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- EMDB-62413: Cryo-EM structure of ChCas12b-sgRNA-extended non-target DNA terna... -

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Basic information

Entry
Database: EMDB / ID: EMD-62413
TitleCryo-EM structure of ChCas12b-sgRNA-extended non-target DNA ternary complex (Complex-D)
Map data
Sample
  • Complex: ChCas12b-sgRNA-extended non-target DNA ternary complex (Complex-D)
    • Protein or peptide: C2c1 CRISPR-Cas endonuclease RuvC-like domain-containing protein
    • RNA: sgRNA
    • DNA: Target DNA strand
    • DNA: Non-target DNA strand
  • Ligand: MAGNESIUM ION
KeywordsCRISPR / Cas12b / gene-editing / RNA BINDING PROTEIN/RNA/DNA / RNA BINDING PROTEIN-RNA-DNA complex
Function / homology: / : / C2c1 CRISPR-Cas endonuclease, RuvC-like domain / C2c1 CRISPR-Cas endonuclease RuvC-like domain-containing protein
Function and homology information
Biological speciesCandidatus Hydrogenedentes bacterium ADurb.Bin170 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.11 Å
AuthorsLi Y / Li J / Pei X / Gan J / Lin J
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32130063 China
CitationJournal: Nucleic Acids Res / Year: 2025
Title: Catalytic-state structure of Candidatus Hydrogenedentes Cas12b revealed by cryo-EM studies.
Authors: Ye Li / Jian Li / Xiaotong Pei / Jingjing Wei / Jianhua Gan / Jinzhong Lin /
Abstract: The CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein) systems are adaptive immune mechanisms that play critical roles in defending archaea and ...The CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein) systems are adaptive immune mechanisms that play critical roles in defending archaea and bacteria against invading entities. These systems can be divided into two classes, with class 2 comprising three types (II, V, and VI). Because of their ability to cleave double-stranded DNA, many class 2 CRISPR-Cas proteins have been harnessed as genome editing tools. Unlike the well-studied type II Cas9 proteins, the structural studies of the type V-B Cas12b proteins are limited, hindering their engineering and broader application. Here, we report four complex structures of ChCas12b, which reveal many unique structural features. The folding of the single guide RNA (sgRNA) of ChCas12b is distinct from that of AacCas12b and BthCas12b. Notably, many of these unique features are involved in ChCas12b-sgRNA interaction, suggesting that they are co-evolved. While ChCas12b shares a conserved two-cation-assisted catalytic mechanism with its homologs, it recognizes a longer guide:target heteroduplex, potentially offering higher fidelity in DNA editing. Altogether, our studies suggested that Cas12b family proteins exhibit significant diversity in their folding, sgRNA and target DNA binding. In the future, it is worth characterizing more representative proteins to identify CRISPR-Cas proteins with higher gene editing ability and fidelity.
History
DepositionNov 14, 2024-
Header (metadata) releaseJul 2, 2025-
Map releaseJul 2, 2025-
UpdateJul 2, 2025-
Current statusJul 2, 2025Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_62413.png

Downloads & links

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Map

FileDownload / File: emd_62413.map.gz / Format: CCP4 / Size: 125 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 320 pix.
= 306.88 Å		0.96 Å/pix.
x 320 pix.
= 306.88 Å		0.96 Å/pix.
x 320 pix.
= 306.88 Å

Surface

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Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.959 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-0.3652279 - 0.74454325
Average (Standard dev.)0.00040958644 (±0.021141326)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 306.88 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_62413_msk_1.map
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AxesZYX

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Half map: #2

Fileemd_62413_half_map_1.map
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Half map: #1

Fileemd_62413_half_map_2.map
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Sample components

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Entire : ChCas12b-sgRNA-extended non-target DNA ternary complex (Complex-D)

EntireName: ChCas12b-sgRNA-extended non-target DNA ternary complex (Complex-D)
Components
  • Complex: ChCas12b-sgRNA-extended non-target DNA ternary complex (Complex-D)
    • Protein or peptide: C2c1 CRISPR-Cas endonuclease RuvC-like domain-containing protein
    • RNA: sgRNA
    • DNA: Target DNA strand
    • DNA: Non-target DNA strand
  • Ligand: MAGNESIUM ION

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Supramolecule #1: ChCas12b-sgRNA-extended non-target DNA ternary complex (Complex-D)

SupramoleculeName: ChCas12b-sgRNA-extended non-target DNA ternary complex (Complex-D)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Candidatus Hydrogenedentes bacterium ADurb.Bin170 (bacteria)

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Macromolecule #1: C2c1 CRISPR-Cas endonuclease RuvC-like domain-containing protein

