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- EMDB-48053: FnCas9 perfect match DNA product state no RuvC -

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Basic information

Entry
Database: EMDB / ID: EMD-48053
TitleFnCas9 perfect match DNA product state no RuvC
Map dataFnCas9 perfect match DNA product state no RuvC
Sample
  • Complex: Ternary complex of FnCas9 with HBB gRNA and HBB DNA
    • Protein or peptide: CRISPR-associated endonuclease Cas9
    • RNA: gRNA
    • DNA: HBB DNA TS 1
    • DNA: HBB DNA NTS
    • DNA: HBB DNA TS 2
  • Ligand: MAGNESIUM ION
KeywordsCRISPR / Cas9 / Hydrolase-RNA-DNA complex
Function / homology
Function and homology information


endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
CRISPR system subtype II-B RNA-guided endonuclease Cas9/Csx12 / : / : / CRISPR-associated endonuclease Cas9 alpha-helical lobe / CRISPR-associated endonuclease Cas9, C-terminal domain / Cas9-type HNH domain / Cas9-type HNH domain profile.
Similarity search - Domain/homology
CRISPR-associated endonuclease Cas9
Similarity search - Component
Biological speciesFrancisella tularensis subsp. novicida (bacteria) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsHibshman GN / Taylor DW
Funding support United States, 2 items
OrganizationGrant numberCountry
Welch FoundationF-1938 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM138348 United States
CitationJournal: Nucleic Acids Res / Year: 2025
Title: Structural basis of a dual-function type II-B CRISPR-Cas9.
Authors: Grace N Hibshman / David W Taylor /
Abstract: Cas9 from Streptococcus pyogenes (SpCas9) revolutionized genome editing by enabling programmable DNA cleavage guided by an RNA. However, SpCas9 tolerates mismatches in the DNA-RNA duplex, which can ...Cas9 from Streptococcus pyogenes (SpCas9) revolutionized genome editing by enabling programmable DNA cleavage guided by an RNA. However, SpCas9 tolerates mismatches in the DNA-RNA duplex, which can lead to deleterious off-target editing. Here, we reveal that Cas9 from Francisella novicida (FnCas9) possesses a unique structural feature-the REC3 clamp-that underlies its intrinsic high-fidelity DNA targeting. Through kinetic and structural analyses, we show that the REC3 clamp forms critical contacts with the PAM-distal region of the R-loop, thereby imposing a novel checkpoint during enzyme activation. Notably, F. novicida encodes a noncanonical small CRISPR-associated RNA (scaRNA) that enables FnCas9 to repress an endogenous bacterial lipoprotein gene, subverting host immune detection. Structures of FnCas9 with scaRNA illustrate how partial R-loop complementarity hinders REC3 clamp docking and prevents cleavage in favor of transcriptional repression. The REC3 clamp is conserved across type II-B CRISPR-Cas9 systems, pointing to a potential path for engineering precise genome editors or developing novel antibacterial strategies. These findings reveal the molecular basis of heightened specificity and virulence enabled by FnCas9, with broad implications for biotechnology and therapeutic development.
History
DepositionNov 22, 2024-
Header (metadata) releaseOct 1, 2025-
Map releaseOct 1, 2025-
UpdateOct 8, 2025-
Current statusOct 8, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_48053.png

Downloads & links

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Map

FileDownload / File: emd_48053.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFnCas9 perfect match DNA product state no RuvC
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 400 pix.
= 333.28 Å		0.83 Å/pix.
x 400 pix.
= 333.28 Å		0.83 Å/pix.
x 400 pix.
= 333.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8332 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.109
Minimum - Maximum-0.2758725 - 0.5174462
Average (Standard dev.)-0.000069444 (±0.010310753)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 333.28 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map B

Fileemd_48053_half_map_1.map
AnnotationHalf Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map A

Fileemd_48053_half_map_2.map
AnnotationHalf Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of FnCas9 with HBB gRNA and HBB DNA

EntireName: Ternary complex of FnCas9 with HBB gRNA and HBB DNA
Components
  • Complex: Ternary complex of FnCas9 with HBB gRNA and HBB DNA
    • Protein or peptide: CRISPR-associated endonuclease Cas9
    • RNA: gRNA
    • DNA: HBB DNA TS 1
    • DNA: HBB DNA NTS
    • DNA: HBB DNA TS 2
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Ternary complex of FnCas9 with HBB gRNA and HBB DNA

SupramoleculeName: Ternary complex of FnCas9 with HBB gRNA and HBB DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Francisella tularensis subsp. novicida (bacteria)
Molecular weightTheoretical: 190.329 KDa

