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- EMDB-60006: Cryo-EM structure of the Cas9d-sgRNA-target DNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-60006
TitleCryo-EM structure of the Cas9d-sgRNA-target DNA complex
Map data
Sample
  • Complex: a protein
    • RNA: RNA (144-MER)
    • DNA: DNA (32-MER)
    • Protein or peptide: a protein
    • DNA: DNA (5'-D(P*CP*GP*GP*GP*TP*CP*AP*AP*AP*TP*G)-3')
  • Ligand: ZINC ION
Keywordsa protein complex / ANTIVIRAL PROTEIN
Biological speciesmetagenome (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.65 Å
AuthorsZhang H / Li X / Liu Z
Funding support China, 1 items
OrganizationGrant numberCountry
Other government China
CitationJournal: Nat Commun / Year: 2025
Title: Insights into the compact CRISPR-Cas9d system.
Authors: Jie Yang / Tongyao Wang / Ying Huang / Zhaoyi Long / Xuzichao Li / Shuqin Zhang / Lingling Zhang / Zhikun Liu / Qian Zhang / Huabing Sun / Minjie Zhang / Hang Yin / Zhongmin Liu / Heng Zhang /
Abstract: Cas9d, the smallest known member of the Cas9 family, employs a compact domain architecture for effective target cleavage. However, the underlying mechanism remains unclear. Here, we present the cryo- ...Cas9d, the smallest known member of the Cas9 family, employs a compact domain architecture for effective target cleavage. However, the underlying mechanism remains unclear. Here, we present the cryo-EM structures of the Cas9d-sgRNA complex in both target-free and target-bound states. Biochemical assays elucidated the PAM recognition and DNA cleavage mechanisms of Cas9d. Structural comparisons revealed that at least 17 base pairs in the guide-target heteroduplex is required for nuclease activity. Beyond its typical role as an adaptor between Cas9 enzymes and targets, the sgRNA also provides structural support and functional regulation for Cas9d. A segment of the sgRNA scaffold interacts with the REC domain to form a functional target recognition module. Upon target binding, this module undergoes a coordinated conformational rearrangement, enabling heteroduplex propagation and facilitating nuclease activity. This hybrid functional module precisely monitors heteroduplex complementarity, resulting in a lower mismatch tolerance compared to SpyCas9. Moreover, structure-guided engineering in both the sgRNA and Cas9d protein led to a more compact Cas9 system with well-maintained nuclease activity. Altogether, our findings provide insights into the target recognition and cleavage mechanisms of Cas9d and shed light on the development of high-fidelity mini-CRISPR tools.
History
DepositionMay 3, 2024-
Header (metadata) releaseFeb 19, 2025-
Map releaseFeb 19, 2025-
UpdateSep 3, 2025-
Current statusSep 3, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_60006.png

Downloads & links

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Map

FileDownload / File: emd_60006.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.)
0.83 Å/pix.
x 300 pix.
= 249. Å		0.83 Å/pix.
x 300 pix.
= 249. Å		0.83 Å/pix.
x 300 pix.
= 249. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-2.7888868 - 3.932924
Average (Standard dev.)-0.0011416143 (±0.065342486)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 249.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_60006_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_60006_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : a protein

EntireName: a protein
Components
  • Complex: a protein
    • RNA: RNA (144-MER)
    • DNA: DNA (32-MER)
    • Protein or peptide: a protein
    • DNA: DNA (5'-D(P*CP*GP*GP*GP*TP*CP*AP*AP*AP*TP*G)-3')
  • Ligand: ZINC ION

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Supramolecule #1: a protein

SupramoleculeName: a protein / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: metagenome (others)

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Macromolecule #1: RNA (144-MER)

MacromoleculeName: RNA (144-MER) / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: metagenome (others)
Molecular weightTheoretical: 51.127133 KDa
SequenceString:
GGUUCGAAAU UAGGUGCGCU UCGCGUUACA GUUAAGGCUC UGAAAAGAGC CUUAAUUGUA AAACGCCUAU ACAGUGAAGG GAUAUACGC UUGGGUUUGU CCAGCCUGAG CCUCUAUGCC AGAAAUGGCG CCUUCAUCGU GGGUUAGGAC AUUUAAUUUU

