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- PDB-9bs6: CryoEM structure of ThermoCas9 in post-cleavage state with a DNA ... -

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Basic information

Entry
Database: PDB / ID: 9bs6
TitleCryoEM structure of ThermoCas9 in post-cleavage state with a DNA containing NNNNCGA PAM
Components
  • CRISPR-associated endonuclease Cas9
  • DNA (25-MER)
  • DNA (5'-D(*AP*GP*CP*TP*TP*CP*GP*TP*GP*TP*AP*TP*AP*C)-3')
  • DNA (5'-D(P*CP*TP*AP*GP*A)-3')
  • DNA (5'-D(P*GP*TP*AP*TP*AP*CP*AP*CP*GP*AP*AP*GP*CP*T)-3')
  • RNA (114-MER)
KeywordsHYDROLASE/DNA/RNA / CRISPR-Cas9 / Cas9 / Epigenetics / endonuclease / HNH / RuvC / metal binding / HYDROLASE-DNA-RNA complex
Function / homologyGUANOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / :
Function and homology information
Biological speciesGeobacillus thermodenitrificans (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsZhao, Y. / Shu, Y.
Funding support United States, 1items
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, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 28, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: DNA (25-MER)
D: DNA (5'-D(P*GP*TP*AP*TP*AP*CP*AP*CP*GP*AP*AP*GP*CP*T)-3')
P: DNA (5'-D(*AP*GP*CP*TP*TP*CP*GP*TP*GP*TP*AP*TP*AP*C)-3')
X: DNA (5'-D(P*CP*TP*AP*GP*A)-3')
A: CRISPR-associated endonuclease Cas9
B: RNA (114-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)199,4689
Polymers198,8966
Non-polymers5723
Water1629
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA chain , 4 types, 4 molecules CDPX

#1: DNA chain DNA (25-MER)


Mass: 7883.092 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: DNA chain DNA (5'-D(P*GP*TP*AP*TP*AP*CP*AP*CP*GP*AP*AP*GP*CP*T)-3')


Mass: 10572.813 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (5'-D(*AP*GP*CP*TP*TP*CP*GP*TP*GP*TP*AP*TP*AP*C)-3')


Mass: 4270.790 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (5'-D(P*CP*TP*AP*GP*A)-3')


Mass: 1808.229 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein / RNA chain , 2 types, 2 molecules AB

#5: Protein CRISPR-associated endonuclease Cas9


Mass: 126563.461 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Geobacillus thermodenitrificans (bacteria)
Gene: cas9-2, cas9, GTHT12_03401 / Production host: Escherichia coli (E. coli)
References: UniProt: A0A1W6VMQ3, Hydrolases; Acting on ester bonds
#6: RNA chain RNA (114-MER)


Mass: 47798.094 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Geobacillus thermodenitrificans (bacteria)
Production host: Escherichia coli (E. coli)

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Non-polymers , 3 types, 12 molecules

#7: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM
#8: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#9: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 9 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Ternary complex of ThermoCas9 bound with RNA and DNA containing NNNNCGA PAM
Type: COMPLEX / Entity ID: #1-#6 / Source: MULTIPLE SOURCES
Source (natural)Organism: Geobacillus thermodenitrificans (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOCONTINUUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 92000 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 14.6 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004312628
ELECTRON MICROSCOPYf_angle_d0.830117821
ELECTRON MICROSCOPYf_chiral_restr0.04722044
ELECTRON MICROSCOPYf_plane_restr0.00581669
ELECTRON MICROSCOPYf_dihedral_angle_d17.24183088

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