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- EMDB-8991: CasX-gRNA-DNA (45T-45NT) State II -

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Basic information

Entry
Database: EMDB / ID: EMD-8991
TitleCasX-gRNA-DNA (45T-45NT) State II
Map dataCasX-gRNA-DNA(45bp) State II
Sample
  • Complex: CasX-gRNA-DNA (45T-45NT) State II
Biological speciesDeltaproteobacteria (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.2 Å
AuthorsLiu JJ / Orlova N
CitationJournal: Nature / Year: 2019
Title: CasX enzymes comprise a distinct family of RNA-guided genome editors.
Authors: Jun-Jie Liu / Natalia Orlova / Benjamin L Oakes / Enbo Ma / Hannah B Spinner / Katherine L M Baney / Jonathan Chuck / Dan Tan / Gavin J Knott / Lucas B Harrington / Basem Al-Shayeb / ...Authors: Jun-Jie Liu / Natalia Orlova / Benjamin L Oakes / Enbo Ma / Hannah B Spinner / Katherine L M Baney / Jonathan Chuck / Dan Tan / Gavin J Knott / Lucas B Harrington / Basem Al-Shayeb / Alexander Wagner / Julian Brötzmann / Brett T Staahl / Kian L Taylor / John Desmarais / Eva Nogales / Jennifer A Doudna /
Abstract: The RNA-guided CRISPR-associated (Cas) proteins Cas9 and Cas12a provide adaptive immunity against invading nucleic acids, and function as powerful tools for genome editing in a wide range of ...The RNA-guided CRISPR-associated (Cas) proteins Cas9 and Cas12a provide adaptive immunity against invading nucleic acids, and function as powerful tools for genome editing in a wide range of organisms. Here we reveal the underlying mechanisms of a third, fundamentally distinct RNA-guided genome-editing platform named CRISPR-CasX, which uses unique structures for programmable double-stranded DNA binding and cleavage. Biochemical and in vivo data demonstrate that CasX is active for Escherichia coli and human genome modification. Eight cryo-electron microscopy structures of CasX in different states of assembly with its guide RNA and double-stranded DNA substrates reveal an extensive RNA scaffold and a domain required for DNA unwinding. These data demonstrate how CasX activity arose through convergent evolution to establish an enzyme family that is functionally separate from both Cas9 and Cas12a.
History
DepositionJul 24, 2018-
Header (metadata) releaseAug 22, 2018-
Map releaseFeb 20, 2019-
UpdateFeb 20, 2019-
Current statusFeb 20, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8991.png

Downloads & links

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Map

FileDownload / File: emd_8991.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCasX-gRNA-DNA(45bp) State II
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.9 Å/pix.
x 256 pix.
= 230.4 Å		0.9 Å/pix.
x 256 pix.
= 230.4 Å		0.9 Å/pix.
x 256 pix.
= 230.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.9 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2 / Movie #1: 0.2
Minimum - Maximum-0.4474112 - 0.7598784
Average (Standard dev.)0.0021774876 (±0.0360821)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 230.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.90.90.9
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z230.400230.400230.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.4470.7600.002

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Supplemental data

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Sample components

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Entire : CasX-gRNA-DNA (45T-45NT) State II

EntireName: CasX-gRNA-DNA (45T-45NT) State II
Components
  • Complex: CasX-gRNA-DNA (45T-45NT) State II

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Supramolecule #1: CasX-gRNA-DNA (45T-45NT) State II

SupramoleculeName: CasX-gRNA-DNA (45T-45NT) State II / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Deltaproteobacteria (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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: 7.5
GridDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 46.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 5.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 73566
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD

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