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- EMDB-24836: Cas9 in complex with 15-17MM DNA, 60 min time-point -

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Basic information

Entry
Database: EMDB / ID: EMD-24836
TitleCas9 in complex with 15-17MM DNA, 60 min time-point
Map data
Sample
  • Complex: Cas9 bound to 15-17MM DNA, 60 min time-point, linear conformation
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
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 / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain / Cas9-type HNH domain profile. ...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 / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.33 Å
AuthorsBravo JPK / Taylor DW / Liu MS / Johnson KA
Funding support United States, 2 items
OrganizationGrant numberCountry
Welch FoundationF-1938 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM138348 United States
CitationJournal: Nature / Year: 2022
Title: Structural basis for mismatch surveillance by CRISPR-Cas9.
Authors: Jack P K Bravo / Mu-Sen Liu / Grace N Hibshman / Tyler L Dangerfield / Kyungseok Jung / Ryan S McCool / Kenneth A Johnson / David W Taylor /
Abstract: CRISPR-Cas9 as a programmable genome editing tool is hindered by off-target DNA cleavage, and the underlying mechanisms by which Cas9 recognizes mismatches are poorly understood. Although Cas9 ...CRISPR-Cas9 as a programmable genome editing tool is hindered by off-target DNA cleavage, and the underlying mechanisms by which Cas9 recognizes mismatches are poorly understood. Although Cas9 variants with greater discrimination against mismatches have been designed, these suffer from substantially reduced rates of on-target DNA cleavage. Here we used kinetics-guided cryo-electron microscopy to determine the structure of Cas9 at different stages of mismatch cleavage. We observed a distinct, linear conformation of the guide RNA-DNA duplex formed in the presence of mismatches, which prevents Cas9 activation. Although the canonical kinked guide RNA-DNA duplex conformation facilitates DNA cleavage, we observe that substrates that contain mismatches distal to the protospacer adjacent motif are stabilized by reorganization of a loop in the RuvC domain. Mutagenesis of mismatch-stabilizing residues reduces off-target DNA cleavage but maintains rapid on-target DNA cleavage. By targeting regions that are exclusively involved in mismatch tolerance, we provide a proof of concept for the design of next-generation high-fidelity Cas9 variants.
History
DepositionSep 9, 2021-
Header (metadata) releaseMar 2, 2022-
Map releaseMar 2, 2022-
UpdateMar 23, 2022-
Current statusMar 23, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0696
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0696
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24836.png

Downloads & links

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Map

FileDownload / File: emd_24836.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.81 Å
Density
Contour LevelBy AUTHOR: 0.0696 / Movie #1: 0.0696
Minimum - Maximum-0.16355745 - 0.43297485
Average (Standard dev.)5.964553e-05 (±0.008451491)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 311.04 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.810.810.81
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z311.040311.040311.040
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.1640.4330.000

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Supplemental data

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Sample components

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Entire : Cas9 bound to 15-17MM DNA, 60 min time-point, linear conformation

EntireName: Cas9 bound to 15-17MM DNA, 60 min time-point, linear conformation
Components
  • Complex: Cas9 bound to 15-17MM DNA, 60 min time-point, linear conformation

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Supramolecule #1: Cas9 bound to 15-17MM DNA, 60 min time-point, linear conformation

SupramoleculeName: Cas9 bound to 15-17MM DNA, 60 min time-point, linear conformation
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Streptococcus pyogenes (bacteria)

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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

BufferpH: 7
VitrificationCryogen name: ETHANE

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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
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 80.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.33 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 222693

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