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Basic information

Entry
Database: EMDB / ID: EMD-65811
TitleCryo-EM structure of AtCas9-sgRNA-underwound DNA (TTGA) ternary complex
Map data
Sample
  • Complex: AtCas9-sgRNA-underwound DNA (TTGA PAM) 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: 2.89 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: Duan M / Meng B / Zhou L / Wu LJ / Tong X / Huang D / Yin H / Liu ZJ / Zhang Y
History
DepositionAug 11, 2025-
Header (metadata) releaseJul 1, 2026-
Map releaseJul 1, 2026-
UpdateJul 1, 2026-
Current statusJul 1, 2026Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_65811.png

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Map

FileReleased
Voxel sizeX=Y=Z: 1.05 Å
Density
Contour LevelBy AUTHOR: 0.18
Minimum - Maximum-3.1127193 - 3.8009696
Average (Standard dev.)0.0021074358 (±0.054329816)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 268.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : AtCas9-sgRNA-underwound DNA (TTGA PAM) ternary complex

EntireName: AtCas9-sgRNA-underwound DNA (TTGA PAM) ternary complex
Components
  • Complex: AtCas9-sgRNA-underwound DNA (TTGA PAM) 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-underwound DNA (TTGA PAM) ternary complex

SupramoleculeName: AtCas9-sgRNA-underwound DNA (TTGA PAM) 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: 9.941408 KDa
SequenceString:
(DT)(DC)(DA)(DA)(DA)(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: 9.73732 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)(DT)(DT) (DT)(DG)(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: 1.8 µm / Nominal defocus min: 1.4000000000000001 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: 2.89 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.7) / Number images used: 1023398
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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