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- EMDB-44859: CryoEM structure of ThermoCas9 in post-cleavage state with a DNA ... -

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Basic information

Entry
Database: EMDB / ID: EMD-44859
TitleCryoEM structure of ThermoCas9 in post-cleavage state with a DNA containing NNNNCGA PAM
Map data
Sample
  • Complex: Ternary complex of ThermoCas9 bound with RNA and DNA containing NNNNCGA PAM
    • DNA: DNA (25-MER)
    • DNA: DNA (5'-D(P*GP*TP*AP*TP*AP*CP*AP*CP*GP*AP*AP*GP*CP*T)-3')
    • DNA: DNA (5'-D(*AP*GP*CP*TP*TP*CP*GP*TP*GP*TP*AP*TP*AP*C)-3')
    • DNA: DNA (5'-D(P*CP*TP*AP*GP*A)-3')
    • Protein or peptide: CRISPR-associated endonuclease Cas9
    • RNA: RNA (114-MER)
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: water
KeywordsCRISPR-Cas9 / Cas9 / Epigenetics / endonuclease / HNH / RuvC / metal binding / HYDROLASE-DNA-RNA complex
Function / homology:
Function and homology information
Biological speciesGeobacillus thermodenitrificans (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsZhao Y / Shu Y
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM099604 United States
CitationJournal: Nature / Year: 2026
Title: Molecular basis for methylation-sensitive editing by Cas9.
Authors: Mitchell O Roth / Yuerong Shu / Yu Zhao / Despoina Trasanidou / Renee D Hoffman / Christian Südfeld / Eugenios Bouzetos / Nikolaos Trasanidis / Michael Zawrotny / Mary K Gelasco / Megan L ...Authors: Mitchell O Roth / Yuerong Shu / Yu Zhao / Despoina Trasanidou / Renee D Hoffman / Christian Südfeld / Eugenios Bouzetos / Nikolaos Trasanidis / Michael Zawrotny / Mary K Gelasco / Megan L Medina / Anuska Das / Jay Rai / Hemant N Goswami / Bing Wang / John van der Oost / Hong Li /
Abstract: The bacterial CRISPR-Cas9 (Cas9) nuclease has become a powerful genome manipulation tool for a wide range of organisms. However, it has yet to fully leverage the pervasive presence of DNA methylation ...The bacterial CRISPR-Cas9 (Cas9) nuclease has become a powerful genome manipulation tool for a wide range of organisms. However, it has yet to fully leverage the pervasive presence of DNA methylation in genomes. Here, to fill this gap, we report biochemical, structural and human genome-editing characterizations of a methylation-sensitive Cas9 (ThermoCas9). ThermoCas9 efficiently binds to and cleaves DNA upstream of its protospacer adjacent motif (PAM) 5'-NNNNCGA-3' or 5'-NNNNCCA-3' in vitro. Methylation of the fifth cytosine in either PAM sequence (CpG or CpC), however, significantly inhibits ThermoCas9 activity. Cryo-electron microscopy structures of ThermoCas9 in pre-cleavage and post-cleavage states at 2.8 Å and 2.2 Å resolution, respectively, reveal the molecular basis for the stringent requirement of the unmethylated cytosine in PAM binding and provide guidance for further enzyme engineering. We demonstrate methylation-sensitive editing by ThermoCas9 in human cell lines with distinct DNA methylation landscapes. Moreover, we demonstrate that a catalytically enhanced ThermoCas9 efficiently targets luminal expression signature genes that are consistently hypomethylated in patients with breast cancer. Owing to its sensitivity to DNA methylation, ThermoCas9 can specifically target cells with disease-related hypomethylation, which adds another layer of precision to genome-editing technologies.
History
DepositionMay 13, 2024-
Header (metadata) releaseMay 28, 2025-
Map releaseMay 28, 2025-
UpdateApr 29, 2026-
Current statusApr 29, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44859.png

Downloads & links

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Map

FileDownload / File: emd_44859.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.)
0.82 Å/pix.
x 300 pix.
= 246. Å		0.82 Å/pix.
x 300 pix.
= 246. Å		0.82 Å/pix.
x 300 pix.
= 246. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.46
Minimum - Maximum-3.7062674 - 5.862869
Average (Standard dev.)0.01034128 (±0.10136296)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 246.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_44859_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_44859_half_map_2.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of ThermoCas9 bound with RNA and DNA containing N...