MacromoleculeName: C2c1 CRISPR-Cas endonuclease RuvC-like domain-containing protein
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Candidatus Hydrogenedentes bacterium ADurb.Bin170 (bacteria)
Molecular weightTheoretical: 167.419828 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: PADDLSTQRA YTLRLQGTDP EDQSWRDALW MTHEAVNAGG RAFGDWLLTL RGGIAHELAD TPVKGKKDIT DELRKKRRIL LALSWLSVE SRRGAPDKFI VAGGEEPAGS RNEKVLQALK EILKRRGLSA EESESWMSDC RASLSAAIRD DAVWVNRSAA F DDAQVRIG ...String:
PADDLSTQRA YTLRLQGTDP EDQSWRDALW MTHEAVNAGG RAFGDWLLTL RGGIAHELAD TPVKGKKDIT DELRKKRRIL LALSWLSVE SRRGAPDKFI VAGGEEPAGS RNEKVLQALK EILKRRGLSA EESESWMSDC RASLSAAIRD DAVWVNRSAA F DDAQVRIG ASLTREDIWD MLDPFFGSRE AYLTPAKKKK EDEDSSEGTG EEKAKDLVQK AGQWLSSRFG TGKGANFDAM AE VYSKISE WAGTAQEGVS GKEGIKNLAD ALAAFSPVSQ NLEGVLKLIS GPGYKSATRN LLGELDSLPV VSRDHLSALH EKA AEDTVK CKESTGTKGR RPYADAILND VEKRCGFTYL TDSDNRSVSI LDTSEFPSDY KWGTARHSEF AVILDHAARR ISVA HSWIK LAEAERDRCE EDAAKVYDLP DKVKEWLDTF CSNRSDISGA QGEGYRIRRK AIEGWKEVVA SWGRSSCITA EDRVA AARA LQDDPEIDKF GAIQLFEILA QDEALCVWHK DGDVAKSPDA QMLIDYVLAS DAESKKRRFK VPAYRHPDAL LHPIFC DFG NSRWDITYDI HGARGKKKAK RGSKKEEAMP RGVAMKLWTG SDVLSVSLRW QSKKLAADLA LDQEAEEVTD TAAVSRA DR LGRAAAGIDR GAGVTIAGLF EEAHWNGRLQ APRQQLEAIA AVRDNQKLSS EERERRIAFM KDRIRWLVTF SAKLRPQG P WHSYAPTQGL QSDPKYWPHS EINKKRKGQA KLILSRLPGL RILSVDLGHR FAAACAVWET MSSEAIQEAC RLANHQLPA PADLYLHLKR TVQKNLIDGE KTVEESTVYR RIGADRLPDG TAHPAPWARL DRQFLIKLQG EEKVREASNE EVWQVHLMES ALGLSFPLI DRLVYAGWGG TEKQAARLEA LREKGWKPTG TPADQDEEGG GYKPSLAVDE LMFSAVRTLR LALKYHGDRA R IAFALTAD YKPMPGDTRY YFSEAKDRSS GADAAEREAK HKDYLLDMLL LWHDLAFSRK WRDEEAKELW NLHIAALPGY QA PAAPIQE EAGQGRKKAR EEARAKMTPA AEALLADGTL REKLHGLWKE RWEKDDAQWK KHLRWMKDGI LPRGGRAATP SIR YVGGLS LTRLATLTEF RRKVQVGFYT RLFPSGEKRE IKEAFGQTAL DALERLREQR VKQLASRIAE AALGAGRVSR TALK QDPKR PEARVDAACH AVIIENLEHY RPEETRTRRE NRGLMNWASS KVKKYLSEAC QLHGLFLREV PAGYTSRQDS RTGAP GMRC QDVTVKTFLN SPFWQKQCVQ AQKNKSTARD RFLCALKEAV AQGGMEEEKK MGPIRVPVPG GEVFVSADAA SPAAKG LQA DLNAAANIGL RALLDPDWPG KWWYVPCDRK TAYPAKEKVE GSAAVDVKQA LPFVLPEEKE NKGKTKGGKK GKGEVMN LW RDVSAEPLMT GQWLDYTAYR KEVENRVIQV LTAQLKARNP LRFGNLGDEE EIPY

UniProtKB: C2c1 CRISPR-Cas endonuclease RuvC-like domain-containing protein

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Macromolecule #2: sgRNA

MacromoleculeName: sgRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Candidatus Hydrogenedentes bacterium ADurb.Bin170 (bacteria)
Molecular weightTheoretical: 37.052035 KDa
SequenceString:
GCUUCUACAG GAGGCGAAAA GACUGCGGAA CGUGUCUUCC CCUUCAAUGG GCGUGGCACC GCAGCGUUGU UCAGUCCUGA UCAACGGAC ACGGAUAUGU UGAAGAACAC CAUGAC

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Macromolecule #3: Target DNA strand

MacromoleculeName: Target DNA strand / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Candidatus Hydrogenedentes bacterium ADurb.Bin170 (bacteria)
Molecular weightTheoretical: 15.569955 KDa
SequenceString:
(DC)(DG)(DC)(DC)(DC)(DT)(DT)(DG)(DC)(DT) (DC)(DA)(DC)(DG)(DT)(DC)(DA)(DT)(DG)(DG) (DT)(DG)(DT)(DT)(DC)(DT)(DT)(DC)(DA) (DA)(DC)(DA)(DT)(DA)(DT)(DC)(DC)(DC)(DA) (DA) (DC)(DG)(DC)(DA)(DT)(DG)(DG)(DT) (DG)(DG)(DC)

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Macromolecule #4: Non-target DNA strand

MacromoleculeName: Non-target DNA strand / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Candidatus Hydrogenedentes bacterium ADurb.Bin170 (bacteria)
Molecular weightTheoretical: 15.68401 KDa
SequenceString:
(DG)(DC)(DC)(DA)(DC)(DC)(DA)(DT)(DG)(DC) (DG)(DT)(DT)(DG)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DG) (DT) (DG)(DA)(DG)(DC)(DA)(DA)(DG)(DG) (DG)(DC)(DG)

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / 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: 7.4
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: 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: 1.5 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: NONE
Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.11 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 47756
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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