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- PDB-8fzt: SpCas9 with dual-guide RNA and target DNA -

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Basic information

Entry
Database: PDB / ID: 8fzt
TitleSpCas9 with dual-guide RNA and target DNA
Components
  • CRISPR-associated endonuclease Cas9/Csn1
  • CrE2
  • E2-NTS
  • E2_TS
  • tracrRNA
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, bridge helix / Bridge helix of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 ...CRISPR-associated endonuclease Cas9, bridge helix / Bridge helix of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 / 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)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.03 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 dual-guide RNA and target DNA
Authors: Korolev, S. / Gagnon, K.
History
DepositionJan 30, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 31, 2023Provider: repository / Type: Initial release
Revision 2.0Jun 19, 2024Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Database references / Derived calculations / Refinement description / Structure summary
Category: atom_site / chem_comp_atom ...atom_site / chem_comp_atom / chem_comp_bond / ndb_struct_na_base_pair / ndb_struct_na_base_pair_step / pdbx_contact_author / pdbx_poly_seq_scheme / pdbx_struct_assembly / pdbx_struct_oper_list / pdbx_unobs_or_zero_occ_residues / pdbx_validate_close_contact / pdbx_validate_rmsd_angle / pdbx_validate_torsion / refine / refine_ls_restr / software / struct_conf / struct_conn / struct_ref_seq
Item: _pdbx_poly_seq_scheme.auth_mon_id / _pdbx_poly_seq_scheme.auth_seq_num ..._pdbx_poly_seq_scheme.auth_mon_id / _pdbx_poly_seq_scheme.auth_seq_num / _pdbx_poly_seq_scheme.pdb_mon_id / _pdbx_poly_seq_scheme.pdb_seq_num / _pdbx_struct_assembly.details / _pdbx_struct_oper_list.symmetry_operation / _refine.B_iso_mean / _refine.pdbx_ls_cross_valid_method / _refine.pdbx_stereochemistry_target_values / _refine_ls_restr.dev_ideal / _refine_ls_restr.number / _refine_ls_restr.type / _struct_conf.beg_auth_comp_id / _struct_conf.beg_auth_seq_id / _struct_conf.beg_label_comp_id / _struct_conf.beg_label_seq_id / _struct_conf.end_auth_comp_id / _struct_conf.end_auth_seq_id / _struct_conf.end_label_comp_id / _struct_conf.end_label_seq_id / _struct_conf.pdbx_PDB_helix_length / _struct_ref_seq.db_align_beg / _struct_ref_seq.db_align_end / _struct_ref_seq.pdbx_auth_seq_align_beg / _struct_ref_seq.pdbx_auth_seq_align_end
Description: Model completeness
Details: Improved geometry of DNA-RNA double helix at bending point
Provider: author / Type: Coordinate replacement

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
E: CRISPR-associated endonuclease Cas9/Csn1
A: CrE2
B: tracrRNA
C: E2_TS
D: E2-NTS


Theoretical massNumber of molelcules
Total (without water)221,3465
Polymers221,3465
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: 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
#2: RNA chain CrE2


Mass: 13370.906 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)
#3: RNA chain tracrRNA


Mass: 22574.494 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria) / References: GenBank: 1403816625
#4: DNA chain E2_TS


Mass: 12334.909 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#5: DNA chain E2-NTS


Mass: 12290.917 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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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 double stranded guide RNA 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.: 1.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
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 FIELD / 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: 11890 / 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

EM software
IDNameVersionCategory
1cryoSPARC3.13particle selection
2EPUimage acquisition
4cryoSPARC3.03CTF correction
7PHENIXmodel fitting
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.03 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 108043 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 86.32 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00314507
ELECTRON MICROSCOPYf_angle_d0.59720224
ELECTRON MICROSCOPYf_chiral_restr0.03882332
ELECTRON MICROSCOPYf_plane_restr0.00432035
ELECTRON MICROSCOPYf_dihedral_angle_d15.94583142

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