+Open data
-Basic information
Entry | Database: PDB / ID: 6vpc | ||||||
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Title | Structure of the SpCas9 DNA adenine base editor - ABE8e | ||||||
Components |
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Keywords | DNA BINDING PROTEIN/DNA/RNA / Base editor / ABE / Cas9 / CRISPR / DNA BINDING PROTEIN-DNA-RNA complex | ||||||
Function / homology | Function and homology information tRNA(adenine34) deaminase / tRNA wobble adenosine to inosine editing / tRNA-specific adenosine-34 deaminase activity / adenosine to inosine editing / maintenance of CRISPR repeat elements / 3'-5' exonuclease activity / DNA endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / protein homodimerization activity ...tRNA(adenine34) deaminase / tRNA wobble adenosine to inosine editing / tRNA-specific adenosine-34 deaminase activity / adenosine to inosine editing / maintenance of CRISPR repeat elements / 3'-5' exonuclease activity / DNA endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / protein homodimerization activity / DNA binding / RNA binding / zinc ion binding / metal ion binding Similarity search - Function | ||||||
Biological species | Streptococcus pyogenes (bacteria) Escherichia coli (E. coli) synthetic construct (others) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å | ||||||
Authors | Knott, G.J. / Lapinaite, A. / Doudna, J.A. | ||||||
Funding support | United States, 1items
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Citation | Journal: Science / Year: 2020 Title: DNA capture by a CRISPR-Cas9-guided adenine base editor. Authors: Audrone Lapinaite / Gavin J Knott / Cody M Palumbo / Enrique Lin-Shiao / Michelle F Richter / Kevin T Zhao / Peter A Beal / David R Liu / Jennifer A Doudna / Abstract: CRISPR-Cas-guided base editors convert A•T to G•C, or C•G to T•A, in cellular DNA for precision genome editing. To understand the molecular basis for DNA adenosine deamination by adenine base ...CRISPR-Cas-guided base editors convert A•T to G•C, or C•G to T•A, in cellular DNA for precision genome editing. To understand the molecular basis for DNA adenosine deamination by adenine base editors (ABEs), we determined a 3.2-angstrom resolution cryo-electron microscopy structure of ABE8e in a substrate-bound state in which the deaminase domain engages DNA exposed within the CRISPR-Cas9 R-loop complex. Kinetic and structural data suggest that ABE8e catalyzes DNA deamination up to ~1100-fold faster than earlier ABEs because of mutations that stabilize DNA substrates in a constrained, transfer RNA-like conformation. Furthermore, ABE8e's accelerated DNA deamination suggests a previously unobserved transient DNA melting that may occur during double-stranded DNA surveillance by CRISPR-Cas9. These results explain ABE8e-mediated base-editing outcomes and inform the future design of base editors. | ||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6vpc.cif.gz | 393.3 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6vpc.ent.gz | 294.8 KB | Display | PDB format |
PDBx/mmJSON format | 6vpc.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/vp/6vpc ftp://data.pdbj.org/pub/pdb/validation_reports/vp/6vpc | HTTPS FTP |
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-Related structure data
Related structure data | 21308MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-RNA chain , 1 types, 1 molecules A
#1: RNA chain | Mass: 26357.629 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria) |
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-Protein , 2 types, 3 molecules BEF
#2: Protein | Mass: 157870.984 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Streptococcus pyogenes (bacteria) / Gene: cas9, E6A91_04015, E6A98_03170 / Production host: Escherichia coli (E. coli) References: UniProt: A0A4U7H5M9, UniProt: Q99ZW2*PLUS, Hydrolases; Acting on ester bonds |
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#5: Protein | Mass: 24657.887 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli) / References: UniProt: P68398*PLUS |
-DNA chain , 2 types, 2 molecules CD
#3: DNA chain | Mass: 15390.882 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
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#4: DNA chain | Mass: 15436.831 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
-Non-polymers , 2 types, 5 molecules
#6: Chemical | #7: Chemical | |
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-Details
Has ligand of interest | Y |
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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: CRISPR-Cas9 ABE8e in a substrate-bound state / Type: COMPLEX Details: ABE8e in a substrate-bound state where the deaminase (TadA-8e) engages DNA exposed within the CRISPR-Cas9 R-loop complex. Entity ID: #1-#5 / Source: MULTIPLE SOURCES |
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Molecular weight | Experimental value: NO |
Source (natural) | Organism: Streptococcus pyogenes (bacteria) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 7.5 Details: 20 mM Tris-HCL (pH 7.5) 200 mM KCl 1 mM DTT 0.25% (v/v) Glycerol |
Specimen | Conc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-2/2 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281.15 K Details: The grids were rapidly plunged into liquid ethane using a FEI Vitrobot MarkIV maintained at 8C and 100% humidity, after being blotted for 4.5s with blot force 8. |
-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 FIELDBright-field microscopy |
Specimen holder | Cryogen: NITROGEN |
Image recording | Electron dose: 48 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5022 |
-Processing
Software |
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | |||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 180927 / Algorithm: EXACT BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT | |||||||||||||||||||||||||||||||||||
Atomic model building |
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Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | |||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 131.89 Å2 | |||||||||||||||||||||||||||||||||||
Refine LS restraints |
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