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- EMDB-8558: Negative stain reconstruction of Cas1-Cas2/3 complex of type I-F ... -

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Basic information

Entry
Database: EMDB / ID: EMD-8558
TitleNegative stain reconstruction of Cas1-Cas2/3 complex of type I-F CRISPR system from Pseudomonas aeruginosa (PA14)
Map dataNegative stain reconstruction of Pseudomonas aeruginosa (PA14) Cas1-Cas2/3 complex
Sample
  • Complex: Complex containing dimer of Cas2/3 protein and tetramer of Cas1 protein.
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / DNA endonuclease activity / helicase activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / defense response to virus / Hydrolases; Acting on ester bonds / hydrolase activity / DNA binding / ATP binding / metal ion binding / identical protein binding
Similarity search - Function
Helicase Cas3, CRISPR-associated, Yersinia-type / : / : / CRISPR-associated nuclease/helicase Cas3, I-F/YPEST, Cas2 domain / CRISPR-associated Cas3 subtype I-F/YPEST-like, C-terminal domain / CRISPR-associated protein Cas1, YPEST subtype / Cas3, HD domain / : / CRISPR-associated Cas3-type HD domain / CRISPR-associated Cas3-type HD domain superfamily ...Helicase Cas3, CRISPR-associated, Yersinia-type / : / : / CRISPR-associated nuclease/helicase Cas3, I-F/YPEST, Cas2 domain / CRISPR-associated Cas3 subtype I-F/YPEST-like, C-terminal domain / CRISPR-associated protein Cas1, YPEST subtype / Cas3, HD domain / : / CRISPR-associated Cas3-type HD domain / CRISPR-associated Cas3-type HD domain superfamily / CRISPR-associated nuclease/helicase Cas3, C-terminal / HD Cas3-type domain profile. / CRISPR-associated endonuclease Cas1, N-terminal domain / : / CRISPR-associated protein Cas1 / CRISPR-associated endonuclease Cas1, C-terminal domain / CRISPR associated protein Cas1 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
CRISPR-associated endonuclease Cas1 / CRISPR-associated nuclease/helicase Cas3 subtype I-F/YPEST
Similarity search - Component
Biological speciesPseudomonas aeruginosa PA14 (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 15.6 Å
AuthorsChowdhury S / Lander GC / Rollins MF / Carter J / Golden MS / Wilkinson RA / Bondy-Denomy J / Wiedenheft B
CitationJournal: Proc Natl Acad Sci U S A / Year: 2017
Title: Cas1 and the Csy complex are opposing regulators of Cas2/3 nuclease activity.
Authors: MaryClare F Rollins / Saikat Chowdhury / Joshua Carter / Sarah M Golden / Royce A Wilkinson / Joseph Bondy-Denomy / Gabriel C Lander / Blake Wiedenheft /
Abstract: The type I-F CRISPR adaptive immune system in (PA14) consists of two CRISPR loci and six CRISPR-associated () genes. Type I-F systems rely on a CRISPR RNA (crRNA)-guided surveillance complex (Csy ...The type I-F CRISPR adaptive immune system in (PA14) consists of two CRISPR loci and six CRISPR-associated () genes. Type I-F systems rely on a CRISPR RNA (crRNA)-guided surveillance complex (Csy complex) to bind foreign DNA and recruit a -acting nuclease (i.e., Cas2/3) for target degradation. In most type I systems, Cas2 and Cas3 are separate proteins involved in adaptation and interference, respectively. However, in I-F systems, these proteins are fused into a single polypeptide. Here we use biochemical and structural methods to show that two molecules of Cas2/3 assemble with four molecules of Cas1 (Cas2/3:Cas1) into a four-lobed propeller-shaped structure, where the two Cas2 domains form a central hub (twofold axis of symmetry) flanked by two Cas1 lobes and two Cas3 lobes. We show that the Cas1 subunits repress Cas2/3 nuclease activity and that foreign DNA recognition by the Csy complex activates Cas2/3, resulting in bidirectional degradation of DNA targets. Collectively, this work provides a structure of the Cas1-2/3 complex and explains how Cas1 and the target-bound Csy complex play opposing roles in the regulation of Cas2/3 nuclease activity.
History
DepositionJan 13, 2017-
Header (metadata) releaseFeb 8, 2017-
Map releaseApr 26, 2017-
UpdateJul 18, 2018-
Current statusJul 18, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0466
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0466
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8558.png

Downloads & links

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Map

FileDownload / File: emd_8558.map.gz / Format: CCP4 / Size: 3.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain reconstruction of Pseudomonas aeruginosa (PA14) Cas1-Cas2/3 complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.1 Å/pix.
x 96 pix.
= 393.6 Å		4.1 Å/pix.
x 96 pix.
= 393.6 Å		4.1 Å/pix.
x 96 pix.
= 393.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0466 / Movie #1: 0.0466
Minimum - Maximum-0.09443157 - 0.18779674
Average (Standard dev.)0.0006248977 (±0.010670355)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions969696
Spacing969696
CellA=B=C: 393.59998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.14.14.1
M x/y/z969696
origin x/y/z0.0000.0000.000
length x/y/z393.600393.600393.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-152-37
NX/NY/NZ998271
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS969696
D min/max/mean-0.0940.1880.001

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Supplemental data

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Sample components

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Entire : Complex containing dimer of Cas2/3 protein and tetramer of Cas1 p...

EntireName: Complex containing dimer of Cas2/3 protein and tetramer of Cas1 protein.
Components
  • Complex: Complex containing dimer of Cas2/3 protein and tetramer of Cas1 protein.

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Supramolecule #1: Complex containing dimer of Cas2/3 protein and tetramer of Cas1 p...

SupramoleculeName: Complex containing dimer of Cas2/3 protein and tetramer of Cas1 protein.
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Pseudomonas aeruginosa PA14 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3)
Molecular weightTheoretical: 386 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

Concentration0.03 mg/mL
BufferpH: 7.5 / Details: 20 mM HEPES, 150 mM KCl
StainingType: NEGATIVE / Material: Uranyl Formate
Details: Sample absorbed on carbon surface was stained with 2% (w/v) Uranyl Formate solution, and finally air-dried after blotting off excess stain.
GridModel: 400 mesh Cu-Rh maxtaform grids (Electron Microscopy Sciences) that were coated with a thin layer of amorphous carbon.
Material: COPPER/RHODIUM / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.02 kPa / Details: 15mA current
DetailsMono dispersed protein solution

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
TemperatureMin: 293.0 K / Max: 295.0 K
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Sampling interval: 15.6 µm / Number grids imaged: 1 / Number real images: 1159 / Average exposure time: 0.479 sec. / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 0.99 µm / Calibrated defocus min: 0.99 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 52000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

DetailsAutomated image acquisition software Leginon was used for data collection.
Particle selectionNumber selected: 221700
Details: Difference of Gaussian (DoG)-based automated particle picker, implemented in Appion processing package was used for particle selection.
CTF correctionSoftware - Name: CTFFIND4
Details: Ctffind4 was used for determining the CTF of each micrograph. Phases of each micrographs were flipped before particle extraction.
Startup modelType of model: OTHER
Details: An initial 3D reference was generated with selected 2D class averages using sxviper program of the SPARX EM data processing package (sparx-em.org)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 15.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 26403
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final 3D classificationNumber classes: 8 / Software - Name: RELION (ver. 1.4)

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