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Yorodumi- EMDB-5860: Structures of Cas9 endonucleases reveal RNA-mediated conformation... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-5860 | |||||||||
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Title | Structures of Cas9 endonucleases reveal RNA-mediated conformational activation | |||||||||
Map data | Reconstruction of Cas9 loaded with crRNA:tracrRNA and bound to target DNA duplex | |||||||||
Sample |
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Keywords | CRISPR-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 | |||||||||
Biological species | Enterobacteria phage lambda (virus) / Streptococcus pyogenes (bacteria) | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 21.0 Å | |||||||||
Authors | Jinek 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 | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_5860.map.gz | 10.6 MB | EMDB map data format | |
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Header (meta data) | emd-5860-v30.xml emd-5860.xml | 15.2 KB 15.2 KB | Display Display | EMDB header |
Images | emd_5860.png | 214 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-5860 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-5860 | HTTPS FTP |
-Related structure data
Related structure data | 5858C 5859C 4cmpC 4cmqC 4ogcC 4ogeC C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_5860.map.gz / Format: CCP4 / Size: 11.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of Cas9 loaded with crRNA:tracrRNA and bound to target DNA duplex | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.18 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Cas9 loaded with crRNA:tracrRNA and bound to target DNA duplex
Entire | Name: Cas9 loaded with crRNA:tracrRNA and bound to target DNA duplex |
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Components |
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-Supramolecule #1000: Cas9 loaded with crRNA:tracrRNA and bound to target DNA duplex
Supramolecule | Name: Cas9 loaded with crRNA:tracrRNA and bound to target DNA duplex type: sample / ID: 1000 Oligomeric state: one Cas9 binds to one crRNA:tracRNA heteroduplex and one target DNA duplex Number unique components: 5 |
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Molecular weight | Theoretical: 230 KDa |
-Macromolecule #1: 55 bp DNA duplex target strand
Macromolecule | Name: 55 bp DNA duplex target strand / type: dna / ID: 1 Details: annealed with 55 bp DNA duplex non-target strand to form a double helix Classification: DNA / Structure: DOUBLE HELIX / Synthetic?: Yes |
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Source (natural) | Organism: Enterobacteria phage lambda (virus) |
Molecular weight | Theoretical: 17 KDa |
Sequence | String: GCTCAATTTT GACAGCCCAC ATGGCATTCC ACTTATCACT GGCATCCTTC CACTC |
-Macromolecule #5: 55 bp DNA duplex non-target strand
Macromolecule | Name: 55 bp DNA duplex non-target strand / type: dna / ID: 5 Details: annealed with 55 bp DNA duplex target strand to form a double helix Classification: DNA / Structure: DOUBLE HELIX / Synthetic?: Yes |
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Source (natural) | Organism: Enterobacteria phage lambda (virus) |
Molecular weight | Theoretical: 17 KDa |
Sequence | String: GAGTGGAAGG ATGCCAGTGA TAAGTGGAAT GCCATGTGGG CTGTCAAAAT TGAGC |
-Macromolecule #2: CRISPR-associated endonuclease Cas9
Macromolecule | Name: CRISPR-associated endonuclease Cas9 / type: protein_or_peptide / ID: 2 / Name.synonym: Cas9 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes |
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Source (natural) | Organism: Streptococcus pyogenes (bacteria) |
Molecular weight | Theoretical: 159 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: Rosetta 2(DE3) |
Sequence | UniProtKB: 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 |
-Macromolecule #3: CRISPR RNA
Macromolecule | Name: CRISPR RNA / type: rna / ID: 3 / Name.synonym: crRNA Details: Forms complete guide RNA by hybridization with tracrRNA. Classification: OTHER / Structure: OTHER / Synthetic?: Yes |
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Source (natural) | Organism: Streptococcus pyogenes (bacteria) |
Molecular weight | Theoretical: 14 KDa |
Sequence | String: GUGAUAAGUG GAAUGCCAUG GUUUUAGAGC UAUGCUGUUU UG |
-Macromolecule #4: trans-activating CRISPR RNA
Macromolecule | Name: trans-activating CRISPR RNA / type: rna / ID: 4 / Name.synonym: tracrRNA Details: Forms complete guide RNA by hybridization with crRNA. Made using T7 RNA polymerase. Classification: OTHER / Structure: OTHER / Synthetic?: No |
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Source (natural) | Organism: Streptococcus pyogenes (bacteria) |
Molecular weight | Theoretical: 24 KDa |
Sequence | String: GGACAGCAUA GCAAGUUAAA AUAAGGCUAG UCCGUUAUCA ACUUGAAAAA GUGGCACCGA GUCGGUGCUU UUU |
-Experimental details
-Structure determination
Method | negative staining |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.01 mg/mL |
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Buffer | pH: 7.5 Details: 20 mM Tris-Cl pH 7.5, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, 5% glycerol |
Staining | Type: 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. |
Grid | Details: glow-discharged 400 mesh continuous carbon grids |
Vitrification | Cryogen name: NONE / Instrument: OTHER |
-Electron microscopy
Microscope | FEI TECNAI 20 |
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Electron beam | Acceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: -1.4 µm / Nominal defocus min: -0.4 µm / Nominal magnification: 100000 |
Sample stage | Specimen holder: Room temperature single tilt / Specimen holder model: SIDE ENTRY, EUCENTRIC |
Temperature | Average: 78 K |
Alignment procedure | Legacy - Astigmatism: Objective astigmatism was corrected at 210,000 times magnification Legacy - Electron beam tilt params: 0 |
Details | Data acquired using Leginon. |
Date | Apr 8, 2013 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 213 / Average electron dose: 20 e/Å2 |
Tilt angle min | 0 |
Tilt angle max | 0 |
-Image processing
CTF correction | Details: whole micrograph |
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Final reconstruction | Algorithm: 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: 24435 |
Details | Particles were selected using template based picking in Appion. |