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- EMDB-8294: Structure of 2x duplex target DNA-bound Cascade/I-C -

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Basic information

Entry
Database: EMDB / ID: EMD-8294
TitleStructure of 2x duplex target DNA-bound Cascade/I-C
Map dataapo Cascade/I-C
Sample
  • Complex: apo-Cascade/I-C
Biological speciesDesulfovibrio vulgaris (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.7 Å
AuthorsHochstrasser ML / Taylor DW / Kornfeld JK / Nogales E / Doudna JA
Funding support United States, 1 items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)1244557 United States
CitationJournal: Mol Cell / Year: 2016
Title: DNA Targeting by a Minimal CRISPR RNA-Guided Cascade.
Authors: Megan L Hochstrasser / David W Taylor / Jack E Kornfeld / Eva Nogales / Jennifer A Doudna /
Abstract: Bacteria employ surveillance complexes guided by CRISPR (clustered, regularly interspaced, short palindromic repeats) RNAs (crRNAs) to target foreign nucleic acids for destruction. Although most ...Bacteria employ surveillance complexes guided by CRISPR (clustered, regularly interspaced, short palindromic repeats) RNAs (crRNAs) to target foreign nucleic acids for destruction. Although most type I and type III CRISPR systems require four or more distinct proteins to form multi-subunit surveillance complexes, the type I-C systems use just three proteins to achieve crRNA maturation and double-stranded DNA target recognition. We show that each protein plays multiple functional and structural roles: Cas5c cleaves pre-crRNAs and recruits Cas7 to position the RNA guide for DNA binding and unwinding by Cas8c. Cryoelectron microscopy reconstructions of free and DNA-bound forms of the Cascade/I-C surveillance complex reveal conformational changes that enable R-loop formation with distinct positioning of each DNA strand. This streamlined type I-C system explains how CRISPR pathways can evolve compact structures that retain full functionality as RNA-guided DNA capture platforms.
History
DepositionJul 18, 2016-
Header (metadata) releaseAug 31, 2016-
Map releaseAug 31, 2016-
UpdateDec 25, 2019-
Current statusDec 25, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.082
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.082
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8294.png

Downloads & links

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Map

FileDownload / File: emd_8294.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationapo Cascade/I-C
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.04 Å/pix.
x 160 pix.
= 326.4 Å		2.04 Å/pix.
x 160 pix.
= 326.4 Å		2.04 Å/pix.
x 160 pix.
= 326.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.04 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.082 / Movie #1: 0.082
Minimum - Maximum-0.10696636 - 0.31502187
Average (Standard dev.)-0.0003334375 (±0.012508408)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 326.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.042.042.04
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z326.400326.400326.400
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ281156
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-0.1070.315-0.000

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Supplemental data

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Sample components

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Entire : apo-Cascade/I-C

EntireName: apo-Cascade/I-C
Components
  • Complex: apo-Cascade/I-C

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Supramolecule #1: apo-Cascade/I-C

SupramoleculeName: apo-Cascade/I-C / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Desulfovibrio vulgaris (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pHMGWA
Molecular weightTheoretical: 342 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

Concentration0.25 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
20.0 mMHEPES
100.0 mMpotassium chlorideKCl
1.0 mMTCEP
GridModel: Protochip C-flat 4/2 / Material: COPPER / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsThe sample was monodisperse.

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Electron microscopy

MicroscopeFEI TITAN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Number real images: 170 / Average exposure time: 18.0 sec. / Average electron dose: 40.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 4.5 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 17000
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 57000
CTF correctionSoftware - Name: CTFFIND (ver. 3)
Startup modelType of model: OTHER / Details: Negative stain reconstruction of sample
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 28000
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: EMAN (ver. 2)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: EMAN (ver. 2)
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 1.4)

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