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Open data
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Basic information
Entry | Database: PDB / ID: 8spq | |||||||||
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Title | SpRY-Cas9:gRNA complex targeting TGG PAM DNA | |||||||||
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Function / homology | ![]() maintenance of CRISPR repeat elements / 3'-5' exonuclease activity / DNA endonuclease activity / defense response to virus / ![]() ![]() ![]() ![]() Similarity search - Function | |||||||||
Biological species | ![]() ![]() ![]() ![]() ![]() ![]() | |||||||||
Method | ![]() ![]() ![]() | |||||||||
![]() | Hibshman, G.N. / Bravo, J.P.K. / Taylor, D.W. | |||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 368 KB | Display | ![]() |
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PDB format | ![]() | 285.4 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 40681MC ![]() 8sqhC ![]() 8srsC ![]() 8t6oC ![]() 8t6pC ![]() 8t6sC ![]() 8t6tC ![]() 8t6xC ![]() 8t6yC ![]() 8t76C ![]() 8t77C ![]() 8t78C ![]() 8t79C ![]() 8t7sC ![]() 8tzzC ![]() 8u3yC M: map data used to model this data C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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1 |
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Components
-DNA chain , 3 types, 3 molecules CDE
#3: DNA chain | Mass: 16865.783 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() ![]() |
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#4: DNA chain | Mass: 5854.799 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() ![]() |
#5: DNA chain | Mass: 5761.732 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() ![]() |
-Protein / RNA chain / Non-polymers , 3 types, 6 molecules AB![](data/chem/img/MG.gif)
![](data/chem/img/MG.gif)
#1: 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.) ![]() ![]() Gene: cas9, csn1, SPy_1046 / Production host: ![]() ![]() ![]() References: UniProt: Q99ZW2, ![]() |
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#2: RNA chain | ![]() Mass: 31702.900 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() ![]() |
#6: Chemical | ChemComp-MG / |
-Details
Has ligand of interest | Y |
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-Experimental details
-Experiment
Experiment | Method: ![]() |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: ![]() |
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Sample preparation
Component | Name: Ternary complex of SpRY-Cas9 with lambda 1 gRNA and lambda 1 DNA Type: COMPLEX / Entity ID: #1-#5 / Source: MULTIPLE SOURCES |
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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![]() ![]() |
Vitrification![]() | Cryogen name: ETHANE |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source![]() ![]() |
Electron lens | Mode: BRIGHT FIELD![]() |
Image recording | Electron dose: 80 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
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Processing
CTF correction![]() | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
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3D reconstruction![]() | Resolution: 3.26 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 30690 / Symmetry type: POINT | ||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||
Displacement parameters | Biso mean: 158.4 Å2 | ||||||||||||||||||||||||
Refine LS restraints |
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