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Macromolecule #2: DNA (32-MER)

MacromoleculeName: DNA (32-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: metagenome (others)
Molecular weightTheoretical: 11.27125 KDa
SequenceString:
(DC)(DA)(DT)(DT)(DT)(DG)(DA)(DC)(DC)(DC) (DG)(DC)(DG)(DA)(DA)(DG)(DC)(DG)(DC)(DA) (DC)(DC)(DT)(DA)(DA)(DT)(DT)(DT)(DC) (DG)(DA)(DA)(DG)(DC)(DC)(DC)(DT)

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Macromolecule #4: DNA (5'-D(P*CP*GP*GP*GP*TP*CP*AP*AP*AP*TP*G)-3')

MacromoleculeName: DNA (5'-D(P*CP*GP*GP*GP*TP*CP*AP*AP*AP*TP*G)-3') / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: metagenome (others)
Molecular weightTheoretical: 11.502379 KDa
SequenceString:
(DA)(DG)(DG)(DG)(DC)(DT)(DT)(DC)(DG)(DA) (DA)(DA)(DT)(DT)(DA)(DG)(DG)(DT)(DG)(DC) (DG)(DC)(DT)(DT)(DC)(DG)(DC)(DG)(DG) (DG)(DT)(DC)(DA)(DA)(DA)(DT)(DG)

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Macromolecule #3: a protein

MacromoleculeName: a protein / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: metagenome (others)
Molecular weightTheoretical: 86.585766 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MERELVLGIA YGGKYTGLAV VDRRHNQVLY ANRLKMRDDV AGILKDRRKQ RGIRRTAQTK KKRLRELKNY LKSIGYNEST ATFETVYSL AHKRGYDYAD MPEEKTSEEI EAMDVEERKQ WEKEKQEWEE TKRNSRHRKE VVKDVHKAMI EGRATEEQIK R VERIFNKQ ...String:
MERELVLGIA YGGKYTGLAV VDRRHNQVLY ANRLKMRDDV AGILKDRRKQ RGIRRTAQTK KKRLRELKNY LKSIGYNEST ATFETVYSL AHKRGYDYAD MPEEKTSEEI EAMDVEERKQ WEKEKQEWEE TKRNSRHRKE VVKDVHKAMI EGRATEEQIK R VERIFNKQ YRPKRFNNRI LTKCKVEDCG VNTPLRKNVR DLLIENIVRF FPIEQSEKDN LKDAVLDKNR REEVKSFFRK HK TDEHIRK QVYDIADNKL SGRTVFCKEH ILEHTEHSKE ERKVFRLAPS LKTKIENVLA VIKDEILPKF TVNKVVMESN NFD IAAKTQ GKKRLAKEEY GKGPREGKET RKEALLRETD GRCIYCGKSI DISNAHDDAI FPRKAGGLNI FANLVACCAV CNEN KKGRT PLESGISPKP EIIAFMKNDL KKKILEDARN INTVDFNKYM SHASIGWRYM RDRLRESAGN KKLPIERQSG IYTAY FRRW WGFKKERGNT LHHALDAVIL ASRKGYSDDG LVDMTLKPKY NKGGEFDPEK HLPEPIEFKM DKGSRGSALH DRNPLS YKK GIITRRFMVT EIECGKEDDV ISETYREKLK EAFKRFDTKK GKCLTDKEAK EAGFCIKKNE LVMSLKCSIK GTGPGQM IR INNNVFKTNV HNVGVDVYLD EKGKKKAYER KNPRLSKHFI EPPPQPNGRV SFTLKRRDMV TVEGEDAIYR IKKLGTSP T IEAVVGSDGK TRTVSATKLT KANSAE

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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 K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING ONLY
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.65 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 171509
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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