Journal: 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
Deposition
Jul 18, 2016
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Header (metadata) release
Aug 31, 2016
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Map release
Aug 31, 2016
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Update
Dec 25, 2019
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Current status
Dec 25, 2019
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Download / File: emd_8295.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation
Structure of 2x duplex DNA target-bound Cascade/I-C
Voxel size
X=Y=Z: 2.04 Å
Density
Contour Level
By AUTHOR: 0.05 / Movie #1: 0.055
Minimum - Maximum
-0.11069547 - 0.31982172
Average (Standard dev.)
0.00080018723 (±0.016988246)
Symmetry
Space group: 1
Details
EMDB XML:
Map geometry
Axis order
X
Y
Z
Origin
0
0
0
Dimensions
160
160
160
Spacing
160
160
160
Cell
A=B=C: 326.4 Å α=β=γ: 90.0 °
CCP4 map header:
mode
Image stored as Reals
Å/pix. X/Y/Z
2.04
2.04
2.04
M x/y/z
160
160
160
origin x/y/z
0.000
0.000
0.000
length x/y/z
326.400
326.400
326.400
α/β/γ
90.000
90.000
90.000
start NX/NY/NZ
0
0
0
NX/NY/NZ
28
11
56
MAP C/R/S
1
2
3
start NC/NR/NS
0
0
0
NC/NR/NS
160
160
160
D min/max/mean
-0.111
0.320
0.001
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Supplemental data
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Sample components
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Entire : 2x duplex DNA target-bound Cascade
Entire
Name: 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
Supramolecule
Name: 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 expression
Organism: Escherichia coli (E. coli)
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
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Sample preparation
Concentration
0.25 mg/mL
Buffer
pH: 7.5 Component:
Concentration
Name
Formula
20.0 mM
HEPES
100.0 mM
potassium chloride
KCl
1.0 mM
TCEP
Grid
Model: Protochips C-flat 4/2 / Material: COPPER / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: PLASMA CLEANING
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details
The sample was monodisperse.
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Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film 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
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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