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- EMDB-61880: Cryo-EM structure of Cas5-HNH Cascade bound with dsDNA -

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Entry
Database: EMDB / ID: EMD-61880
TitleCryo-EM structure of Cas5-HNH Cascade bound with dsDNA
Map datasharp map
Sample
  • Complex: Cas5-HNH Cascade complex bound with DNA
    • Protein or peptide: CRISPR-associated protein Cse1 (CRISPR_cse1)
    • Protein or peptide: CRISPR-associated protein Cse2 (CRISPR_cse2)
    • Protein or peptide: CRISPR system Cascade subunit CasC
    • Protein or peptide: CRISPR system Cascade subunit CasD
    • DNA: DNA (54-MER)
    • Protein or peptide: CRISPR-associated endoribonuclease Cse3
    • RNA: RNA (61-MER)
    • DNA: non-target DNA
  • Ligand: MAGNESIUM ION
KeywordsCryo-EM strcuture / Cascade / CRISPR-Cas / IMMUNE SYSTEM / DNA BINDING PROTEIN
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / hydrolase activity / RNA binding / zinc ion binding
Similarity search - Function
CRISPR-associated protein, CT1975 / CRISPR-associated protein Cse2 / Cse2 superfamily / CT1975-like protein / CRISPR-associated protein Cse2 (CRISPR_cse2) / CRISPR-associated protein, CasD / CRISPR-associated protein Cse1 / CRISPR-associated protein Cse1 (CRISPR_cse1) / CRISPR-associated protein Cse3 / CRISPR associated protein ...CRISPR-associated protein, CT1975 / CRISPR-associated protein Cse2 / Cse2 superfamily / CT1975-like protein / CRISPR-associated protein Cse2 (CRISPR_cse2) / CRISPR-associated protein, CasD / CRISPR-associated protein Cse1 / CRISPR-associated protein Cse1 (CRISPR_cse1) / CRISPR-associated protein Cse3 / CRISPR associated protein / CRISPR_assoc / HNH endonuclease / HNH endonuclease / CRISPR-associated protein, Cas5 / CRISPR-associated protein (Cas_Cas5) / CRISPR-associated protein Cas5, N-terminal / HNH nucleases / HNH nuclease
Similarity search - Domain/homology
CRISPR system Cascade subunit CasC / CRISPR-associated endoribonuclease Cse3 / CRISPR system Cascade subunit CasD / CRISPR-associated protein Cse2 (CRISPR_cse2) / CRISPR-associated protein Cse1 (CRISPR_cse1)
Similarity search - Component
Biological speciesCandidatus Cloacimonetes bacterium ADurb.Bin088 (bacteria) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.93 Å
AuthorsLiu YN / Zhang H / Zhu H
Funding support China, 1 items
OrganizationGrant numberCountry
Chinese Academy of Sciences China
CitationJournal: Nat Commun / Year: 2025
Title: Structural basis for RNA-guided DNA degradation by Cas5-HNH/Cascade complex.
Authors: Yanan Liu / Lin Wang / Qian Zhang / Pengyu Fu / Lingling Zhang / Ying Yu / Heng Zhang / Hongtao Zhu /
Abstract: Type I-E CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated proteins) system is one of the most extensively studied RNA-guided adaptive immune systems in ...Type I-E CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated proteins) system is one of the most extensively studied RNA-guided adaptive immune systems in prokaryotes, providing defense against foreign genetic elements. Unlike the previously characterized Cas3 nuclease, which exhibits progressive DNA cleavage in the typical type I-E system, a recently identified HNH-comprising Cascade system enables precise DNA cleavage. Here, we present several near-atomic cryo-electron microscopy (cryo-EM) structures of the Candidatus Cloacimonetes bacterium Cas5-HNH/Cascade complex, both in its DNA-bound and unbound states. Our analysis reveals extensive interactions between the HNH domain and adjacent subunits, including Cas6 and Cas11, with mutations in these key interactions significantly impairing enzymatic activity. Upon DNA binding, the Cas5-HNH/Cascade complex adopts a more compact conformation, with subunits converging toward the center of nuclease, leading to its activation. Notably, we also find that divalent ions such as zinc, cobalt, and nickel down-regulate enzyme activity by destabilizing the Cascade complex. Together, these findings offer structural insights into the assembly and activation of the Cas5-HNH/Cascade complex.
History
DepositionOct 11, 2024-
Header (metadata) releaseDec 11, 2024-
Map releaseDec 11, 2024-
UpdateJun 18, 2025-
Current statusJun 18, 2025Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_61880.png

