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- PDB-9ar5: Structure of Acidothermus cellulolyticus Cas9 bound with methylat... -

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Basic information

Entry
Database: PDB / ID: 9ar5
TitleStructure of Acidothermus cellulolyticus Cas9 bound with methylated DNA
Components
  • CRISPR-associated endonuclease, Csn1 family
  • DNA (25-MER)
  • DNA/RNA (5'-D(P*TP*AP*CP*A)-R(P*(5MC))-D(P*CP*AP*AP*GP*CP*T)-3')
  • RNA (92-MER)
KeywordsDNA BINDING PROTEIN/DNA/RNA / CRISPR-Cas9 / Methylated DNA / AceCas9 / HNH rotation / DNA BINDING PROTEIN-DNA-RNA complex
Function / homology
Function and homology information


endonuclease activity / defense response to virus / DNA binding / RNA binding / zinc ion binding
Similarity search - Function
CRISPR-associated endonuclease Cas9, beta-hairpin domain / Cas9 C-terminal domain / Cas9, topo homolgy domain / CRISPR-associated endonuclease Cas9 beta-hairpin domain / Topo homolgy domain in CRISPR-associated endonuclease Cas9 / Cas9 C-terminal domain / : / Cas9, alpha-helical lobe domain / Cas9 alpha-helical lobe domain / HNH endonuclease ...CRISPR-associated endonuclease Cas9, beta-hairpin domain / Cas9 C-terminal domain / Cas9, topo homolgy domain / CRISPR-associated endonuclease Cas9 beta-hairpin domain / Topo homolgy domain in CRISPR-associated endonuclease Cas9 / Cas9 C-terminal domain / : / Cas9, alpha-helical lobe domain / Cas9 alpha-helical lobe domain / HNH endonuclease / HNH endonuclease / RuvC endonuclease subdomain 3 / RuvC endonuclease subdomain 3 / HNH nucleases / CRISPR-associated endonuclease Cas9 / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / CRISPR-associated endonuclease, Csn1 family
Similarity search - Component
Biological speciesAcidothermus cellulolyticus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.88 Å
AuthorsDas, A. / Rai, J. / Wang, B. / Li, H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM099604 United States
CitationJournal: To be Published
Title: Molecular Basis for Epigenetic-sensitive Editing by Cas9
Authors: Roth, M.O. / Shu, Y. / Zhao, Y. / Trasanidou, D. / Hoffman, R.D. / Trasanidis, N. / Gelasco, M.K. / Medina, M.L. / Das, D. / Rai, J. / Wang, B. / Li, H.
History
DepositionFeb 22, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 26, 2025Provider: repository / Type: Initial release
Revision 1.0Feb 26, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Feb 26, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 26, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 26, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 26, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: CRISPR-associated endonuclease, Csn1 family
B: RNA (92-MER)
X: DNA (25-MER)
D: DNA/RNA (5'-D(P*TP*AP*CP*A)-R(P*(5MC))-D(P*CP*AP*AP*GP*CP*T)-3')


Theoretical massNumber of molelcules
Total (without water)172,7664
Polymers172,7664
Non-polymers00
Water00
1


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

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Components

#1: Protein CRISPR-associated endonuclease, Csn1 family


Mass: 127498.094 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Acidothermus cellulolyticus (bacteria) / Gene: Acel_1951 / Production host: Escherichia coli (E. coli) / References: UniProt: A0LWB3
#2: RNA chain RNA (92-MER)


Mass: 34230.328 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Acidothermus cellulolyticus (bacteria)
#3: DNA chain DNA (25-MER)


Mass: 7704.969 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Acidothermus cellulolyticus (bacteria)
#4: DNA chain DNA/RNA (5'-D(P*TP*AP*CP*A)-R(P*(5MC))-D(P*CP*AP*AP*GP*CP*T)-3')


Mass: 3332.214 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Acidothermus cellulolyticus (bacteria)
Has ligand of interestY
Has protein modificationY

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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: CRISPR-Cas9 / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Source (natural)Organism: Acidothermus cellulolyticus (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: 1000 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (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: 2.88 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 275004 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 44.84 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003211920
ELECTRON MICROSCOPYf_angle_d0.603316766
ELECTRON MICROSCOPYf_chiral_restr0.03761929
ELECTRON MICROSCOPYf_plane_restr0.00511715
ELECTRON MICROSCOPYf_dihedral_angle_d16.59912692

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