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- EMDB-65812: Cryo-EM structure of AtCas9-sgRNA-B-form DNA ternary complex -

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Basic information

Entry
Database: EMDB / ID: EMD-65812
TitleCryo-EM structure of AtCas9-sgRNA-B-form DNA ternary complex
Map data
Sample
  • Complex: AtCas9-sgRNA-B-form DNA ternary complex
    • Protein or peptide: CRISPR-associated endonuclease Cas9
    • RNA: sgRNA
    • DNA: TS DNA
    • DNA: NTS DNA
KeywordsComplex / Endonuclease / Immunity / RNA BINDING PROTEIN/RNA/DNA / RNA BINDING PROTEIN-RNA-DNA complex
Biological speciesAlicyclobacillus tengchongensis (bacteria) / Alicyclobacillus tolerans (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.15 Å
AuthorsMeng B / Duan M / Wu LJ / Liu ZJ / Zhang Y
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Nat Struct Mol Biol / Year: 2026
Title: Structural basis of AtCas9 recognition of PAM mutants in underwound DNA topology.
Authors: Min Duan / Bing Meng / Lei Zhou / Lijie Wu / Xiaohan Tong / Dongchao Huang / Hao Yin / Zhi-Jie Liu / Ying Zhang /
Abstract: The CRISPR-Cas9 system locates targets through guide RNA pairing and recognition of a protospacer-adjacent motif (PAM). Although PAM specificity is sequence-determined, DNA topology can relax PAM ...The CRISPR-Cas9 system locates targets through guide RNA pairing and recognition of a protospacer-adjacent motif (PAM). Although PAM specificity is sequence-determined, DNA topology can relax PAM requirements and enable near-PAMless cleavage by the type II-C Alicyclobacillus tengchongensis Cas9 (AtCas9). However, the structural mechanism underlying this regulation remains unknown. Here we report cryogenic-electron microscopy (cryo-EM) structures of AtCas9 bound to B-form DNA or a 340 bp underwound minicircle DNA containing wild-type or mutant PAMs. Despite PAM sequences differences, all three underwound complexes adopt an almost identical architecture distinct from the B-form DNA-bound state. On B-form DNA, AtCas9 recognizes the PAM through base-specific hydrogen bonds and steric exclusion, conferring preference for NCNNN and NRNNA (R = A/G). By contrast, underwound DNA widens the PAM major groove and promotes sequence-independent backbone contacts, explaining the near-PAMless cleavage. These findings uncover a topology-dependent mechanism of PAM recognition and establish a cryo-EM platform using underwound minicircle DNA for structural studies under native-like topological states.
History
DepositionAug 11, 2025-
Header (metadata) releaseJul 1, 2026-
Map releaseJul 1, 2026-
UpdateJul 22, 2026-
Current statusJul 22, 2026Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_65812.png

Downloads & links

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Map

FileDownload / File: emd_65812.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.03 Å/pix.
x 256 pix.
= 262.4 Å		1.03 Å/pix.
x 256 pix.
= 262.4 Å		1.03 Å/pix.
x 256 pix.
= 262.4 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.025 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-1.4666407 - 2.0062747
Average (Standard dev.)-0.00016283154 (±0.039945383)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 262.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_65812_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_65812_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : AtCas9-sgRNA-B-form DNA ternary complex

EntireName: AtCas9-sgRNA-B-form DNA ternary complex
Components
  • Complex: AtCas9-sgRNA-B-form DNA ternary complex
    • Protein or peptide: CRISPR-associated endonuclease Cas9
    • RNA: sgRNA
    • DNA: TS DNA
    • DNA: NTS DNA

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Supramolecule #1: AtCas9-sgRNA-B-form DNA ternary complex

SupramoleculeName: AtCas9-sgRNA-B-form DNA ternary complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Alicyclobacillus tengchongensis (bacteria)

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Macromolecule #1: CRISPR-associated endonuclease Cas9

