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- PDB-8t77: SpRY-Cas9:gRNA complex bound to non-target DNA with 6 bp R-loop -

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Basic information

Entry
Database: PDB / ID: 8t77
TitleSpRY-Cas9:gRNA complex bound to non-target DNA with 6 bp R-loop
Components
  • CRISPR-associated endonuclease Cas9/Csn1
  • NTS
  • TS
  • gRNA
KeywordsIMMUNE SYSTEM / SpRY-Cas9 / CRISPR / Cas9 / R-loop
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 / Cas9 RuvC domain / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain ...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 / Cas9 RuvC domain / 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)
Escherichia phage Lambda (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.25 Å
AuthorsHibshman, G.N. / Bravo, 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: Nat Commun / Year: 2024
Title: Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9.
Authors: Grace N Hibshman / Jack P K Bravo / Matthew M Hooper / Tyler L Dangerfield / Hongshan Zhang / Ilya J Finkelstein / Kenneth A Johnson / David W Taylor /
Abstract: CRISPR-Cas9 is a powerful tool for genome editing, but the strict requirement for an NGG protospacer-adjacent motif (PAM) sequence immediately next to the DNA target limits the number of editable ...CRISPR-Cas9 is a powerful tool for genome editing, but the strict requirement for an NGG protospacer-adjacent motif (PAM) sequence immediately next to the DNA target limits the number of editable genes. Recently developed Cas9 variants have been engineered with relaxed PAM requirements, including SpG-Cas9 (SpG) and the nearly PAM-less SpRY-Cas9 (SpRY). However, the molecular mechanisms of how SpRY recognizes all potential PAM sequences remains unclear. Here, we combine structural and biochemical approaches to determine how SpRY interrogates DNA and recognizes target sites. Divergent PAM sequences can be accommodated through conformational flexibility within the PAM-interacting region, which facilitates tight binding to off-target DNA sequences. Nuclease activation occurs ~1000-fold slower than for Streptococcus pyogenes Cas9, enabling us to directly visualize multiple on-pathway intermediate states. Experiments with SpG position it as an intermediate enzyme between Cas9 and SpRY. Our findings shed light on the molecular mechanisms of PAMless genome editing.
History
DepositionJun 19, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 1, 2024Provider: repository / Type: Initial release
Revision 1.1May 15, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CRISPR-associated endonuclease Cas9/Csn1
B: gRNA
C: TS
D: NTS


Theoretical massNumber of molelcules
Total (without water)195,5024
Polymers195,5024
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/Csn1 / SpCas9 / SpyCas9


Mass: 159149.406 Da / Num. of mol.: 1
Mutation: A61R, L1111R, D1135L, S1136W, G1218K, E1219Q, N1317R, A1322R, R1333P, R1335Q, T1337R
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 gRNA


Mass: 28724.102 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)
#3: DNA chain TS


Mass: 4895.174 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia phage Lambda (virus)
#4: DNA chain NTS


Mass: 2732.839 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia phage Lambda (virus)

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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 SpRY-Cas9 with gRNA and non-target DNA with 6 bp R-loop
Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 0.21154 MDa / Experimental value: YES
Source (natural)Organism: Streptococcus pyogenes (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

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.25 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 114471 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00511183
ELECTRON MICROSCOPYf_angle_d0.98315601
ELECTRON MICROSCOPYf_dihedral_angle_d19.172638
ELECTRON MICROSCOPYf_chiral_restr0.0541819
ELECTRON MICROSCOPYf_plane_restr0.0071562

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