Downloads & links

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Map

FileDownload / File: emd_61880.map.gz / Format: CCP4 / Size: 175 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharp map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.81 Å/pix.
x 358 pix.
= 291. Å		0.81 Å/pix.
x 358 pix.
= 291. Å		0.81 Å/pix.
x 358 pix.
= 291. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.81285 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.04
Minimum - Maximum-0.14373367 - 16.149560000000001
Average (Standard dev.)0.01158541 (±0.66223747)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions358358358
Spacing358358358
CellA=B=C: 291.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: raw map

Fileemd_61880_additional_1.map
Annotationraw map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_61880_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_61880_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram (log scale)

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Sample components

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Entire : Cas5-HNH Cascade complex bound with DNA

EntireName: Cas5-HNH Cascade complex bound with DNA
Components
  • Complex: Cas5-HNH Cascade complex bound with DNA
    • Protein or peptide: CRISPR-associated protein Cse1 (CRISPR_cse1)
    • Protein or peptide: CRISPR-associated protein Cse2 (CRISPR_cse2)
    • Protein or peptide: CRISPR system Cascade subunit CasC
    • Protein or peptide: CRISPR system Cascade subunit CasD
    • DNA: DNA (54-MER)
    • Protein or peptide: CRISPR-associated endoribonuclease Cse3
    • RNA: RNA (61-MER)
    • DNA: non-target DNA
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Cas5-HNH Cascade complex bound with DNA

SupramoleculeName: Cas5-HNH Cascade complex bound with DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8
Source (natural)Organism: Candidatus Cloacimonetes bacterium ADurb.Bin088 (bacteria)
Molecular weightTheoretical: 400 kDa/nm

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Macromolecule #1: CRISPR-associated protein Cse1 (CRISPR_cse1)

MacromoleculeName: CRISPR-associated protein Cse1 (CRISPR_cse1) / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Candidatus Cloacimonetes bacterium ADurb.Bin088 (bacteria)
Molecular weightTheoretical: 60.665359 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MYCAAVCFPQ TKYQQRGTAL KKPLVGLRKM GVEAAAWNTL KVTRDRPKLT FPDLITPQSK FLDNDLWLKY KVPIEQKEHV MYNLLCDNW VNVVYLSGKP DRISLVQTLK DAHCLQLAYS NPMDRFTVFR FLLALGYWCF ANTNVEPEPD KPLPVSWIPW L EENKEYFE ...String:
MYCAAVCFPQ TKYQQRGTAL KKPLVGLRKM GVEAAAWNTL KVTRDRPKLT FPDLITPQSK FLDNDLWLKY KVPIEQKEHV MYNLLCDNW VNVVYLSGKP DRISLVQTLK DAHCLQLAYS NPMDRFTVFR FLLALGYWCF ANTNVEPEPD KPLPVSWIPW L EENKEYFE LFGDGKRFFQ ADPSSRIRAI TDLIHEIPTA HNLCHFKHVT DYIDGLCEAC CIKGLLRLPV FTTVGGRGIG AG INNTPPF YLLWHANDLA GMLAQNWQPW DNMGIPAWLG SFQKESREVG LLAGMTWLPR KVYLHDPVPG QAACCSCGLP SEA LVYSCS IEVEPVPKGL EWKDPHGVYT DQGKSLQSKI KLMSNDRYTF ADRDWYSPLF SYLHAEGNSR QGKLWLVGFA SDKA KSIDI WDKIIELEGT DTNDELLAQL ANRATALNAM RKKPLRGDFK KSVGTPQIAD IIPHAENRIA INAGKMTENR GYSWQ DADT EYGELLTKVA YSLEPAQTVD ARLKRGNFIS RKPWPIIPES KTKPAEGDQN E

UniProtKB: CRISPR-associated protein Cse1 (CRISPR_cse1)

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Macromolecule #2: CRISPR-associated protein Cse2 (CRISPR_cse2)

