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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: EMD-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
Sample
  • Complex: Csm complex with crRNA from T.onnurineus
Biological speciesThermococcus onnurineus NA1 (archaea)
Methodsingle particle reconstruction / negative staining / Resolution: 25.0 Å
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.
History
DepositionNov 2, 2016-
Header (metadata) releaseDec 7, 2016-
Map releaseApr 5, 2017-
UpdateAug 2, 2017-
Current statusAug 2, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • 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

emd_3454.png

Downloads & links

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Map

FileDownload / File: emd_3454.map.gz / Format: CCP4 / Size: 18.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCsm complex with crRNA from T.onnurineus
Projections & slices

Image control

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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.5 / Movie #1: 0.5
Minimum - Maximum-0.8233378 - 2.4102547
Average (Standard dev.)0.007941852 (±0.1374122)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-85-85-85
Dimensions170170170
Spacing170170170
CellA=B=C: 351.9 Å
α=β=γ: 90.0 °

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
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
Components
  • Complex: Csm complex with crRNA from T.onnurineus

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Supramolecule #1: Csm complex with crRNA from T.onnurineus

SupramoleculeName: Csm complex with crRNA from T.onnurineus / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Thermococcus onnurineus NA1 (archaea)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pRSFDuet-1, pETDuel-1, pACYCDuet-1
Molecular weightExperimental: 280 KDa

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.025 mg/mL
BufferpH: 8
StainingType: NEGATIVE / Material: Uranyl acetate

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Electron microscopy

MicroscopeJEOL 2100F
Image recordingFilm or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm
Sample stageSpecimen holder model: JEOL

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Image processing

CTF correctionSoftware - Name: EMAN (ver. 2.12) / Software - details: e2ctf.py
Final reconstructionNumber classes used: 50 / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN (ver. 2.12) / Software - details: e2refine_easy.py / Number images used: 7644
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
FSC plot (resolution estimation)

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