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- EMDB-21308: Structure of the SpCas9 DNA adenine base editor - ABE8e -

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Basic information

Entry
Database: EMDB / ID: EMD-21308
TitleStructure of the SpCas9 DNA adenine base editor - ABE8e
Map data
Sample
  • Complex: CRISPR-Cas9 ABE8e in a substrate-bound state
    • RNA: Cas9 (SpCas9) single-guide RNA (sgRNA)
    • Protein or peptide: CRISPR-associated endonuclease Cas9
    • DNA: DNA target strand (TS)
    • DNA: DNA non-target strand (NTS)
    • Protein or peptide: t-RNA adenine deaminase A v8e (TadA-8e)
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION
KeywordsBase 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
MafB19-like deaminase / tRNA-specific adenosine deaminase / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, bridge helix / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / Bridge helix of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / APOBEC/CMP deaminase, zinc-binding / Cytidine and deoxycytidylate deaminases zinc-binding region signature. ...MafB19-like deaminase / tRNA-specific adenosine deaminase / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, bridge helix / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / Bridge helix of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / APOBEC/CMP deaminase, zinc-binding / Cytidine and deoxycytidylate deaminases zinc-binding region signature. / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cytidine and deoxycytidylate deaminase domain / Cytidine and deoxycytidylate deaminases domain profile. / Cas9-type HNH domain / Cas9-type HNH domain profile. / Cytidine deaminase-like / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsKnott GJ / Lapinaite A
Funding support United States, 1 items
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, 2020-
Header (metadata) releaseJul 29, 2020-
Map releaseJul 29, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6vpc
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21308.png

Downloads & links

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Map

FileDownload / File: emd_21308.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.94 Å
Density
Contour LevelBy AUTHOR: 0.008 / Movie #1: 0.012
Minimum - Maximum-0.07627928 - 0.16077888
Average (Standard dev.)0.000020004078 (±0.003749351)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 240.64 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.940.940.94
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z240.640240.640240.640
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0760.1610.000

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Supplemental data

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Half map: #1

Fileemd_21308_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_21308_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : CRISPR-Cas9 ABE8e in a substrate-bound state

EntireName: CRISPR-Cas9 ABE8e in a substrate-bound state
Components
  • Complex: CRISPR-Cas9 ABE8e in a substrate-bound state
    • RNA: Cas9 (SpCas9) single-guide RNA (sgRNA)
    • Protein or peptide: CRISPR-associated endonuclease Cas9
    • DNA: DNA target strand (TS)
    • DNA: DNA non-target strand (NTS)
    • Protein or peptide: t-RNA adenine deaminase A v8e (TadA-8e)
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION

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Supramolecule #1: CRISPR-Cas9 ABE8e in a substrate-bound state

SupramoleculeName: CRISPR-Cas9 ABE8e in a substrate-bound state / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Details: ABE8e in a substrate-bound state where the deaminase (TadA-8e) engages DNA exposed within the CRISPR-Cas9 R-loop complex.
Source (natural)Organism: Streptococcus pyogenes (bacteria)

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

MacromoleculeName: Cas9 (SpCas9) single-guide RNA (sgRNA) / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 26.357629 KDa
SequenceString:
GGUUCCACUU UCUUAGACUG AGUUUUAGAG CUAGAAAUAG CAAGUUAAAA UAAGGCUAGU CCGUUAUCAA CUUGAAAAAG UG

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Macromolecule #2: CRISPR-associated endonuclease Cas9

