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- EMDB-3454: RNA activation-independent DNA targeting by a Csm complex of the ... -

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Basic information

Entry
Database: EMDB / ID: 3454
TitleRNA activation-independent DNA targeting by a Csm complex of the Type III CRISPR-Cas system
Map dataCsm complex with crRNA from T.onnurineus
SampleCsm complex with crRNA from T.onnurineus:
SourceThermococcus onnurineus NA1 (archaea)
Methodsingle particle reconstruction / 25 Å resolution
AuthorsJung TY / Song JJ / Hebert H / Woo EJ / Oh BH / Park KH / An Y
CitationJournal: EMBO Rep. / Year: 2017
Title: RNA activation-independent DNA targeting of the Type III CRISPR-Cas system by a Csm complex.
Authors: Kwang-Hyun Park / Yan An / Tae-Yang Jung / In-Young Baek / Haemin Noh / Woo-Chan Ahn / Hans Hebert / Ji-Joon Song / Jeong-Hoon Kim / Byung-Ha Oh / Eui-Jeon Woo
Abstract: The CRISPR-Cas system is an adaptive and heritable immune response that destroys invading foreign nucleic acids. The effector complex of the Type III CRISPR-Cas system targets RNA and DNA in a ...The CRISPR-Cas system is an adaptive and heritable immune response that destroys invading foreign nucleic acids. The effector complex of the Type III CRISPR-Cas system targets RNA and DNA in a transcription-coupled manner, but the exact mechanism of DNA targeting by this complex remains elusive. In this study, an effector Csm holocomplex derived from is reconstituted with a minimalistic combination of Csm12345, and shows RNA targeting and RNA-activated single-stranded DNA (ssDNA) targeting activities. Unexpectedly, in the absence of an RNA transcript, it cleaves ssDNA containing a sequence complementary to the bound crRNA guide region in a manner dependent on the HD domain of the Csm1 subunit. This nuclease activity is blocked by a repeat tag found in the host CRISPR loci. The specific cleavage of ssDNA without a target RNA suggests a novel ssDNA targeting mechanism of the Type III system, which could facilitate the efficient and complete degradation of foreign nucleic acids.
DateDeposition: Nov 2, 2016 / Header (metadata) release: Dec 7, 2016 / Map release: Apr 5, 2017 / Last update: Aug 2, 2017

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_3454.map.gz (map file in CCP4 format, 19653 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
170 pix
2.07 Å/pix.
= 351.9 Å
170 pix
2.07 Å/pix.
= 351.9 Å
170 pix
2.07 Å/pix.
= 351.9 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.07 Å
Density
Contour Level:0.5 (by author), 0.5 (movie #1):
Minimum - Maximum-0.8233378 - 2.4102547
Average (Standard dev.)0.007941852 (0.1374122)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions170170170
Origin-85-85-85
Limit848484
Spacing170170170
CellA=B=C: 351.9 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.072.072.07
M x/y/z170170170
origin x/y/z0.0000.0000.000
length x/y/z351.900351.900351.900
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS-85-85-85
NC/NR/NS170170170
D min/max/mean-0.8232.4100.008

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Supplemental data

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Sample components

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Entire Csm complex with crRNA from T.onnurineus

EntireName: Csm complex with crRNA from T.onnurineus / Number of components: 1
MassExperimental: 280 kDa

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Component #1: protein, Csm complex with crRNA from T.onnurineus

ProteinName: Csm complex with crRNA from T.onnurineus / Recombinant expression: No
MassExperimental: 280 kDa
SourceSpecies: Thermococcus onnurineus NA1 (archaea)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pRSFDuet-1, pETDuel-1, pACYCDuet-1

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle
Sample solutionSpecimen conc.: 0.025 mg/ml / pH: 8
VitrificationCryogen name: NONE

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Electron microscopy imaging

ImagingMicroscope: JEOL 2100F
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 30 e/Å2 / Illumination mode: FLOOD BEAM
LensCs: 2 mm / Imaging mode: BRIGHT FIELD
Specimen HolderModel: JEOL
CameraDetector: TVIPS TEMCAM-F415 (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 7644
3D reconstructionSoftware: EMAN / Resolution: 25 Å / Resolution method: FSC 0.5 CUT-OFF
FSC plot
(resolution estimation)

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