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- EMDB-5858: Structures of Cas9 endonucleases reveal RNA-mediated conformation... -

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Basic information

Entry
Database: EMDB / ID: EMD-5858
TitleStructures of Cas9 endonucleases reveal RNA-mediated conformational activation
Map dataReconstruction of apo-Cas9
Sample
  • Sample: apo-Cas9
  • Protein or peptide: CRISPR-associated endonuclease Cas9
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 / CRISPR-associated endonuclease Cas9 / HNH endonuclease ...: / : / 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 / 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: 19.0 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: 5.04
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 5.04
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_5858.map.gz / Format: CCP4 / Size: 11.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of apo-Cas9
Voxel sizeX=Y=Z: 2.18 Å
Density
Contour LevelBy AUTHOR: 5.04 / Movie #1: 5.04
Minimum - Maximum-8.26776505 - 18.654703139999999
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-72-72-72
Dimensions144144144
Spacing144144144
CellA=B=C: 313.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.182.182.18
M x/y/z144144144
origin x/y/z0.0000.0000.000
length x/y/z313.920313.920313.920
α/β/γ90.00090.00090.000
start NX/NY/NZ-95-75153
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS-72-72-72
NC/NR/NS144144144
D min/max/mean-8.26818.655-0.000

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Supplemental data

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Sample components

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Entire : apo-Cas9

EntireName: apo-Cas9
Components
  • Sample: apo-Cas9
  • Protein or peptide: CRISPR-associated endonuclease Cas9

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Supramolecule #1000: apo-Cas9

SupramoleculeName: apo-Cas9 / type: sample / ID: 1000 / Oligomeric state: monomer of apo-Cas9 / Number unique components: 1
Molecular weightExperimental: 160 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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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
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: -1.5 µm / Nominal defocus min: -0.5 µm / Nominal magnification: 100000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
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.
DateMar 19, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 281 / Average electron dose: 20 e/Å2
Tilt angle min0
Tilt angle max0

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Image processing

CTF correctionDetails: whole micrograph
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 19.0 Å / Resolution method: OTHER / Software - Name: EMAN2, SPARX / Details: Image pre-processing performed in Appion. / Number images used: 18000
DetailsParticles were selected using template based picking in Appion.

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