EntireName: Ternary complex of ThermoCas9 bound with RNA and DNA containing NNNNCGA PAM
Components
  • Complex: Ternary complex of ThermoCas9 bound with RNA and DNA containing NNNNCGA PAM
    • DNA: DNA (25-MER)
    • DNA: DNA (5'-D(P*GP*TP*AP*TP*AP*CP*AP*CP*GP*AP*AP*GP*CP*T)-3')
    • DNA: DNA (5'-D(*AP*GP*CP*TP*TP*CP*GP*TP*GP*TP*AP*TP*AP*C)-3')
    • DNA: DNA (5'-D(P*CP*TP*AP*GP*A)-3')
    • Protein or peptide: CRISPR-associated endonuclease Cas9
    • RNA: RNA (114-MER)
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: Ternary complex of ThermoCas9 bound with RNA and DNA containing N...

SupramoleculeName: Ternary complex of ThermoCas9 bound with RNA and DNA containing NNNNCGA PAM
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Geobacillus thermodenitrificans (bacteria)

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Macromolecule #1: DNA (25-MER)

MacromoleculeName: DNA (25-MER) / type: dna / ID: 1 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 7.883092 KDa
SequenceString:
(DC)(DA)(DG)(DG)(DA)(DT)(DC)(DT)(DT)(DG) (DC)(DC)(DA)(DT)(DC)(DC)(DT)(DA)(DC)(DC) (DT)(DC)(DT)(DA)(DG)(DA)

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Macromolecule #2: DNA (5'-D(P*GP*TP*AP*TP*AP*CP*AP*CP*GP*AP*AP*GP*CP*T)-3')

MacromoleculeName: DNA (5'-D(P*GP*TP*AP*TP*AP*CP*AP*CP*GP*AP*AP*GP*CP*T)-3')
type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 10.572813 KDa
SequenceString:
(DG)(DG)(DT)(DA)(DG)(DG)(DA)(DT)(DG)(DG) (DC)(DA)(DA)(DG)(DA)(DT)(DC)(DC)(DT)(DG) (DG)(DT)(DA)(DT)(DA)(DC)(DA)(DC)(DG) (DA)(DA)(DG)(DC)(DT)

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Macromolecule #3: DNA (5'-D(*AP*GP*CP*TP*TP*CP*GP*TP*GP*TP*AP*TP*AP*C)-3')

MacromoleculeName: DNA (5'-D(*AP*GP*CP*TP*TP*CP*GP*TP*GP*TP*AP*TP*AP*C)-3')
type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 4.27079 KDa
SequenceString:
(DA)(DG)(DC)(DT)(DT)(DC)(DG)(DT)(DG)(DT) (DA)(DT)(DA)(DC)

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Macromolecule #4: DNA (5'-D(P*CP*TP*AP*GP*A)-3')

MacromoleculeName: DNA (5'-D(P*CP*TP*AP*GP*A)-3') / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 1.808229 KDa
SequenceString:
(DT)(DC)(DT)(DA)(DG)(DA)

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Macromolecule #5: CRISPR-associated endonuclease Cas9

