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- EMDB-4691: Reconstruction of the CRISPR-associated hexameric RNase Csx1 from... -

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Basic information

Entry
Database: EMDB / ID: EMD-4691
TitleReconstruction of the CRISPR-associated hexameric RNase Csx1 from Sulfolobus islandicus
Map dataCryo-EM reconstruction of the Csx1 hexamer
Sample
  • Complex: Csx1 hexamer
Function / homologyCRISPR system endoribonuclease Csx1-like / CRISPR system endoribonuclease Csx1, HEPN domain / CRISPR-associated (Cas) DxTHG family
Function and homology information
Biological speciesSulfolobus islandicus REY15A (acidophilic)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.62 Å
AuthorsMontoya G / Sofos N / Molina R / Stella S
CitationJournal: Nat Commun / Year: 2019
Title: Structure of Csx1-cOA complex reveals the basis of RNA decay in Type III-B CRISPR-Cas.
Authors: Rafael Molina / Stefano Stella / Mingxia Feng / Nicholas Sofos / Vykintas Jauniskis / Irina Pozdnyakova / Blanca López-Méndez / Qunxin She / Guillermo Montoya /
Abstract: Type III CRISPR-Cas multisubunit complexes cleave ssRNA and ssDNA. These activities promote the generation of cyclic oligoadenylate (cOA), which activates associated CRISPR-Cas RNases from the ...Type III CRISPR-Cas multisubunit complexes cleave ssRNA and ssDNA. These activities promote the generation of cyclic oligoadenylate (cOA), which activates associated CRISPR-Cas RNases from the Csm/Csx families, triggering a massive RNA decay to provide immunity from genetic invaders. Here we present the structure of Sulfolobus islandicus (Sis) Csx1-cOA complex revealing the allosteric activation of its RNase activity. SisCsx1 is a hexamer built by a trimer of dimers. Each dimer forms a cOA binding site and a ssRNA catalytic pocket. cOA undergoes a conformational change upon binding in the second messenger binding site activating ssRNA degradation in the catalytic pockets. Activation is transmitted in an allosteric manner through an intermediate HTH domain, which joins the cOA and catalytic sites. The RNase functions in a sequential cooperative fashion, hydrolyzing phosphodiester bonds in 5'-C-C-3'. The degradation of cOA by Ring nucleases deactivates SisCsx1, suggesting that this enzyme could be employed in biotechnological applications.
History
DepositionMar 12, 2019-
Header (metadata) releaseOct 16, 2019-
Map releaseOct 16, 2019-
UpdateOct 16, 2019-
Current statusOct 16, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.13
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 2.13
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4691.png

Downloads & links

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Map

FileDownload / File: emd_4691.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM reconstruction of the Csx1 hexamer
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1 Å/pix.
x 400 pix.
= 400. Å		1 Å/pix.
x 400 pix.
= 400. Å		1 Å/pix.
x 400 pix.
= 400. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.13 / Movie #1: 2.13
Minimum - Maximum-6.2350516 - 12.365875000000001
Average (Standard dev.)0.010751821 (±0.4619843)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 400.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z111
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z400.000400.000400.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-6.23512.3660.011

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Supplemental data

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Sample components

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Entire : Csx1 hexamer

EntireName: Csx1 hexamer
Components
  • Complex: Csx1 hexamer

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Supramolecule #1: Csx1 hexamer

SupramoleculeName: Csx1 hexamer / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Sulfolobus islandicus REY15A (acidophilic) / Location in cell: cytoplasm
Molecular weightExperimental: 320 KDa

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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

Concentration0.5 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
20.0 mMTris-HCl
GridModel: Quantifoil, UltrAuFoil / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 2 / Number real images: 2061 / Average electron dose: 50.0 e/Å2
Details: 808 exposures received 60 electrons pr square Angstrom. 1253 exposures received 40 electrons pr square Angstrom.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: -3.0 µm / Nominal defocus min: -2.1 µm / Nominal magnification: 96000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 93232
CTF correctionSoftware - Name: CTFFIND / Software - details: as implemented in cistem
Startup modelType of model: NONE / Details: A model was generated ab initio in cisTEM.
Final reconstructionNumber classes used: 7 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.62 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 1.0.0-beta) / Number images used: 43700
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cisTEM (ver. 1.0.0-beta)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cisTEM (ver. 1.0.0-beta)
Final 3D classificationNumber classes: 20 / Avg.num./class: 3500 / Software - Name: cisTEM (ver. 1.0.0-beta)

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