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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 / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Francisella tularensis subsp. novicida (bacteria)
Molecular weightTheoretical: 190.756188 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNFKILPIAI DLGVKNTGVF SAFYQKGTSL ERLDNKNGKV YELSKDSYTL LMNNRTARRH QRRGIDRKQL VKRLFKLIWT EQLNLEWDK DTQQAISFLF NRRGFSFITD GYSPEYLNIV PEQVKAILMD IFDDYNGEDD LDSYLKLATE QESKISEIYN K LMQKILEF ...String:
MNFKILPIAI DLGVKNTGVF SAFYQKGTSL ERLDNKNGKV YELSKDSYTL LMNNRTARRH QRRGIDRKQL VKRLFKLIWT EQLNLEWDK DTQQAISFLF NRRGFSFITD GYSPEYLNIV PEQVKAILMD IFDDYNGEDD LDSYLKLATE QESKISEIYN K LMQKILEF KLMKLCTDIK DDKVSTKTLK EITSYEFELL ADYLANYSES LKTQKFSYTD KQGNLKELSY YHHDKYNIQE FL KRHATIN DRILDTLLTD DLDIWNFNFE KFDFDKNEEK LQNQEDKDHI QAHLHHFVFA VNKIKSEMAS GGRHRSQYFQ EIT NVLDEN NHQEGYLKNF CENLHNKKYS NLSVKNLVNL IGNLSNLELK PLRKYFNDKI HAKADHWDEQ KFTETYCHWI LGEW RVGVK DQDKKDGAKY SYKDLCNELK QKVTKAGLVD FLLELDPCRT IPPYLDNNNR KPPKCQSLIL NPKFLDNQYP NWQQY LQEL KKLQSIQNYL DSFETDLKVL KSSKDQPYFV EYKSSNQQIA SGQRDYKDLD ARILQFIFDR VKASDELLLN EIYFQA KKL KQKASSELEK LESSKKLDEV IANSQLSQIL KSQHTNGIFE QGTFLHLVCK YYKQRQRARD SRLYIMPEYR YDKKLHK YN NTGRFDDDNQ LLTYCNHKPR QKRYQLLNDL AGVLQVSPNF LKDKIGSDDD LFISKWLVEH IRGFKKACED SLKIQKDN R GLLNHKINIA RNTKGKCEKE IFNLICKIEG SEDKKGNYKH GLAYELGVLL FGEPNEASKP EFDRKIKKFN SIYSFAQIQ QIAFAERKGN ANTCAVCSAD NAHRMQQIKI TEPVEDNKDK IILSAKAQRL PAIPTRIVDG AVKKMATILA KNIVDDNWQN IKQVLSAKH QLHIPIITES NAFEFEPALA DVKGKSLKDR RKKALERISP ENIFKDKNNR IKEFAKGISA YSGANLTDGD F DGAKEELD HIIPRSHKKY GTLNDEANLI CVTRGDNKNK GNRIFCLRDL ADNYKLKQFE TTDDLEIEKK IADTIWDANK KD FKFGNYR SFINLTPQEQ KAFRHALFLA DENPIKQAVI RAINNRNRTF VNGTQRYFAE VLANNIYLRA KKENLNTDKI SFD YFGIPT IGNGRGIAEI RQLYEKVDSD IQAYAKGDKP QASYSHLIDA MLAFCIAADE HRNDGSIGLE IDKNYSLYPL DKNT GEVFT KDIFSQIKIT DNEFSDKKLV RKKAIEGFNT HRQMTRDGIY AENYLPILIH KELNEVRKGY TWKNSEEIKI FKGKK YDIQ QLNNLVYCLK FVDKPISIDI QISTLEELRN ILTTNNIAAT AEYYYINLKT QKLHEYYIEN YNTALGYKKY SKEMEF LRS LAYRSERVKI KSIDDVKQVL DKDSNFIIGK ITLPFKKEWQ RLYREWQNTT IKDDYEFLKS FFNVKSITKL HKKVRKD FS LPISTNEGKF LVKRKTWDNN FIYQILNDSD SRADGTKPFI PAFDISKNEI VEAIIDSFTS KNIFWLPKNI ELQKVDNK N IFAIDTSKWF EVETPSDLRD IGIATIQYKI DNNSRPKVRV KLDYVIDDDS KINYFMNHSL LKSRYPDKVL EILKQSTII EFESSGFNKT IKEMLGMKLA GIYNETSNN

UniProtKB: CRISPR-associated endonuclease Cas9

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Macromolecule #2: gRNA

MacromoleculeName: gRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Francisella tularensis subsp. novicida (bacteria)
Molecular weightTheoretical: 30.039703 KDa
SequenceString:
AGUAACGGCA GACUUCUCCU CGUUUCAGUU GCGCCGAAAG GCGCUCUGUA AUCAUUUAAA AGUAUUUUGA ACGGACCUCU GUUUGACAC GUCUG

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Macromolecule #3: HBB DNA TS 1

MacromoleculeName: HBB DNA TS 1 / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 3.647393 KDa
SequenceString:
(DC)(DC)(DG)(DA)(DT)(DA)(DC)(DC)(DT)(DG) (DA)(DG)

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Macromolecule #4: HBB DNA NTS

MacromoleculeName: HBB DNA NTS / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 2.795847 KDa
SequenceString:
(DA)(DG)(DG)(DT)(DA)(DT)(DC)(DG)(DG)

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Macromolecule #5: HBB DNA TS 2

MacromoleculeName: HBB DNA TS 2 / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 5.53159 KDa
SequenceString:
(DG)(DA)(DG)(DA)(DA)(DG)(DT)(DC)(DT)(DG) (DC)(DC)(DG)(DT)(DT)(DA)(DC)(DT)

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / 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: GATAN K3 (6k x 4k) / Average electron dose: 80.0 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: 1.5 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: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 48457
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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