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- EMDB-61853: Cryo-EM structure of FrCas9 in complex with sgRNA and 26-nt TS an... -

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Basic information

Entry
Database: EMDB / ID: EMD-61853
TitleCryo-EM structure of FrCas9 in complex with sgRNA and 26-nt TS and 4-nt NTS substrates
Map data
Sample
  • Complex: Ternary complex of FrCas9-sgRNA bound to 26-nt TS and 4-nt NTS
    • Protein or peptide: CRISPR-associated endonuclease Cas9
    • RNA: sgRNA
    • DNA: DNA (26-mer)
  • Ligand: water
KeywordsFrCas9 / high fidelity / off-target / nuclease protein / complex / HYDROLASE / HYDROLASE-RNA-DNA complex
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease
Similarity search - Domain/homology
CRISPR-associated endonuclease Cas9
Similarity search - Component
Biological speciesFaecalibaculum rodentium (bacteria) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.46 Å
AuthorsChen SD / Yang M / Liu SQ
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Cell Genom / Year: 2025
Title: Structural and functional bases of F. rodentium Cas9 provide insights into CRISPR-Cas protein engineering.
Authors: Mei Yang / Siqi Liu / Guanqiao Chen / Xi Liu / Dapeng Sun / Jingjing Zhang / Yumei Wang / Shoudeng Chen / Rui Tian / Zheng Hu /
Abstract: The Faecalibaculum rodentium (Fr) CRISPR-Cas9 system exhibits enhanced gene-editing precision and efficiency compared to SpCas9, with distinctive advantages in targeting the TATA box in eukaryotic ...The Faecalibaculum rodentium (Fr) CRISPR-Cas9 system exhibits enhanced gene-editing precision and efficiency compared to SpCas9, with distinctive advantages in targeting the TATA box in eukaryotic promoters. However, the underlying molecular mechanisms remained unexplored. Here, we present cryo-electron microscopy structures of the FrCas9-single guide RNA (sgRNA)-DNA complex in both the R-loop expansion and pre-catalytic states, shedding light on its specialized recognition of the 5'-NRTA-3' protospacer adjacent motif (PAM) and the unusual overwinding of the sgRNA-DNA heteroduplex. Our investigations into the structure and extensive mutational analyses reveal that the phosphate lock loop plays a pivotal role in finely adjusting FrCas9's off-target sensitivity and catalytic efficiency. Remarkably, targeted residue substitutions in the phosphate lock loop and the PAM-distal region were found to synergistically enhance both the editing precision and efficiency of FrCas9. These findings advance our understanding of Cas9's accuracy and potency mechanisms while providing a molecular foundation for the rational design and development of next-generation CRISPR technologies.
History
DepositionOct 10, 2024-
Header (metadata) releaseSep 17, 2025-
Map releaseSep 17, 2025-
UpdateOct 29, 2025-
Current statusOct 29, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_61853.png

Downloads & links

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Map

FileDownload / File: emd_61853.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 300 pix.
= 249. Å		0.83 Å/pix.
x 300 pix.
= 249. Å		0.83 Å/pix.
x 300 pix.
= 249. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03
Minimum - Maximum-0.02188705 - 1.9732403
Average (Standard dev.)0.0016527603 (±0.030850025)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 249.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_61853_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_61853_half_map_2.map
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 FrCas9-sgRNA bound to 26-nt TS and 4-nt NTS

EntireName: Ternary complex of FrCas9-sgRNA bound to 26-nt TS and 4-nt NTS
Components
  • Complex: Ternary complex of FrCas9-sgRNA bound to 26-nt TS and 4-nt NTS
    • Protein or peptide: CRISPR-associated endonuclease Cas9
    • RNA: sgRNA
    • DNA: DNA (26-mer)
  • Ligand: water

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Supramolecule #1: Ternary complex of FrCas9-sgRNA bound to 26-nt TS and 4-nt NTS