MacromoleculeName: CRISPR-associated endonuclease Cas9 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Geobacillus thermodenitrificans (bacteria)
Molecular weightTheoretical: 126.563461 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKYKIGLDIG ITSIGWAVIN LDIPRIEDLG VRIFDRAENP KTGESLALPR RLARSARRRL RRRKHRLERI RRLFVREGIL TKEELNKLF EKKHEIDVWQ LRVEALDRKL NNDELARILL HLAKRRGFRS NRKSERTNKE NSTMLKHIEE NQSILSSYRT V AEMVVKDP ...String:
MKYKIGLDIG ITSIGWAVIN LDIPRIEDLG VRIFDRAENP KTGESLALPR RLARSARRRL RRRKHRLERI RRLFVREGIL TKEELNKLF EKKHEIDVWQ LRVEALDRKL NNDELARILL HLAKRRGFRS NRKSERTNKE NSTMLKHIEE NQSILSSYRT V AEMVVKDP KFSLHKRNKE DNYTNTVARD DLEREIKLIF AKQREYGNIV CTEAFEHEYI SIWASQRPFA SKDDIEKKVG FC TFEPKEK RAPKATYTFQ SFTVWEHINK LRLVSPGGIR ALTDDERRLI YKQAFHKNKI TFHDVRTLLN LPDDTRFKGL LYD RNTTLK ENEKVRFLEL GAYHKIRKAI DSVYGKGAAK SFRPIDFDTF GYALTMFKDD TDIRSYLRNE YEQNGKRMEN LADK VYDEE LIEELLNLSF SKFGHLSLKA LRNILPYMEQ GEVYSTACER AGYTFTGPKK KQKTVLLPNI PPIANPVVMR ALTQA RKVV NAIIKKYGSP VSIHIELARE LSQSFDERRK MQKEQEGNRK KNETAIRQLV EYGLTLNPTG LDIVKFKLWS EQNGKC AYS LQPIEIERLL EPGYTEVDHV IPYSRSLDDS YTNKVLVLTK ENREKGNRTP AEYLGLGSER WQQFETFVLT NKQFSKK KR DRLLRLHYDE NEENEFKNRN LNDTRYISRF LANFIREHLK FADSDDKQKV YTVNGRITAH LRSRWNFNKN REESNLHH A VDAAIVACTT PSDIARVTAF YQRREQNKEL SKKTDPQFPQ PWPHFADELQ ARLSKNPKES IKALNLGNYD NEKLESLQP VFVSRMPKRS ITGAAHQETL RRYIGIDERS GKIQTVVKKK LSEIQLDKTG HFPMYGKESD PRTYEAIRQR LLEHNNDPKK AFQEPLYKP KKNGELGPII RTIKIIDTTN QVIPLNDGKT VAYNSNIVRV DVFEKDGKYY CVPIYTIDMM KGILPNKAIE P NKPYSEWK EMTEDYTFRF SLYPNDLIRI EFPREKTIKT AVGEEIKIKD LFAYYQTIDS SNGGLSLVSH DNNFSLRSIG SR TLKRFEK YQVDVLGNIY KVRGEKRVGV ASSSHSKAGE TIRPL

UniProtKB: UNIPROTKB: A0A1W6VMQ3

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Macromolecule #6: RNA (114-MER)

MacromoleculeName: RNA (114-MER) / type: rna / ID: 6 / Number of copies: 1
Source (natural)Organism: Geobacillus thermodenitrificans (bacteria)
Molecular weightTheoretical: 47.798094 KDa
SequenceString:
GGUAGGAUGG CAAGAUCCUG GUAGUCAUAG UUCCCCUGGA AACAGGGUUA CUAUGAUAAG GGCUUUCUGC CUAUAGGCAG ACUGACCCG UGGCGUUGGG GAUCGCCUAU CGCCCGCUUU CUUCGGGCAU UCCCCACUCU UAGGCGUUUU

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Macromolecule #7: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 7 / Number of copies: 1 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM

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Macromolecule #8: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 8 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #9: water

MacromoleculeName: water / type: ligand / ID: 9 / Number of copies: 9 / 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: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOCONTINUUM (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: 0.8 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: PDB ENTRY
PDB model - PDB ID:

8d2q

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 92000
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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