+
Open data
-
Basic information
Entry | Database: PDB / ID: 8t6o | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Title | SpRY-Cas9:gRNA complex targeting TAC PAM DNA with 0 bp R-loop | |||||||||
![]() |
| |||||||||
![]() | IMMUNE SYSTEM / SpRY-Cas9 / CRISPR / Cas9 / R-loop | |||||||||
Function / homology | ![]() 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 | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å | |||||||||
![]() | Hibshman, G.N. / Bravo, J.P.K. / Taylor, D.W. | |||||||||
Funding support | ![]()
| |||||||||
![]() | ![]() 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 |
|
-
Structure visualization
Structure viewer | Molecule: ![]() ![]() |
---|
-
Downloads & links
-
Download
PDBx/mmCIF format | ![]() | 362.1 KB | Display | ![]() |
---|---|---|---|---|
PDB format | ![]() | 281 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
---|
-Related structure data
Related structure data | ![]() 41073MC ![]() 8spqC ![]() 8sqhC ![]() 8srsC ![]() 8t6pC ![]() 8t6sC ![]() 8t6tC ![]() 8t6xC ![]() 8t6yC ![]() 8t76C ![]() 8t77C ![]() 8t78C ![]() 8t79C ![]() 8t7sC ![]() 8tzzC ![]() 8u3yC M: map data used to model this data C: citing same article ( |
---|---|
Similar structure data | Similarity search - Function & homology ![]() |
-
Links
-
Assembly
Deposited unit | ![]()
|
---|---|
1 |
|
-
Components
#1: DNA chain | Mass: 16970.938 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() |
---|---|
#2: Protein | 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.) ![]() ![]() ![]() References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds |
#3: RNA chain | Mass: 27979.586 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() |
#4: DNA chain | Mass: 16920.820 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
---|---|
EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-
Sample preparation
Component | Name: Ternary complex of SpRY-Cas9 with gRNA and NAC PAM DNA after one minute of DNA incubation with 0 bp R-loop Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES |
---|---|
Molecular weight | Value: 0.21154 MDa / Experimental value: YES |
Source (natural) | Organism: ![]() |
Source (recombinant) | Organism: ![]() ![]() |
Buffer solution | pH: 7.5 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE |
-
Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
---|---|
Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm |
Image recording | Electron dose: 80 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
-
Processing
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
3D reconstruction | Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 99758 / Symmetry type: POINT | ||||||||||||||||||||||||
Refine LS restraints |
|