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- EMDB-65771: SuperFi Cas9 - 22nt sgRNA - DNA ternary complex -

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Basic information

Entry
Database: EMDB / ID: EMD-65771
TitleSuperFi Cas9 - 22nt sgRNA - DNA ternary complex
Map data
Sample
  • Complex: SuperFi Cas9 - 22nt sgRNA - DNA ternary complex Class A
    • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1
    • DNA: DNA (50-MER)
    • DNA: DNA (50-MER)
    • RNA: RNA (102-MER)
KeywordsComplex / cas / SuperFi cas9 / IMMUNE SYSTEM
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / 3'-5' exonuclease activity / DNA 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, bridge helix / Bridge helix of CRISPR-associated endonuclease Cas9 / 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 ...CRISPR-associated endonuclease Cas9, bridge helix / Bridge helix of CRISPR-associated endonuclease Cas9 / 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 / Ribonuclease H superfamily
Similarity search - Domain/homology
CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes (bacteria) / Streptococcus pyogenes serotype M1 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsZheng R / Ma LJ
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Struct Mol Biol / Year: 2026
Title: Improving the efficiency of high-fidelity Cas9 by enhancing PAM-distal interactions.
Authors: Rong Zheng / Zhike Lu / Rongwei Wei / Young-Cheul Shin / Jiang Du / Qingfeng Zhang / Jianbo Li / Xiaoqi Wang / Yi Wei / Botao Liu / Yang Chen / Lihong Ding / Heng Zhang / Hui Chen / Jing Huang / Lijia Ma /
Abstract: Engineering CRISPR enzymes for high fidelity often impairs cleavage activity. Meanwhile, a mechanistic understanding of why high-fidelity mutations reduce Cas9's cleavage activity remains unclear, ...Engineering CRISPR enzymes for high fidelity often impairs cleavage activity. Meanwhile, a mechanistic understanding of why high-fidelity mutations reduce Cas9's cleavage activity remains unclear, presenting a challenge in balancing nuclease specificity and efficiency for clinical applications. In this study, we show that extending the spacer region to 21 or 22 nucleotides restores the impaired cleavage activity of SuperFi-Cas9, a high-fidelity Cas9 variant with 7 mutations in the RuvC domain at the protospacer adjacent motif (PAM)-distal region. Cryo-electron microscopy structures and mutational analyses reveal that the negatively charged mutations in a protruding loop of the RuvC domain create repulsive forces that destabilize the nuclease-single guide (sg)RNA-DNA complex. Spacer extension enhances interactions in the PAM-distal region, effectively restoring cleavage activity and balancing editing efficiency with specificity. In addition, we develop a deep learning model, AIdit-SuperFi, to predict optimal sgRNA length for high-fidelity genome editing. Our findings introduce a straightforward strategy to enhance CRISPR complex stability and provide mechanistic insights into the impaired cleavage activity of engineered high-fidelity Cas9, presenting a pathway toward precise and efficient genome editing and clinical translation of CRISPR technologies.
History
DepositionAug 9, 2025-
Header (metadata) releaseDec 31, 2025-
Map releaseDec 31, 2025-
UpdateApr 1, 2026-
Current statusApr 1, 2026Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_65771.png

Downloads & links

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Map

FileDownload / File: emd_65771.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 192 pix.
= 206.208 Å		1.07 Å/pix.
x 192 pix.
= 206.208 Å		1.07 Å/pix.
x 192 pix.
= 206.208 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.074 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.173
Minimum - Maximum-0.13761558 - 0.49701726
Average (Standard dev.)0.0028568879 (±0.0319079)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 206.20801 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_65771_msk_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_65771_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_65771_half_map_2.map
Projections & Slices
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Sample components

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Entire : SuperFi Cas9 - 22nt sgRNA - DNA ternary complex Class A

EntireName: SuperFi Cas9 - 22nt sgRNA - DNA ternary complex Class A
Components
  • Complex: SuperFi Cas9 - 22nt sgRNA - DNA ternary complex Class A
    • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1
    • DNA: DNA (50-MER)
    • DNA: DNA (50-MER)
    • RNA: RNA (102-MER)

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Supramolecule #1: SuperFi Cas9 - 22nt sgRNA - DNA ternary complex Class A

SupramoleculeName: SuperFi Cas9 - 22nt sgRNA - DNA ternary complex Class A
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Streptococcus pyogenes (bacteria)

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Macromolecule #1: CRISPR-associated endonuclease Cas9/Csn1

