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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: To be Published
Title: CryoEM structure of ThermoCas9 in post-cleavage state with a DNA containing NNNNCGA PAM
Authors: Shu, Y. / Zhao, Y.
History
DepositionMay 13, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 28, 2025Provider: repository / Type: Initial release
Revision 1.0May 28, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0May 28, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 28, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 28, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0May 28, 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

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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