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- PDB-9ar6: CryoEM structure of ThermoCas9 in pre-cleavage state bound with t... -

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Basic information

Entry
Database: PDB / ID: 9ar6
TitleCryoEM structure of ThermoCas9 in pre-cleavage state bound with the DNA containing NNNNCCA PAM
Components
  • CRISPR-associated endonuclease Cas9
  • non-target DNA
  • single guide RNA
  • target-strand DNA
KeywordsDNA BINDING PROTEIN/DNA/RNA / CRISPR-Cas9 / Cas9 / Epigenetics / endonuclease / HNH / RuvC / metal binding / RNA / DNA BINDING PROTEIN-DNA-RNA complex
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / :
Function and homology information
Biological speciesGeobacillus thermodenitrificans (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.77 Å
AuthorsZhao, Y. / Shu, Y. / 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. / Shu, Y. / Trasanidou, D. / Zhao, Y. / van der Oost, J. / 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 Cas9
D: non-target DNA
P: target-strand DNA
B: single guide RNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)189,6266
Polymers189,5804
Non-polymers462
Water362
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 , 2 types, 2 molecules DP

#2: DNA chain non-target DNA


Mass: 3615.395 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Geobacillus thermodenitrificans (bacteria)
#3: DNA chain target-strand DNA


Mass: 12238.864 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Geobacillus thermodenitrificans (bacteria)

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Protein / RNA chain , 2 types, 2 molecules AB

#1: 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
#4: RNA chain single guide RNA


Mass: 47162.719 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Geobacillus thermodenitrificans (bacteria)

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Non-polymers , 2 types, 4 molecules

#5: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Na / Feature type: SUBJECT OF INVESTIGATION
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2 / 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 with single guide RNA and target DNA
Type: COMPLEX / Entity ID: #1-#4 / 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: 820 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k 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: 2.77 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 63887 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 96.96 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.005712752
ELECTRON MICROSCOPYf_angle_d0.775617943
ELECTRON MICROSCOPYf_chiral_restr0.04882063
ELECTRON MICROSCOPYf_plane_restr0.00621723
ELECTRON MICROSCOPYf_dihedral_angle_d16.57253044

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