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- EMDB-5859: Structures of Cas9 endonucleases reveal RNA-mediated conformation... -

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Basic information

Entry
Database: EMDB / ID: EMD-5859
TitleStructures of Cas9 endonucleases reveal RNA-mediated conformational activation
Map dataReconstruction of Cas9 loaded with crRNA:tracrRNA
Sample
  • Sample: Cas9 loaded with crRNA:tracrRNA
  • Protein or peptide: CRISPR-associated endonuclease Cas9
  • RNA: CRISPR RNA
  • RNA: trans-activating CRISPR RNA
KeywordsCRISPR-Cas / crRNA / tracrRNA / Cas9 / genome engineering / bacterial immunity
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / 3'-5' exonuclease activity / DNA 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, PAM-interacting domain / CRISPR-associated endonuclease Cas9, bridge helix / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / Bridge helix of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / Cas9 RuvC domain / CRISPR-associated endonuclease Cas9 ...: / : / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, bridge helix / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / Bridge helix of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / Cas9 RuvC domain / CRISPR-associated endonuclease Cas9 / HNH endonuclease / 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 / negative staining / Resolution: 21.0 Å
AuthorsJinek M / Jiang F / Taylor DW / Sternberg SH / Kaya E / Ma E / Anders C / Hauer M / Zhou K / Lin S ...Jinek M / Jiang F / Taylor DW / Sternberg SH / Kaya E / Ma E / Anders C / Hauer M / Zhou K / Lin S / Kaplan M / Iavarone AT / Charpentier E / Nogales E / Doudna JA
CitationJournal: Science / Year: 2014
Title: Structures of Cas9 endonucleases reveal RNA-mediated conformational activation.
Authors: Martin Jinek / Fuguo Jiang / David W Taylor / Samuel H Sternberg / Emine Kaya / Enbo Ma / Carolin Anders / Michael Hauer / Kaihong Zhou / Steven Lin / Matias Kaplan / Anthony T Iavarone / ...Authors: Martin Jinek / Fuguo Jiang / David W Taylor / Samuel H Sternberg / Emine Kaya / Enbo Ma / Carolin Anders / Michael Hauer / Kaihong Zhou / Steven Lin / Matias Kaplan / Anthony T Iavarone / Emmanuelle Charpentier / Eva Nogales / Jennifer A Doudna /
Abstract: Type II CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA ...Type II CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA-induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation.
History
DepositionJan 13, 2014-
Header (metadata) releaseFeb 12, 2014-
Map releaseFeb 19, 2014-
UpdateMar 26, 2014-
Current statusMar 26, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 5.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 5.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5859.png

Downloads & links

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Map

FileDownload / File: emd_5859.map.gz / Format: CCP4 / Size: 5.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of Cas9 loaded with crRNA:tracrRNA
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.8 Å/pix.
x 112 pix.
= 313.6 Å		2.8 Å/pix.
x 112 pix.
= 313.6 Å		2.8 Å/pix.
x 112 pix.
= 313.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.8 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 5.39 / Movie #1: 5.5
Minimum - Maximum-6.77789021 - 19.1176548
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-56-56-56
Dimensions112112112
Spacing112112112
CellA=B=C: 313.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.82.82.8
M x/y/z112112112
origin x/y/z0.0000.0000.000
length x/y/z313.600313.600313.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-95-75153
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS-56-56-56
NC/NR/NS112112112
D min/max/mean-6.77819.118-0.000

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Supplemental data

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Sample components

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Entire : Cas9 loaded with crRNA:tracrRNA

EntireName: Cas9 loaded with crRNA:tracrRNA
Components
  • Sample: Cas9 loaded with crRNA:tracrRNA
  • Protein or peptide: CRISPR-associated endonuclease Cas9
  • RNA: CRISPR RNA
  • RNA: trans-activating CRISPR RNA

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Supramolecule #1000: Cas9 loaded with crRNA:tracrRNA

SupramoleculeName: Cas9 loaded with crRNA:tracrRNA / type: sample / ID: 1000
Oligomeric state: one Cas9 binds to one crRNA:tracRNA heteroduplex
Number unique components: 3
Molecular weightTheoretical: 196 KDa

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Macromolecule #1: CRISPR-associated endonuclease Cas9

MacromoleculeName: CRISPR-associated endonuclease Cas9 / 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)
Molecular weightTheoretical: 159 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: Rosetta 2(DE3)
SequenceUniProtKB: CRISPR-associated endonuclease Cas9/Csn1
GO: defense response to virus, maintenance of CRISPR repeat elements, 3'-5' exonuclease activity, DNA endonuclease activity, RNA binding, DNA binding
InterPro: INTERPRO: IPR010145, INTERPRO: IPR025978, HNH nuclease

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Macromolecule #2: CRISPR RNA

MacromoleculeName: CRISPR RNA / type: rna / ID: 2 / Name.synonym: crRNA
Details: Forms complete guide RNA by hybridization with tracrRNA.
Classification: OTHER / Structure: OTHER / Synthetic?: Yes
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 14 KDa
SequenceString:
GUGAUAAGUG GAAUGCCAUG GUUUUAGAGC UAUGCUGUUU UG

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Macromolecule #3: trans-activating CRISPR RNA

MacromoleculeName: trans-activating CRISPR RNA / type: rna / ID: 3 / Name.synonym: tracrRNA
Details: Forms complete guide RNA by hybridization with crRNA. Made using T7 RNA polymerase.
Classification: OTHER / Structure: OTHER / Synthetic?: No
Source (natural)Organism: Streptococcus pyogenes (bacteria)
Molecular weightTheoretical: 24 KDa
SequenceString:
GGACAGCAUA GCAAGUUAAA AUAAGGCUAG UCCGUUAUCA ACUUGAAAAA GUGGCACCGA GUCGGUGCUU UUU

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.01 mg/mL
BufferpH: 7.5
Details: 20 mM Tris-Cl pH 7.5, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, 5% glycerol
StainingType: NEGATIVE
Details: After adsorption for 1 min, we stained the samples consecutively with six droplets of 2% (w/v) uranyl acetate solution, gently blotted off the residual stain, and air-dried the sample in a fume hood.
GridDetails: glow-discharged 400 mesh continuous carbon grids
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 20
TemperatureAverage: 78 K
Alignment procedureLegacy - Astigmatism: Objective astigmatism was corrected at 210,000 times magnification
Legacy - Electron beam tilt params: 0
DetailsData acquired using Leginon.
DateMay 10, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 297 / Average electron dose: 20 e/Å2
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.2 mm / Nominal defocus max: -1.4 µm / Nominal defocus min: -0.5 µm / Nominal magnification: 80000
Sample stageSpecimen holder: Room temperature single tilt / Specimen holder model: SIDE ENTRY, EUCENTRIC

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Image processing

DetailsParticles were selected using template based picking in Appion.
CTF correctionDetails: whole micrograph
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 21.0 Å / Resolution method: OTHER / Software - Name: EMAN2, SPARX / Details: Image pre-processing performed in Appion. / Number images used: 36982

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