SupramoleculeName: Ternary complex of FrCas9-sgRNA bound to 26-nt TS and 4-nt NTS
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Faecalibaculum rodentium (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 / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Faecalibaculum rodentium (bacteria)
Molecular weightTheoretical: 159.661672 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MCTKESEKLN KNADYYIGLD MGTSSAGWAV SDSEYNLIRR KGKDLWGVRQ FEEAKTAAER RGFRVARRRK QRQQVRNRLL SEEFQNEIT KIDSGFLKRM EDSRFVISDK RVPEKYTLFN DSGYTDVEYY NQYPTIYHLR KALIESNERF DIRLVFLGIH S LFQHPGHF ...String:
MCTKESEKLN KNADYYIGLD MGTSSAGWAV SDSEYNLIRR KGKDLWGVRQ FEEAKTAAER RGFRVARRRK QRQQVRNRLL SEEFQNEIT KIDSGFLKRM EDSRFVISDK RVPEKYTLFN DSGYTDVEYY NQYPTIYHLR KALIESNERF DIRLVFLGIH S LFQHPGHF LDKGDVDTDN TGPEELIQFL EDCMNEIQIS IPLVSNQKVL TDILTDSRIT RRDKEQQILE ILQPNKESKK AV SQFVKVL TGQKAKLGDL IMMEDKDTEE YKYSFSFREK TLEEILPDIE GVIDGLALEY IESIYSLYSW SLLNSYMKDT LTG HYYSYL AEARVAAYDK HHSDLVKLKT LFREYIPEEY DNFFRKMEKA NYSHYIGSTE YDGEKRCRTA KAKQEDFYKS INKM LEKIP ECSEKTEIQK EIIEGTFLLK QTGPQNGFVP NQLQLKELRK ILQNASKHYP FLTEKDERDM TAIDRIEALF SFRIP YYIG PLKNTDNQGH GWAVRRDGHE QIPVRPWNFE EIIDESASAD LFIKNLVNSC TYLRTEKVLP KSSLLYQEFE VLNELN NLR INGMYPDEIQ PGLKRMIFEQ CFYSGKKVTG KKLQLFLRSV LTNSSTEEFV LTGIDKDFKS SLSSYKKFCE LFGVKTL ND TQKVMAEQII EWSTVYGDSR KFLKRKLEDN YPELTDQQIR RIAGFKFSEW GNLSRAFLEM EGYKDEAGNP VTIIRALR D TQKNLMQLLS NDSAFAKKLQ ELNDYVTRDI WSIEPDDLDG MYLSAPVRRM IWQTFLILRE VVDTIGYSPK KIFMEMARG EQEKKRTASR KKQLIDLYKE AGMKNDELFG DLESLEEAQL RSKKLYLYFR QMGRDIYSGK LIDFMDVLHG NRYDIDAIHP QSKKKDDSL ENNLVLTSKD FNNHIKQDVY PIPEQIQSRQ KGFWAMLLKQ GFMSQEKYNR LMRTTPFTDE ELAEFVNRQL V ETRQGTKA IISLINQCFP DSEVVYVKAG NTSDFRQRFD IPKSRDLNNY HHAVDAYLNI VVGNVYDTKF TKNPINFIKK MR KSGNLHS YSLRRMYDFN VQRGDQTAWV AENDTTLKTV KKTAFKTSPM VTKRTYERKG GLADSVLIAA KKAKPGVHLP VKT SDSRFA NQVSTYGGYD NVKGSHFFLV EHQQKKKTIR SIENVPIHLK EKLKTKEELE HYCAQVLGMV QPDVRLTRIP MYSL LLIDG YYYYLTGRTG GNLSLSNAVE LCLPAKEQAH IRMISKIAGG RSTDALSAEA KDDFRKKNLR LYDELAEKHR STIFS KRKN PIGPKLLKYR EAFVKQTIEN QCKVILQILK LTSTNCKTSA DLKLIGGSGQ EGVMSISKLL RAEKYAEFYL ICQSPS GIY ETRKNLLTI

UniProtKB: CRISPR-associated endonuclease Cas9

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Macromolecule #2: sgRNA

MacromoleculeName: sgRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 40.071625 KDa
SequenceString:
UGCAAGACAU AGAAAUAACC UGGUUUGAGU GUCUUGUUAA UUGAAAAAUU AACAAGAUGA GUUCAAAUCA GGCUCCUAGA GAGAUCCGA ACUUACCUUC AUGGCGGGCA UUGUGCCCUU UUUUUU

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Macromolecule #3: DNA (26-mer)

MacromoleculeName: DNA (26-mer) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 7.943136 KDa
SequenceString:
(DT)(DA)(DC)(DA)(DC)(DA)(DG)(DG)(DT)(DT) (DA)(DT)(DT)(DT)(DC)(DT)(DA)(DT)(DG)(DT) (DC)(DT)(DT)(DG)(DC)(DA)

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 1 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON III (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: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: NONE
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6o0z

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.46 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 219843
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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