MacromoleculeName: CRISPR-associated protein Cse2 (CRISPR_cse2) / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Candidatus Cloacimonetes bacterium ADurb.Bin088 (bacteria)
Molecular weightTheoretical: 20.300639 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MNRGTVDFIA SLENLKEGDL GILRKLRGAR LDEKLPGFDL FSALWWPLRQ KNQRAPKREV AWLIAKLFAE FRFEQREGAT LPILMGGIC RKLEPKKELP RVLARFDQLA SLDIMQMEEP LSVIMGILRK HQQVCLDWVG LTDVLSFWEQ EPVKREWSDS F IKAYKINK EDSDVD

UniProtKB: CRISPR-associated protein Cse2 (CRISPR_cse2)

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Macromolecule #3: CRISPR system Cascade subunit CasC

MacromoleculeName: CRISPR system Cascade subunit CasC / type: protein_or_peptide / ID: 3 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Candidatus Cloacimonetes bacterium ADurb.Bin088 (bacteria)
Molecular weightTheoretical: 41.794367 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MLIEIHMIQN HSPANLNRDD LGAPKTCYFG GVLRSRISSQ CIKRSIRTSN DFKALLGGVR TRRLADLIQQ EAGETECWKK AQEILNKCG FKNKDDNTKM LVFMSKDKIK DLARIVLDNS LGLTEAAQQV ANVIAQATLA PDIALCGRML EPNDKDKDKK V KWSNTTVE ...String:
MLIEIHMIQN HSPANLNRDD LGAPKTCYFG GVLRSRISSQ CIKRSIRTSN DFKALLGGVR TRRLADLIQQ EAGETECWKK AQEILNKCG FKNKDDNTKM LVFMSKDKIK DLARIVLDNS LGLTEAAQQV ANVIAQATLA PDIALCGRML EPNDKDKDKK V KWSNTTVE AALQVAHAIS THIARPEIDY FVAADDVPGE DAGAGHIGES MFASACFYKY FSIDWEQLVK NLKGDTNLAA HT VGAFLLA AAKTNPSGKQ NSFAAHNYPD GILVEFKNSP ISYANAFVRP VSVVKESDLV EQSIGQLSNY VNDIRLGYYD EQS PVIGFW FSPNNRYPLG YKHSKLASRN IGNLNELVGA VLDYIGGFKW EEVQKSKAYI GG

UniProtKB: CRISPR system Cascade subunit CasC

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Macromolecule #4: CRISPR system Cascade subunit CasD

MacromoleculeName: CRISPR system Cascade subunit CasD / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Candidatus Cloacimonetes bacterium ADurb.Bin088 (bacteria)
Molecular weightTheoretical: 43.554316 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSAPPNTLFL RLEGALQSWG SNEAKFALRR TADAPTKSGV LGLLCAAMGI GRAEAADSWL PKLANLRMGV RIDRPGIRWW DFHTVGAGQ RMRMAELKAP KKPSMVGAAL AETLTPSKVK TRAETLLSRR EYLADASFLV ALQGEPELVA KLSAALAKPV W AIYLGRKS ...String:
MSAPPNTLFL RLEGALQSWG SNEAKFALRR TADAPTKSGV LGLLCAAMGI GRAEAADSWL PKLANLRMGV RIDRPGIRWW DFHTVGAGQ RMRMAELKAP KKPSMVGAAL AETLTPSKVK TRAETLLSRR EYLADASFLV ALQGEPELVA KLSAALAKPV W AIYLGRKS CPPSRPVCEH PPGFYNTLEE ALSAVPLQKR WHNEPLPQIL PCVMDWIPGY DGEHAPDDAE IHYDLPVSFQ PP RHLPRFV IRRELVVGED VQVSRETGTS VWRPKGTRAD YNNSEYKKVR AERLVMDHAA CMVCKAPATT VQHVNYRRAG GKE IPEDLR ALCRLCADAC TMLEYGSGMT TNRIDPCDPI WRERILAKRK EIVEFRSRGQ RFRKMKPEEE NG

UniProtKB: CRISPR system Cascade subunit CasD

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Macromolecule #6: CRISPR-associated endoribonuclease Cse3

