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- PDB-8jtj: Cryo-EM structure of GeoCas9-sgRNA-dsDNA ternary complex -

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Basic information

Entry
Database: PDB / ID: 8jtj
TitleCryo-EM structure of GeoCas9-sgRNA-dsDNA ternary complex
Components
  • CRISPR-associated endonuclease Cas9
  • DNA (29-MER)
  • DNA (5'-D(P*GP*GP*GP*CP*GP*CP*GP*AP*A)-3')
  • RNA (139-MER)
KeywordsDNA BINDING PROTEIN/RNA/DNA / RNA-binding / DNA-binding / complex / DNA BINDING PROTEIN-RNA-DNA complex
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
Cas9, alpha-helical lobe domain / Cas9 alpha-helical lobe domain / RuvC endonuclease subdomain 3 / RuvC endonuclease subdomain 3 / CRISPR-associated endonuclease Cas9 / HNH endonuclease / 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 Cas9
Similarity search - Component
Biological speciesGeobacillus stearothermophilus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.08 Å
AuthorsShen, P.P. / Liu, B.B. / Li, X. / Zhang, L.L. / Chen, C.-C. / Guo, R.-T.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: To Be Published
Title: Cryo-EM structure of GeoCas9-sgRNA-DNA ternary complex
Authors: Shen, P.P. / Liu, B.B. / Li, X. / Zhang, L.L. / Chen, C.-C. / Guo, R.-T.
History
DepositionJun 21, 2023Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Jun 5, 2024Provider: 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
B: RNA (139-MER)
C: DNA (29-MER)
D: DNA (5'-D(P*GP*GP*GP*CP*GP*CP*GP*AP*A)-3')


Theoretical massNumber of molelcules
Total (without water)184,4394
Polymers184,4394
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 Cas9


Mass: 128171.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Geobacillus stearothermophilus (bacteria)
Gene: cas9 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A150MP45
#2: RNA chain RNA (139-MER)


Mass: 44697.402 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Geobacillus stearothermophilus (bacteria)
Production host: Escherichia coli BL21(DE3) (bacteria)
#3: DNA chain DNA (29-MER)


Mass: 8764.607 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Geobacillus stearothermophilus (bacteria)
#4: DNA chain DNA (5'-D(P*GP*GP*GP*CP*GP*CP*GP*AP*A)-3')


Mass: 2805.849 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Geobacillus stearothermophilus (bacteria)
Has ligand of interestY

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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: type II CRISPR RNA-guided endonuclease Cas9 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.13 MDa / Experimental value: NO
Source (natural)Organism: Geobacillus stearothermophilus (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Details: 20 mM Tris-HCl, 0.1 M KCl, 5 mM MgCl2, 1 mM DTT, 0.5% Glycerol
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: 2400 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 52 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.08 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 389973 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00312163
ELECTRON MICROSCOPYf_angle_d0.6517069
ELECTRON MICROSCOPYf_dihedral_angle_d17.3322822
ELECTRON MICROSCOPYf_chiral_restr0.0381950
ELECTRON MICROSCOPYf_plane_restr0.0051668

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