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- PDB-6vpc: Structure of the SpCas9 DNA adenine base editor - ABE8e -

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Basic information

Entry
Database: PDB / ID: 6vpc
TitleStructure of the SpCas9 DNA adenine base editor - ABE8e
Components
  • CRISPR-associated endonuclease Cas9
  • Cas9 (SpCas9) single-guide RNA (sgRNA)
  • DNA non-target strand (NTS)
  • DNA target strand (TS)
  • t-RNA adenine deaminase A v8e (TadA-8e)
KeywordsDNA 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
tRNA-specific adenosine deaminase / MafB19-like deaminase / Cytidine and deoxycytidylate deaminase zinc-binding region / CRISPR-associated endonuclease Cas9, bridge helix / Bridge helix of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / : ...tRNA-specific adenosine deaminase / MafB19-like deaminase / Cytidine and deoxycytidylate deaminase zinc-binding region / CRISPR-associated endonuclease Cas9, bridge helix / Bridge helix of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / : / Cas9 RuvC domain / APOBEC/CMP deaminase, zinc-binding / Cytidine and deoxycytidylate deaminases zinc-binding region signature. / Cytidine and deoxycytidylate deaminase domain / Cytidine and deoxycytidylate deaminases domain profile. / Cytidine deaminase-like / HNH endonuclease / CRISPR-associated endonuclease Cas9 / 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 / tRNA-specific adenosine deaminase / CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes (bacteria)
Escherichia coli (E. coli)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsKnott, G.J. / Lapinaite, A. / Doudna, J.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)RM1HG009490 United States
CitationJournal: 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
DepositionFeb 3, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 29, 2020Provider: repository / Type: Initial release
Revision 1.1Aug 12, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Cas9 (SpCas9) single-guide RNA (sgRNA)
B: CRISPR-associated endonuclease Cas9
C: DNA target strand (TS)
D: DNA non-target strand (NTS)
E: t-RNA adenine deaminase A v8e (TadA-8e)
F: t-RNA adenine deaminase A v8e (TadA-8e)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)264,57611
Polymers264,3726
Non-polymers2045
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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RNA chain , 1 types, 1 molecules A

#1: RNA chain Cas9 (SpCas9) single-guide RNA (sgRNA)


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 CRISPR-associated endonuclease Cas9


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
#5: Protein t-RNA adenine deaminase A v8e (TadA-8e)


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

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DNA chain , 2 types, 2 molecules CD

#3: DNA chain DNA target strand (TS)


Mass: 15390.882 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA non-target strand (NTS)


Mass: 15436.831 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 2 types, 5 molecules

#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg
#7: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestY

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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: 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
Molecular weightExperimental value: NO
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Details: 20 mM Tris-HCL (pH 7.5) 200 mM KCl 1 mM DTT 0.25% (v/v) Glycerol
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-2/2
VitrificationInstrument: 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.

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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
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 48 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5022

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.17.1_3660refinement
PHENIX1.17.1_3660refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
7Coot0.8.9.1model fitting
9cryoSPARC2.11.0initial Euler assignment
10RELION3final Euler assignment
12RELION33D reconstruction
13PHENIX1.17.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 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
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
14UN3

4un3

14UN31PDBexperimental model
24UN3

4un3

14UN31PDBexperimental model
35F9R

5f9r

15F9R2PDBexperimental model
41Z3A

1z3a

11Z3A3PDBexperimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 131.89 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00216926
ELECTRON MICROSCOPYf_angle_d0.543123523
ELECTRON MICROSCOPYf_chiral_restr0.03582686
ELECTRON MICROSCOPYf_plane_restr0.00442463
ELECTRON MICROSCOPYf_dihedral_angle_d17.49356722

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