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- EMDB-3276: Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage -

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Basic information

Entry
Database: EMDB / ID: 3276
TitleStructures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage
Map dataReconstruction of sgRNA-bound Cas9
SampleCas9 bound to single guide-RNA
  • CRISPR-associated endonuclease Cas9/Csn1
  • nucleic-acidNucleic acid
KeywordsCRISPR-Cas / Cas9 / genome editing
Function / homologyHNH endonuclease / REC lobe of CRISPR-associated endonuclease Cas9 / Cas9-type HNH domain profile. / Cas9-type HNH domain / CRISPR-associated endonuclease Cas9, REC lobe / CRISPR-associated endonuclease Cas9, bridge helix / CRISPR-associated endonuclease Cas9, PAM-interacting domain / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9 / HNH nuclease ...HNH endonuclease / REC lobe of CRISPR-associated endonuclease Cas9 / Cas9-type HNH domain profile. / Cas9-type HNH domain / CRISPR-associated endonuclease Cas9, REC lobe / CRISPR-associated endonuclease Cas9, bridge helix / CRISPR-associated endonuclease Cas9, PAM-interacting domain / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9 / HNH nuclease / Bridge helix of CRISPR-associated endonuclease Cas9 / in:IPR025978: / CRISPR-associated endonuclease Cas9, bridge helix / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / CRISPR-associated endonuclease Cas9 / HNH nuclease / nucleic acid phosphodiester bond hydrolysis / maintenance of CRISPR repeat elements / endodeoxyribonuclease activity / 3'-5' exonuclease activity / defense response to virus / endonuclease activity / Hydrolases, Acting on ester bonds / RNA binding / DNA binding / metal ion binding / CRISPR-associated endonuclease Cas9/Csn1
Function and homology information
SourceStreptococcus pyogenes (bacteria)
Methodsingle particle reconstruction / cryo EM / 4.5 Å resolution
AuthorsJiang F / Taylor DW / Chen JS / Kornfeld JE / Zhou K / Thompson AJ / Nogales E / Doudna JA
CitationJournal: Science / Year: 2016
Title: Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage.
Authors: Fuguo Jiang / David W Taylor / Janice S Chen / Jack E Kornfeld / Kaihong Zhou / Aubri J Thompson / Eva Nogales / Jennifer A Doudna
Abstract: Bacterial adaptive immunity and genome engineering involving the CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) protein Cas9 begin with RNA-guided DNA unwinding ...Bacterial adaptive immunity and genome engineering involving the CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) protein Cas9 begin with RNA-guided DNA unwinding to form an RNA-DNA hybrid and a displaced DNA strand inside the protein. The role of this R-loop structure in positioning each DNA strand for cleavage by the two Cas9 nuclease domains is unknown. We determine molecular structures of the catalytically active Streptococcus pyogenes Cas9 R-loop that show the displaced DNA strand located near the RuvC nuclease domain active site. These protein-DNA interactions, in turn, position the HNH nuclease domain adjacent to the target DNA strand cleavage site in a conformation essential for concerted DNA cutting. Cas9 bends the DNA helix by 30°, providing the structural distortion needed for R-loop formation.
DateDeposition: Dec 10, 2015 / Header (metadata) release: Jan 27, 2016 / Map release: Jan 27, 2016 / Last update: Mar 9, 2016

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.01
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_3276.map.gz (map file in CCP4 format, 93313 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
288 pix
1.01 Å/pix.
= 290.88 Å
288 pix
1.01 Å/pix.
= 290.88 Å
288 pix
1.01 Å/pix.
= 290.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.01 Å
Density
Contour Level:0.01 (by emdb), 0.01 (movie #1):
Minimum - Maximum-0.02166877 - 0.05766536
Average (Standard dev.)0.00000727 (0.00168026)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions288288288
Origin000
Limit287287287
Spacing288288288
CellA=B=C: 290.88 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.011.011.01
M x/y/z288288288
origin x/y/z0.0000.0000.000
length x/y/z290.880290.880290.880
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS288288288
D min/max/mean-0.0220.0580.000

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Supplemental data

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Sample components

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Entire Cas9 bound to single guide-RNA

EntireName: Cas9 bound to single guide-RNA / Number of components: 2
MassTheoretical: 194 kDa

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Component #1: protein, CRISPR-associated endonuclease Cas9/Csn1

ProteinName: CRISPR-associated endonuclease Cas9/Csn1 / a.k.a: Cas9 / Oligomeric Details: Monomer / Number of Copies: 1 / Recombinant expression: Yes
MassTheoretical: 158 kDa
SourceSpecies: Streptococcus pyogenes (bacteria) / Strain: serotype M1
Source (engineered)Expression System: Escherichia coli (E. coli) / Strain: BL21
External referencesGene Ontology: maintenance of CRISPR repeat elements, defense response to virus, nucleic acid phosphodiester bond hydrolysis, DNA binding, RNA binding, endonuclease activity
UniProt: CRISPR-associated endonuclease Cas9/Csn1
InterPro: CRISPR-associated endonuclease Cas9, CRISPR-associated endonuclease Cas9, bridge helix, CRISPR-associated endonuclease Cas9, PAM-interacting domain, CRISPR-associated endonuclease Cas9, REC lobe, InterPro: IPR025978, HNH nuclease

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Component #2: nucleic-acid, single guide-RNA

Nucleic-acidName: single guide-RNA / a.k.a: sgRNASubgenomic mRNA / Class: RNA / Structure: OTHER / Synthetic: Yes
Sequence:
GGCGCAUAAA GAUGAGACGC GUUUUAGAGC UAUGCUGUUU UGAAAAAAAC AGCAUAGCAA GUUAAAAUAA GGCUAGUCCG UUAUCAACUU GAAAAAGUGG CACCGAGUCG GUGCUUCG
MassTheoretical: 36 kDa
SourceSpecies: Streptococcus pyogenes (bacteria) / Strain: serotype M1

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.25 mg/ml
Buffer solution: 30mM Tris 8.0, 150mM NaCl, 20mM EDTA, 5mM DTT and 0.1% glycerol
pH: 8
Support film4/2 C-flat grids with a thin-layer of carbon over the holes
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 100 K / Humidity: 100 %
Method: Grids were rapidly plunged into liquid ethane using an FEI Vitrobot MarkIV maintained at 4 degrees C after being blotted for 4-4.5 seconds with a blotting force of 15-20.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: Jun 29, 2015
Details: Data acquired using Leginon. We collected a 6 s exposure fractionated into 20, 300 ms frames with a dose of 8 electrons per square Angstrom per second.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 48 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 29000 X (nominal)
Astigmatism: Objective astigmatism was corrected at 210,000 times magnification
Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 2000 - 4500 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 5600

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 135000
3D reconstructionAlgorithm: 3D Autorefine / Software: Relion / CTF correction: CTFFind3 / Resolution: 4.5 Å / Resolution method: FSC 0.143, gold-standard

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