+Open data
-Basic information
Entry | Database: PDB / ID: 7v59 | |||||||||
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Title | Cryo-EM structure of spyCas9-sgRNA-DNA dimer | |||||||||
Components |
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Keywords | RNA BINDING PROTEIN/RNA/DNA / Complex / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA-DNA complex | |||||||||
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 | |||||||||
Biological species | Streptococcus pyogenes serotype M1 (bacteria) Streptococcus pyogenes (bacteria) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.26 Å | |||||||||
Authors | Liu, J. / Deng, P. | |||||||||
Funding support | China, 2items
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Citation | Journal: Chem Sci / Year: 2021 Title: Nonspecific interactions between SpCas9 and dsDNA sites located downstream of the PAM mediate facilitated diffusion to accelerate target search. Authors: Mengyi Yang / Ruirui Sun / Pujuan Deng / Yuzhuo Yang / Wenjuan Wang / Jun-Jie Gogo Liu / Chunlai Chen / Abstract: RNA-guided Cas9 (SpCas9) is a sequence-specific DNA endonuclease that works as one of the most powerful genetic editing tools. However, how Cas9 locates its target among huge amounts of dsDNAs ...RNA-guided Cas9 (SpCas9) is a sequence-specific DNA endonuclease that works as one of the most powerful genetic editing tools. However, how Cas9 locates its target among huge amounts of dsDNAs remains elusive. Here, combining biochemical and single-molecule fluorescence assays, we revealed that Cas9 uses both three-dimensional and one-dimensional diffusion to find its target with high efficiency. We further observed surprising apparent asymmetric target search regions flanking PAM sites on dsDNA under physiological salt conditions, which accelerates the target search efficiency of Cas9 by ∼10-fold. Illustrated by a cryo-EM structure of the Cas9/sgRNA/dsDNA dimer, non-specific interactions between DNA ∼8 bp downstream of the PAM site and lysines within residues 1151-1156 of Cas9, especially lys1153, are the key elements to mediate the one-dimensional diffusion of Cas9 and cause asymmetric target search regions flanking the PAM. Disrupting these non-specific interactions, such as mutating these lysines to alanines, diminishes the contribution of one-dimensional diffusion and reduces the target search rate by several times. In addition, low ionic concentrations or mutations on PAM recognition residues that modulate interactions between Cas9 and dsDNA alter apparent asymmetric target search behaviors. Together, our results reveal a unique searching mechanism of Cas9 under physiological salt conditions, and provide important guidance for both and applications of Cas9. | |||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7v59.cif.gz | 626.3 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7v59.ent.gz | 486.2 KB | Display | PDB format |
PDBx/mmJSON format | 7v59.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7v59_validation.pdf.gz | 904.8 KB | Display | wwPDB validaton report |
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Full document | 7v59_full_validation.pdf.gz | 974.3 KB | Display | |
Data in XML | 7v59_validation.xml.gz | 82.9 KB | Display | |
Data in CIF | 7v59_validation.cif.gz | 126.7 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/v5/7v59 ftp://data.pdbj.org/pub/pdb/validation_reports/v5/7v59 | HTTPS FTP |
-Related structure data
Related structure data | 31721MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 158111.234 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Streptococcus pyogenes serotype M1 (bacteria) Gene: cas9, csn1, SPy_1046 / Production host: Escherichia coli (E. coli) References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds #2: RNA chain | Mass: 37137.090 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria) #3: DNA chain | Mass: 15040.633 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria) |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Ternary complex of spyCas9 with sgRNA and DNA / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT |
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Source (natural) | Organism: Streptococcus pyogenes (bacteria) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
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 |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD |
Image recording | Electron dose: 53 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
-Processing
Software | Name: PHENIX / Version: 1.14_3260: / Classification: refinement |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION |
3D reconstruction | Resolution: 5.26 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 150080 / Symmetry type: POINT |