAAAACUCGC CUCAGAAGAU AGGGAGAGUC UAAACGGACG UGGAAGUCGA GGCGCUCUUC GGGGUGCUGA GACUGUGGA AGCGUCAAGA CCACCUGCGA GUCAUCGUAG AGGGUCACCG UAGAUGAGUA AUCAUCUGCC CAUCUAUUGC A UUAUGCAC ...String:
CUUUUUGACG AAAAACUCGC CUCAGAAGAU AGGGAGAGUC UAAACGGACG UGGAAGUCGA GGCGCUCUUC GGGGUGCUGA GACUGUGGA AGCGUCAAGA CCACCUGCGA GUCAUCGUAG AGGGUCACCG UAGAUGAGUA AUCAUCUGCC CAUCUAUUGC A UUAUGCAC GCGAAAGCGU GUGCAUGGGU GGUUCCCGGU UCAGGUGAAA GUGAAA

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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

Concentration0.277 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
500.0 mMNaClsodium chloride
10.0 mMMgCl2Magnesium chloride
20.0 mMTris-HClTris (Hydroxymethyl) Aminomethane Hydrochloride
1.0 mMDTTDithiothreitol

Details: 10mM MgCl2, 20mM Tris-HCl,500nM NaCl, 1mM DTT
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 32 / Number real images: 5296 / Average exposure time: 5.59 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 1.2 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: 2.95 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 879820
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: NOT APPLICABLE

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