MacromoleculeName: CRISPR-associated endonuclease Cas9 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 157.870984 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: KYSIGLAIGT NSVGWAVITD EYKVPSKKFK VLGNTDRHSI KKNLIGALLF DSGETAEATR LKRTARRRYT RRKNRICYLQ EIFSNEMAK VDDSFFHRLE ESFLVEEDKK HERHPIFGNI VDEVAYHEKY PTIYHLRKKL VDSTDKADLR LIYLALAHMI K FRGHFLIE ...String:
KYSIGLAIGT NSVGWAVITD EYKVPSKKFK VLGNTDRHSI KKNLIGALLF DSGETAEATR LKRTARRRYT RRKNRICYLQ EIFSNEMAK VDDSFFHRLE ESFLVEEDKK HERHPIFGNI VDEVAYHEKY PTIYHLRKKL VDSTDKADLR LIYLALAHMI K FRGHFLIE GDLNPDNSDV DKLFIQLVQT YNQLFEENPI NASGVDAKAI LSARLSKSRR LENLIAQLPG EKKNGLFGNL IA LSLGLTP NFKSNFDLAE DAKLQLSKDT YDDDLDNLLA QIGDQYADLF LAAKNLSDAI LLSDILRVNT EITKAPLSAS MIK RYDEHH QDLTLLKALV RQQLPEKYKE IFFDQSKNGY AGYIDGGASQ EEFYKFIKPI LEKMDGTEEL LVKLNREDLL RKQR TFDNG SIPHQIHLGE LHAILRRQED FYPFLKDNRE KIEKILTFRI PYYVGPLARG NSRFAWMTRK SEETITPWNF EEVVD KGAS AQSFIERMTN FDKNLPNEKV LPKHSLLYEY FTVYNELTKV KYVTEGMRKP AFLSGEQKKA IVDLLFKTNR KVTVKQ LKE DYFKKIECFD SVEISGVEDR FNASLGTYHD LLKIIKDKDF LDNEENEDIL EDIVLTLTLF EDREMIEERL KTYAHLF DD KVMKQLKRRR YTGWGRLSRK LINGIRDKQS GKTILDFLKS DGFANRNFMQ LIHDDSLTFK EDIQKAQVSG QGDSLHEH I ANLAGSPAIK KGILQTVKVV DELVKVMGRH KPENIVIEMA RENQTTQKGQ KNSRERMKRI EEGIKELGSQ ILKEHPVEN TQLQNEKLYL YYLQNGRDMY VDQELDINRL SDYDVDAIVP QSFLKDDSID NKVLTRSDKN RGKSDNVPSE EVVKKMKNYW RQLLNAKLI TQRKFDNLTK AERGGLSELD KAGFIKRQLV ETRQITKHVA QILDSRMNTK YDENDKLIRE VKVITLKSKL V SDFRKDFQ FYKVREINNY HHAHDAYLNA VVGTALIKKY PKLESEFVYG DYKVYDVRKM IAKSEQEIGK ATAKYFFYSN IM NFFKTEI TLANGEIRKR PLIETNGETG EIVWDKGRDF ATVRKVLSMP QVNIVKKTEV QTGGFSKESI LPKRNSDKLI ARK KDWDPK KYGGFDSPTV AYSVLVVAKV EKGKSKKLKS VKELLGITIM ERSSFEKNPI DFLEAKGYKE VKKDLIIKLP KYSL FELEN GRKRMLASAG ELQKGNELAL PSKYVNFLYL ASHYEKLKGS PEDNEQKQLF VEQHKHYLDE IIEQISEFSK RVILA DANL DKVLSAYNKH RDKPIREQAE NIIHLFTLTN LGAPAAFKYF DTTIDRKRYT STKEVLDATL IHQSITGLYE TRIDLS Q

UniProtKB: CRISPR-associated endonuclease Cas9

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

MacromoleculeName: t-RNA adenine deaminase A v8e (TadA-8e) / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 24.657887 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH MKRTADGSEF ESPKKKRKVS EVEFSHEYWM RHALTLAKRA RDEREVPVGA VLVLNNRVIG EGWNRAIGLH DPTAHAEIM ALRQGGLVMQ NYRLIDATLY VTFEPCVMCA GAMIHSRIGR VVFGVRNSKR GAAGSLMNVL NYPGMNHRVE I TEGILADE ...String:
MGSSHHHHHH MKRTADGSEF ESPKKKRKVS EVEFSHEYWM RHALTLAKRA RDEREVPVGA VLVLNNRVIG EGWNRAIGLH DPTAHAEIM ALRQGGLVMQ NYRLIDATLY VTFEPCVMCA GAMIHSRIGR VVFGVRNSKR GAAGSLMNVL NYPGMNHRVE I TEGILADE CAALLCDFYR MPRQVFNAQK KAQSSINSGG SSGGSSGSET PGTSESATPE SSGGSSGGS

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Macromolecule #3: DNA target strand (TS)

MacromoleculeName: DNA target strand (TS) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 15.390882 KDa
SequenceString:
(DC)(DT)(DA)(DA)(DT)(DC)(DG)(DC)(DC)(DC) (DT)(DC)(DA)(DG)(DT)(DC)(DT)(DA)(DA)(DG) (DA)(DA)(DA)(DG)(DT)(DG)(DG)(DA)(DA) (DC)(DA)(DC)(DG)(DG)(DT)(DC)(DG)(DG)(DA) (DG) (DC)(DC)(DA)(DC)(DC)(DG)(DA)(DT) (DC)(DG)

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Macromolecule #4: DNA non-target strand (NTS)

MacromoleculeName: DNA non-target strand (NTS) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 15.436831 KDa
SequenceString:
(DC)(DG)(DA)(DT)(DC)(DG)(DG)(DT)(DG)(DG) (DC)(DT)(DC)(DC)(DG)(DA)(DC)(DC)(DG)(DT) (DG)(DT)(DT)(DC)(DC)(8AZ)(DC)(DT)(DT) (DT)(DC)(DT)(DT)(DA)(DG)(DA)(DC)(DT)(DG) (DA)(DG)(DG)(DG)(DC)(DG)(DA)(DT)(DT) (DA)(DG)

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 3 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 7 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.5
Details: 20 mM Tris-HCL (pH 7.5) 200 mM KCl 1 mM DTT 0.25% (v/v) Glycerol
GridModel: C-flat-2/2 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281.15 K / Instrument: FEI VITROBOT MARK IV
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

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 5022 / Average electron dose: 48.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4un3

Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 2.11.0)
Final 3D classificationAvg.num./class: 180927
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: EXACT BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 180927

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Atomic model buiding 1

Initial model
PDB IDChain

4un3

source_name: PDB, initial_model_type: experimental model

4un3

source_name: PDB, initial_model_type: experimental model

5f9r

source_name: PDB, initial_model_type: experimental model

1z3a

source_name: PDB, initial_model_type: experimental model
Output model

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

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