MacromoleculeName: CRISPR-associated endoribonuclease Cse3 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Candidatus Cloacimonetes bacterium ADurb.Bin088 (bacteria)
Molecular weightTheoretical: 31.218768 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MIYLSRLLID TGGNPDRPRP GRKWLDNIYN VHRRLSMAFP SGLRREQDPH FLKPFSPNDF QKTPFLFRVD NNIDGNDKRA IIIVQSVLE PDWDYCFQNA LDFLAAPPET KEYNPEFKAG QLLRFRLRVN ASVRRHIPEM VQQDGQTIET GKILHKRVSL T WDASSTPD ...String:
MIYLSRLLID TGGNPDRPRP GRKWLDNIYN VHRRLSMAFP SGLRREQDPH FLKPFSPNDF QKTPFLFRVD NNIDGNDKRA IIIVQSVLE PDWDYCFQNA LDFLAAPPET KEYNPEFKAG QLLRFRLRVN ASVRRHIPEM VQQDGQTIET GKILHKRVSL T WDASSTPD QALADWLAAK SPKLGFTLQR CELLQLGWVY GSKPEPKNVK VKEQGQGYWR EHKYNPLRFR AALLEGVLEV DD PKLFLKT LSSGIGKAKS FGFGLLSVLP IRNDG

UniProtKB: CRISPR-associated endoribonuclease Cse3

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Macromolecule #5: DNA (54-MER)

MacromoleculeName: DNA (54-MER) / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 16.524588 KDa
SequenceString: (DG)(DC)(DT)(DT)(DG)(DA)(DC)(DA)(DT)(DG) (DT)(DG)(DT)(DG)(DC)(DT)(DA)(DA)(DG)(DC) (DG)(DC)(DA)(DC)(DC)(DT)(DA)(DA)(DT) (DT)(DT)(DC)(DC)(DT)(DG)(DA)(DC)(DG)(DG) (DC) (DA)(DA)(DT)(DC)(DC)(DT) ...String:
(DG)(DC)(DT)(DT)(DG)(DA)(DC)(DA)(DT)(DG) (DT)(DG)(DT)(DG)(DC)(DT)(DA)(DA)(DG)(DC) (DG)(DC)(DA)(DC)(DC)(DT)(DA)(DA)(DT) (DT)(DT)(DC)(DC)(DT)(DG)(DA)(DC)(DG)(DG) (DC) (DA)(DA)(DT)(DC)(DC)(DT)(DT)(DA) (DC)(DC)(DA)(DG)(DC)(DT)

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Macromolecule #8: non-target DNA

MacromoleculeName: non-target DNA / type: dna / ID: 8 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 16.75175 KDa
SequenceString: (DA)(DG)(DC)(DT)(DG)(DG)(DT)(DA)(DA)(DG) (DG)(DA)(DT)(DT)(DG)(DC)(DC)(DG)(DT)(DC) (DA)(DG)(DG)(DA)(DA)(DA)(DT)(DT)(DA) (DG)(DG)(DT)(DG)(DC)(DG)(DC)(DT)(DT)(DA) (DG) (DC)(DA)(DC)(DA)(DC)(DA) ...String:
(DA)(DG)(DC)(DT)(DG)(DG)(DT)(DA)(DA)(DG) (DG)(DA)(DT)(DT)(DG)(DC)(DC)(DG)(DT)(DC) (DA)(DG)(DG)(DA)(DA)(DA)(DT)(DT)(DA) (DG)(DG)(DT)(DG)(DC)(DG)(DC)(DT)(DT)(DA) (DG) (DC)(DA)(DC)(DA)(DC)(DA)(DT)(DG) (DT)(DC)(DA)(DA)(DG)(DC)

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Macromolecule #7: RNA (61-MER)

MacromoleculeName: RNA (61-MER) / type: rna / ID: 7 / Number of copies: 1
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 19.697695 KDa
SequenceString:
GUGAACCGGA UUGCCGUCAG GAAAUUAGGU GCGCUUAGCA GUAUUCCCCA CGCAUGUGGG G

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Macromolecule #9: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 9 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 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: FEI FALCON IV (4k x 4k) / Average electron dose: 48.62 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.2 µm / Nominal defocus min: 1.0 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: 2.93 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 73951
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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