MacromoleculeName: CRISPR-associated endonuclease Cas9 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Alicyclobacillus tolerans (bacteria)
Molecular weightTheoretical: 132.20775 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAYRLGLAIG ITSVGWAVVA LEKDESGLKP VRIQDLGVRI FDKAEDSKTG ASLALPRREA RSARRRTRRR RHRLWRVKRL LEQHGILSM EQIEALYAQR TSSPDVYALR VAGLDRCLIA EEIARVLIHI AHRRGFQSNR KSEIKDSDAG KLLKAVQENE N LMQSKGYR ...String:
MAYRLGLAIG ITSVGWAVVA LEKDESGLKP VRIQDLGVRI FDKAEDSKTG ASLALPRREA RSARRRTRRR RHRLWRVKRL LEQHGILSM EQIEALYAQR TSSPDVYALR VAGLDRCLIA EEIARVLIHI AHRRGFQSNR KSEIKDSDAG KLLKAVQENE N LMQSKGYR TVAEMLVSEA TKTDAEGKLV HGKKHGYVSN VRNKAGEYRH TVSRQAIVDE VRKIFAAQRA LGNDVMSEEL ED SYLKILC SQRNFDDGPG GDSPYGHGSV SPDGVRQSIY ERMVGSCTFE TGEKRAPRSS YSFERFQLLT KVVNLRIYRQ QED GGRYPC ELTQTERARV IDCAYEQTKI TYGKLRKLLD MKDTESFAGL TYGLNRSRNK TEDTVFVEMK FYHEVRKALQ RAGV FIQDL SIETLDQIGW ILSVWKSDDN RRKKLSTLGL SDNVIEELLP LNGSKFGHLS LKAIRKILPF LEDGYSYDVA CELAG YQFQ GKTEYVKQRL LPPLGEGEVT NPVVRRALSQ AIKVVNAVIR KHGSPESIHI ELARELSKNL DERRKIEKAQ KENQKN NEQ IKDEIREILG SAHVTGRDIV KYKLFKQQQE FCMYSGEKLD VTRLFEPGYA EVDAIIPYGI SFDDSYDNKV LVKTEQA RQ KGNRTPLEYL RDKPEQKAKF IALVESIPLS QKKKNHLLMD KRAIDLEQEG FRERNLSDTR YITRALMNHI QAWLLFDE T ASTRSKRVVC VNGAVTAYMR ARWGLTKDRN AGDKHHAADA VVVACIGDSL IQRVTKYDKF KRNALADRNR YVQQVSKSE GITQYVDKET GEVFTWESFD ERKFLPNEPL EPWPFFRDEL LARLSDDPSK NIRAIGLLTY SETEQIDPIF VSRMPTRKVT GAAHKETIR SPRIVKVDDN KGTEIQVVVS KVALTELKLT KDGEIKDYFR PEDDPRLYNT LRERLVQFGG DAKAAFKEPV Y KISKDGSV RTPVRKVKIQ EKLTLGVPVH GGRGIAENGG MVRIDVFAKD GKYYFVPIYV ADVLKRELPN RLATAHKPYS EW RVVDDSY QFKFSLYPND AVMIKPSREV DITYKDRKEP VGLRLMYFAG ADISGATLTL RTHDNSGELK GLGIQGLEVF EKY VVGPLG DTHPVYKERR MPFRVERKMN LEHHHHHH

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Macromolecule #2: sgRNA

MacromoleculeName: sgRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Alicyclobacillus tolerans (bacteria)
Molecular weightTheoretical: 37.424258 KDa
SequenceString:
CAUACACUCU ACCCCAACGA AUAAGUCAUA GUUCCCUCAG AAACGAGGUU GCUAUGAUAA GGCCGAGCAA CAGGCUCGUG CCGCAAAGC ACUGACCCCA AUGGGUCAUC UACUUUUU

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Macromolecule #3: TS DNA

MacromoleculeName: TS DNA / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Alicyclobacillus tolerans (bacteria)
Molecular weightTheoretical: 10.910996 KDa
SequenceString:
(DC)(DT)(DG)(DT)(DA)(DT)(DG)(DG)(DT)(DA) (DC)(DT)(DT)(DA)(DT)(DT)(DC)(DG)(DT)(DT) (DG)(DG)(DG)(DG)(DT)(DA)(DG)(DA)(DG) (DT)(DG)(DT)(DA)(DT)(DG)

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Macromolecule #4: NTS DNA

MacromoleculeName: NTS DNA / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Alicyclobacillus tolerans (bacteria)
Molecular weightTheoretical: 10.622894 KDa
SequenceString:
(DC)(DA)(DT)(DA)(DC)(DA)(DC)(DT)(DC)(DT) (DA)(DC)(DC)(DC)(DC)(DA)(DA)(DC)(DG)(DA) (DA)(DT)(DA)(DA)(DG)(DT)(DA)(DC)(DC) (DA)(DT)(DA)(DC)(DA)(DG)

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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 (6k 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: 2.5 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 AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.15 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.7) / Number images used: 302040
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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