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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: EMD-3276
TitleStructures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage
Map dataReconstruction of sgRNA-bound Cas9
Sample
  • Sample: Cas9 bound to single guide-RNA
  • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1
  • RNA: single guide-RNA
KeywordsCRISPR-Cas / Cas9 / genome editing
Function / homology
Function and homology information


: / maintenance of CRISPR repeat elements / 3'-5' exonuclease activity / DNA endonuclease activity / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
: / CRISPR-associated endonuclease Cas9, bridge helix / CRISPR-associated endonuclease Cas9, bridge helix / Bridge helix of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 ...: / CRISPR-associated endonuclease Cas9, bridge helix / CRISPR-associated endonuclease Cas9, bridge helix / Bridge helix of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9 / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.5 Å
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.
History
DepositionDec 10, 2015-
Header (metadata) releaseJan 27, 2016-
Map releaseJan 27, 2016-
UpdateMar 9, 2016-
Current statusMar 9, 2016Processing site: PDBe / Status: Released

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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

emd_3276.png

Downloads & links

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Map

FileDownload / File: emd_3276.map.gz / Format: CCP4 / Size: 89 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of sgRNA-bound Cas9
Projections & slices

Image control

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

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.01 / Movie #1: 0.01
Minimum - Maximum-0.02166877 - 0.05766536
Average (Standard dev.)0.00000727 (±0.00168026)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 290.88 Å
α=β=γ: 90.0 °

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
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
Components
  • Sample: Cas9 bound to single guide-RNA
  • Protein or peptide: CRISPR-associated endonuclease Cas9/Csn1
  • RNA: single guide-RNA

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Supramolecule #1000: Cas9 bound to single guide-RNA

SupramoleculeName: Cas9 bound to single guide-RNA / type: sample / ID: 1000 / Number unique components: 2
Molecular weightTheoretical: 194 KDa

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Macromolecule #1: CRISPR-associated endonuclease Cas9/Csn1

MacromoleculeName: CRISPR-associated endonuclease Cas9/Csn1 / type: protein_or_peptide / ID: 1 / Name.synonym: Cas9 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Streptococcus pyogenes (bacteria) / Strain: serotype M1
Molecular weightTheoretical: 158 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21
SequenceUniProtKB: CRISPR-associated endonuclease Cas9/Csn1
GO: maintenance of CRISPR repeat elements, defense response to virus, GO: 0090305, DNA binding, RNA binding, endonuclease activity
InterPro: CRISPR-associated endonuclease Cas9, CRISPR-associated endonuclease Cas9, bridge helix, CRISPR-associated endonuclease Cas9, PAM-interacting domain, CRISPR-associated endonuclease Cas9, REC ...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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Macromolecule #2: single guide-RNA

MacromoleculeName: single guide-RNA / type: rna / ID: 2 / Name.synonym: sgRNA / Classification: OTHER / Structure: OTHER / Synthetic?: Yes
Source (natural)Organism: Streptococcus pyogenes (bacteria) / Strain: serotype M1
Molecular weightTheoretical: 36 KDa
SequenceString:
GGCGCAUAAA GAUGAGACGC GUUUUAGAGC UAUGCUGUUU UGAAAAAAAC AGCAUAGCAA GUUAAAAUAA GGCUAGUCCG UUAUCAACUU GAAAAAGUGG CACCGAGUCG GUGCUUCG

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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: 8
Details: 30mM Tris 8.0, 150mM NaCl, 20mM EDTA, 5mM DTT and 0.1% glycerol
GridDetails: 4/2 C-flat grids with a thin-layer of carbon over the holes
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 100 K / Instrument: FEI VITROBOT MARK IV
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

MicroscopeFEI TITAN KRIOS
Alignment procedureLegacy - Astigmatism: Objective astigmatism was corrected at 210,000 times magnification
DetailsData 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.
DateJun 29, 2015
Image recordingCategory: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number real images: 5600 / Average electron dose: 48 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 29000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: CTFFind3
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 4.5 Å / Resolution method: OTHER / Software - Name: Relion / Number images used: 135000

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