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- PDB-8g1i: SpCas9 with sgRNA and target DNA -

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Basic information

Entry
Database: PDB / ID: 8g1i
TitleSpCas9 with sgRNA and target DNA
Components
  • CRISPR-associated endonuclease Cas9/Csn1
  • DNA E2_NTS
  • DNA E2_TS
  • sgRNASubgenomic mRNA
KeywordsHYDROLASE / Cas9 / RNA / DNA
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / 3'-5' exonuclease activity / DNA endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, bridge helix / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / Bridge helix of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain / Cas9-type HNH domain profile. ...CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, bridge helix / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / Bridge helix of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / 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) / CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes (bacteria)
Renilla reniformis (sea pansy)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.12 Å
AuthorsKorolev, S. / Gagnon, K.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: To Be Published
Title: SpCas9 with sgRNA and target DNA
Authors: Korolev, S. / Gagnon, K.
History
DepositionFeb 2, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 17, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: sgRNA
A: CRISPR-associated endonuclease Cas9/Csn1
C: DNA E2_TS
D: DNA E2_NTS


Theoretical massNumber of molelcules
Total (without water)216,9444
Polymers216,9444
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: RNA chain sgRNA / Subgenomic mRNA


Mass: 31543.791 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)
#2: Protein CRISPR-associated endonuclease Cas9/Csn1 / SpCas9 / SpyCas9


Mass: 160774.281 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes (bacteria) / Gene: cas9, csn1, SPy_1046 / Production host: Escherichia coli (E. coli)
References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds
#3: DNA chain DNA E2_TS


Mass: 12334.909 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Renilla reniformis (sea pansy)
#4: DNA chain DNA E2_NTS


Mass: 12290.917 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Renilla reniformis (sea pansy)

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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-associated endonuclease Cas9 complex with sgRNA and target DNA
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 1.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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 FIELDBright-field microscopy / Nominal defocus max: 2400 nm / Nominal defocus min: 1000 nm / C2 aperture diameter: 50 µm
Image recordingAverage exposure time: 4.28 sec. / Electron dose: 51.37 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5008 / Details: Movies were collected in counted-mode
EM imaging opticsChromatic aberration corrector: CEOS manufactured Cc corrector
Image scansWidth: 4096 / Height: 4096

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARC3.13particle selection
2EPUimage acquisition
4cryoSPARC3.13CTF correction
7PHENIX1.20.1model fitting
11cryoSPARC4.0.3classification
13PHENIX1.20.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.12 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 64788 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00314153
ELECTRON MICROSCOPYf_angle_d0.58619703
ELECTRON MICROSCOPYf_dihedral_angle_d16.6853013
ELECTRON MICROSCOPYf_chiral_restr0.0392267
ELECTRON MICROSCOPYf_plane_restr0.0051994

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