MacromoleculeName: CRISPR-associated endonuclease Cas9/Csn1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Streptococcus pyogenes serotype M1 (bacteria)
Molecular weightTheoretical: 158.502375 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDKKYSIGLD IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE ATRLKRTARR RYTRRKNRIC YLQEIFSNE MAKVDDSFFH RLEESFLVEE DKKHERHPIF GNIVDEVAYH EKYPTIYHLR KKLVDSTDKA DLRLIYLALA H MIKFRGHF ...String:
MDKKYSIGLD IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE ATRLKRTARR RYTRRKNRIC YLQEIFSNE MAKVDDSFFH RLEESFLVEE DKKHERHPIF GNIVDEVAYH EKYPTIYHLR KKLVDSTDKA DLRLIYLALA H MIKFRGHF LIEGDLNPDN SDVDKLFIQL VQTYNQLFEE NPINASGVDA KAILSARLSK SRRLENLIAQ LPGEKKNGLF GN LIALSLG LTPNFKSNFD LAEDAKLQLS KDTYDDDLDN LLAQIGDQYA DLFLAAKNLS DAILLSDILR VNTEITKAPL SAS MIKRYD EHHQDLTLLK ALVRQQLPEK YKEIFFDQSK NGYAGYIDGG ASQEEFYKFI KPILEKMDGT EELLVKLNRE DLLR KQRTF DNGSIPHQIH LGELHAILRR QEDFYPFLKD NREKIEKILT FRIPYYVGPL ARGNSRFAWM TRKSEETITP WNFEE VVDK GASAQSFIER MTNFDKNLPN EKVLPKHSLL YEYFTVYNEL TKVKYVTEGM RKPAFLSGEQ KKAIVDLLFK TNRKVT VKQ LKEDYFKKIE CFDSVEISGV EDRFNASLGT YHDLLKIIKD KDFLDNEENE DILEDIVLTL TLFEDREMIE ERLKTYA HL FDDKVMKQLK RRRYTGWGRL SRKLINGIRD KQSGKTILDF LKSDGFANRN FMQLIHDDSL TFKEDIQKAQ VSGQGDSL H EHIANLAGSP AIKKGILQTV KVVDELVKVM GRHKPENIVI EMARENQTTQ KGQKNSRERM KRIEEGIKEL GSQILKEHP VENTQLQNEK LYLYYLQNGR DMYVDQELDI NRLSDYDVDH IVPQSFLKDD SIDNKVLTRS DKNRGKSDNV PSEEVVKKMK NYWRQLLNA KLITQRKFDN LTKAERGGLS ELDKAGFIKR QLVETRQITK HVAQILDSRM NTKYDENDKL IREVKVITLK S KLVSDFRK DFQFYKVREI NNYHHAHDAY LNAVVGTALI KKYPKLESEF VDGDDKVDDD DKMIAKSEDE IGDATAKYFF YS NIMNFFK TEITLANGEI RKRPLIETNG ETGEIVWDKG RDFATVRKVL SMPQVNIVKK TEVQTGGFSK ESILPKRNSD KLI ARKKDW DPKKYGGFDS PTVAYSVLVV AKVEKGKSKK LKSVKELLGI TIMERSSFEK NPIDFLEAKG YKEVKKDLII KLPK YSLFE LENGRKRMLA SAGELQKGNE LALPSKYVNF LYLASHYEKL KGSPEDNEQK QLFVEQHKHY LDEIIEQISE FSKRV ILAD ANLDKVLSAY NKHRDKPIRE QAENIIHLFT LTNLGAPAAF KYFDTTIDRK RYTSTKEVLD ATLIHQSITG LYETRI DLS QLGGD

UniProtKB: CRISPR-associated endonuclease Cas9/Csn1

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Macromolecule #2: DNA (50-MER)

MacromoleculeName: DNA (50-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 15.438878 KDa
SequenceString:
(DA)(DG)(DC)(DT)(DA)(DT)(DG)(DA)(DG)(DT) (DG)(DG)(DC)(DC)(DC)(DC)(DT)(DG)(DT)(DG) (DG)(DA)(DG)(DA)(DG)(DA)(DA)(DG)(DC) (DC)(DT)(DG)(DT)(DC)(DC)(DT)(DG)(DG)(DA) (DA) (DG)(DC)(DC)(DA)(DG)(DC)(DC)(DC) (DC)(DA)

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Macromolecule #3: DNA (50-MER)

MacromoleculeName: DNA (50-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 15.371812 KDa
SequenceString:
(DT)(DG)(DG)(DG)(DG)(DC)(DT)(DG)(DG)(DC) (DT)(DT)(DC)(DC)(DA)(DG)(DG)(DA)(DC)(DA) (DG)(DG)(DC)(DT)(DT)(DC)(DT)(DC)(DT) (DC)(DC)(DA)(DC)(DA)(DG)(DG)(DG)(DG)(DC) (DC) (DA)(DC)(DT)(DC)(DA)(DT)(DA)(DG) (DC)(DT)

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Macromolecule #4: RNA (102-MER)

MacromoleculeName: RNA (102-MER) / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 32.815465 KDa
SequenceString:
CAGGACAGGC UUCUCUCCAC AGGUUUUAGA GCUAGAAAUA GCAAGUUAAA AUAAGGCUAG UCCGUUAUCA ACUUGAAAAA GUGGCACCG AGUCGGUGCU UUU

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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
Component:
ConcentrationFormulaName
150.0 mMNaClNaCl
20.0 mMC8H18N2O4SHEPES pH7.4
10.0 mMMgCl2MgCl2
0.5 mMC9H15O6PC9H15O6P
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recording#0 - Image recording ID: 1 / #0 - Film or detector model: FEI FALCON IV (4k x 4k) / #0 - Average electron dose: 1.25 e/Å2 / #1 - Image recording ID: 2 / #1 - Film or detector model: FEI FALCON IV (4k x 4k) / #1 - Average electron dose: 1.02 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER / 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

Image recording ID1
CTF correctionType: NONE
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 43148
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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