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- PDB-8umf: Structure of PsCas9 in complex with gRNA and DNA in product state -

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Basic information

Entry
Database: PDB / ID: 8umf
TitleStructure of PsCas9 in complex with gRNA and DNA in product state
Components
  • (DNA (80-MER)) x 2
  • Cas9
  • RNA (121-MER)
KeywordsIMMUNE SYSTEM / Cas9 / CRISPR
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesParasutterella secunda (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsBravo, J.P.K. / Taylor, D.W.
Funding support United States, 2items
OrganizationGrant numberCountry
Welch FoundationF-1938 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM138348 United States
CitationJournal: To Be Published
Title: Structure-guided engineering of PsCas9 yields a high fidelity and activity enzyme for in vivo gene editing
Authors: Bravo, J.P.K. / Taylor, D.W.
History
DepositionOct 17, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 2, 2024Provider: repository / Type: Initial release
Revision 1.0Oct 2, 2024Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Oct 2, 2024Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Oct 2, 2024Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Oct 2, 2024Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Oct 2, 2024Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1May 14, 2025Group: Data collection / Structure summary / Category: em_admin / em_software / pdbx_entry_details
Item: _em_admin.last_update / _em_software.name / _pdbx_entry_details.has_protein_modification
Revision 1.1May 14, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cas9
B: RNA (121-MER)
C: DNA (80-MER)
D: DNA (80-MER)
E: DNA (80-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)284,20712
Polymers284,0375
Non-polymers1707
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 Cas9


Mass: 168224.266 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Parasutterella secunda (bacteria) / Production host: Escherichia coli (E. coli)
#2: RNA chain RNA (121-MER)


Mass: 41934.762 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (80-MER)


Mass: 24816.900 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (80-MER)


Mass: 24530.600 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
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: PsCas9 in with gRNA and dsDNA, product state / Type: COMPLEX / Entity ID: #1-#4 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.21154 MDa / Experimental value: YES
Source (natural)Organism: Parasutterella secunda (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: 1500 nm
Image recordingElectron dose: 80 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 77059 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00515431
ELECTRON MICROSCOPYf_angle_d0.78521629
ELECTRON MICROSCOPYf_dihedral_angle_d19.0213476
ELECTRON MICROSCOPYf_chiral_restr0.0472510
ELECTRON MICROSCOPYf_plane_restr0.0052138

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