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- EMDB-63066: Cryo-EM structure of a type II-D CRISPR-Cas9 in complex with sing... -

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Basic information

Entry
Database: EMDB / ID: EMD-63066
TitleCryo-EM structure of a type II-D CRISPR-Cas9 in complex with single-guided RNA and double-stranded DNA
Map data
Sample
  • Complex: Ternary complex of NsCas9-sgRNA-dsDNA
    • Protein or peptide: HNH nuclease domain-containing protein
    • RNA: sgRNA (156-MER)
    • DNA: DNA (41-MER)
    • DNA: DNA (41-MER)
  • Ligand: ZINC ION
  • Ligand: water
KeywordsComplex / CRISPR / HYDROLASE
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
HNH nuclease domain-containing protein
Similarity search - Component
Biological speciesNitrospirae bacterium RBG_13_39_12 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.86 Å
AuthorsWang K / Wang Y
Funding support China, 1 items
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, 2025-
Header (metadata) releaseAug 20, 2025-
Map releaseAug 20, 2025-
UpdateAug 20, 2025-
Current statusAug 20, 2025Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_63066.png

Downloads & links

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Map

FileDownload / File: emd_63066.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 300 pix.
= 246. Å		0.82 Å/pix.
x 300 pix.
= 246. Å		0.82 Å/pix.
x 300 pix.
= 246. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04
Minimum - Maximum-0.25915954 - 0.51986295
Average (Standard dev.)-0.00050221354 (±0.012588706)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 246.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_63066_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_63066_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 : Ternary complex of NsCas9-sgRNA-dsDNA

EntireName: Ternary complex of NsCas9-sgRNA-dsDNA
Components
  • Complex: Ternary complex of NsCas9-sgRNA-dsDNA
    • Protein or peptide: HNH nuclease domain-containing protein
    • RNA: sgRNA (156-MER)
    • DNA: DNA (41-MER)
    • DNA: DNA (41-MER)
  • Ligand: ZINC ION
  • Ligand: water

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Supramolecule #1: Ternary complex of NsCas9-sgRNA-dsDNA

SupramoleculeName: Ternary complex of NsCas9-sgRNA-dsDNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Nitrospirae bacterium RBG_13_39_12 (bacteria)

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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: Nitrospirae bacterium RBG_13_39_12 (bacteria)
Molecular weightTheoretical: 88.439391 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MEKELVLGID YGGKYTGLAV VNQKNNQVLY ARTVKMRDDV TDILAGRREQ RSLRRTLQTK KKRLRELKNY LESIGGIYEE SSGTFTIEP FRTVYSLAHK RGYDYADLPE EKTSEEIEAM DAKERKQWEK EKKELEETQR NSRHRDEVLR DVRNVMTEGN L SEEQIIKV ...String:
MEKELVLGID YGGKYTGLAV VNQKNNQVLY ARTVKMRDDV TDILAGRREQ RSLRRTLQTK KKRLRELKNY LESIGGIYEE SSGTFTIEP FRTVYSLAHK RGYDYADLPE EKTSEEIEAM DAKERKQWEK EKKELEETQR NSRHRDEVLR DVRNVMTEGN L SEEQIIKV ESIFNKQYRH KRFNNRILTK CKVCGKNTPL RINVRELLLE NIVRYLPLQN KERELLKLTI LKGHQQDINE IF KHFRKVY KITLNQKDWP GKNLIDIARN QLRGRLLFCK VHFPENEKYV SIEKKTFRLA PSLKTKIENV LSVIKDDILP NFT LNNVVM ESNNFDIAAK TKGKKRLLKE EYSKGHRESG ETRKEALLRE TDSRCIYCGK GIDLSNAHED HIFPRKAGGI NIFG NLVAC CSVCNEEKRG RTPLESGILP KPEIVSFITN DLKKKILEDA QYINTLDFNK YMSHASIGWR HMRDRLRELT GNKEL LIKR QSGIYTAYFR KWWGFIKERG NHGHHALDAV ILASKKSYAE DGKVDMTIKP CGEDGKEFDI ERHLSEMKEF RRDKGG KSA PLHDRNPLSF KNDIITRRFM VTEIECGKEA VIISEEYRKK LTEAFKRFGI AKGKYLTDEQ AKDAGFYLRK NGEGVMS LK CEVKGTGYNQ MIRIKNNIFK TNVHNVGVAV FLDEKGKKRA CELKNPRLSK HFVKPAEQVK GKVIFILKRG NMVTVEGE E MIYRVKKLGT SPVIEAIVGS DGKTRTVSAT KLLKINHTKK V

UniProtKB: HNH nuclease domain-containing protein

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Macromolecule #2: sgRNA (156-MER)

MacromoleculeName: sgRNA (156-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Nitrospirae bacterium RBG_13_39_12 (bacteria)
Molecular weightTheoretical: 50.211645 KDa
SequenceString:
GGUUAUAACU GAUAUAUGGG GUUACAGUUA AGGCUCUUUG AAAAAAGAGC CUUAAUUGUA AAACGCCUAU ACGGUGAGGG UAUGUACGU UUGGGUUUGU CCAGCCUAAA CCUCUACGCC AGAAAUGGCA CCUUCAUUGU GGGUUAGGAC AAUUUAA

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Macromolecule #3: DNA (41-MER)

MacromoleculeName: DNA (41-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 12.489061 KDa
SequenceString:
(DT)(DC)(DT)(DA)(DG)(DA)(DG)(DG)(DA)(DT) (DC)(DC)(DC)(DC)(DC)(DC)(DA)(DT)(DA)(DT) (DA)(DT)(DC)(DA)(DG)(DT)(DT)(DA)(DT) (DA)(DA)(DC)(DC)(DG)(DA)(DA)(DT)(DT)(DC) (DA) (DC)

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Macromolecule #4: DNA (41-MER)

MacromoleculeName: DNA (41-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 12.560096 KDa
SequenceString:
(DG)(DT)(DG)(DA)(DA)(DT)(DT)(DC)(DC)(DC) (DA)(DA)(DT)(DA)(DT)(DT)(DG)(DA)(DC)(DT) (DA)(DT)(DA)(DT)(DA)(DC)(DC)(DC)(DG) (DG)(DG)(DA)(DT)(DC)(DC)(DT)(DC)(DT)(DA) (DG) (DA)

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 6 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / 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: 1.4000000000000001 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: NONE
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 215731
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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