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- EMDB-8295: Structure of 2x duplex DNA target-bound Cascade/I-C -

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Basic information

Entry
Database: EMDB / ID: EMD-8295
TitleStructure of 2x duplex DNA target-bound Cascade/I-C
Map dataStructure of 2x duplex DNA target-bound Cascade/I-C
Sample
  • Complex: 2x duplex DNA target-bound Cascade
Biological speciesDesulfovibrio vulgaris (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.8 Å
AuthorsHochstrasser JL / 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: 0.055
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.055
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

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Map

FileDownload / File: emd_8295.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of 2x duplex DNA target-bound Cascade/I-C
Voxel sizeX=Y=Z: 2.04 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.055
Minimum - Maximum-0.11069547 - 0.31982172
Average (Standard dev.)0.00080018723 (±0.016988246)
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-0.1110.3200.001

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Supplemental data

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Sample components

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Entire : 2x duplex DNA target-bound Cascade

EntireName: 2x duplex DNA target-bound Cascade
Components
  • Complex: 2x duplex DNA target-bound Cascade

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Supramolecule #1: 2x duplex DNA target-bound Cascade

SupramoleculeName: 2x duplex DNA target-bound Cascade / type: complex / ID: 1 / Parent: 0 / Details: apo-Cascade/I-C bound to 75 bp duplex DNA
Source (natural)Organism: Desulfovibrio vulgaris (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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: Protochips 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 KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 4.5 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 27000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 4200 / Average exposure time: 6.0 sec. / Average electron dose: 48.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 3)
Startup modelType of model: OTHER / Details: Negative stain model of complex
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: EMAN (ver. 2)
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 1.4)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: EMAN (ver. 2)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 8.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 17000

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