|Entry||Database: EMDB / ID: 3276|
|Title||Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage|
|Map data||Reconstruction of sgRNA-bound Cas9|
|Sample||Cas9 bound to single guide-RNA|
|Keywords||CRISPR-Cas / Cas9 / genome editing|
|Function / homology||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 ...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
|Source||Streptococcus pyogenes (bacteria)|
|Method||single particle reconstruction / cryo EM / 4.5 Å resolution|
|Authors||Jiang F / Taylor DW / Chen JS / Kornfeld JE / Zhou K / Thompson AJ / Nogales E / Doudna JA|
|Citation||Journal: 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.
|Date||Deposition: Dec 10, 2015 / Header (metadata) release: Jan 27, 2016 / Map release: Jan 27, 2016 / Last update: Mar 9, 2016|
|Structure viewer||EM map: |
Downloads & links
|File||emd_3276.map.gz (map file in CCP4 format, 93313 KB)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.01 Å|
CCP4 map header:
-Entire Cas9 bound to single guide-RNA
|Entire||Name: Cas9 bound to single guide-RNA / Number of components: 2|
|Mass||Theoretical: 194 kDa|
-Component #1: protein, CRISPR-associated endonuclease Cas9/Csn1
|Protein||Name: CRISPR-associated endonuclease Cas9/Csn1 / a.k.a: Cas9 / Oligomeric Details: Monomer / Number of Copies: 1 / Recombinant expression: Yes|
|Mass||Theoretical: 158 kDa|
|Source||Species: Streptococcus pyogenes (bacteria) / Strain: serotype M1|
|Source (engineered)||Expression System: Escherichia coli (E. coli) / Strain: BL21|
|External references||Gene 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
-Component #2: nucleic-acid, single guide-RNA
|Nucleic-acid||Name: single guide-RNA / a.k.a: sgRNASubgenomic mRNA / Class: RNA / Structure: OTHER / Synthetic: Yes|
GGCGCAUAAA GAUGAGACGC GUUUUAGAGC UAUGCUGUUU UGAAAAAAAC AGCAUAGCAA GUUAAAAUAA GGCUAGUCCG UUAUCAACUU GAAAAAGUGG CACCGAGUCG GUGCUUCG
|Mass||Theoretical: 36 kDa|
|Source||Species: Streptococcus pyogenes (bacteria) / Strain: serotype M1|
|Specimen||Specimen state: particle / Method: cryo EM|
|Sample solution||Specimen conc.: 0.25 mg/ml|
Buffer solution: 30mM Tris 8.0, 150mM NaCl, 20mM EDTA, 5mM DTT and 0.1% glycerol
|Support film||4/2 C-flat grids with a thin-layer of carbon over the holes|
|Vitrification||Instrument: 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.
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: 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 gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 48 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 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 Holder||Model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Camera||Detector: GATAN K2 SUMMIT (4k x 4k)|
|Image acquisition||Number of digital images: 5600|
|Processing||Method: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 135000|
|3D reconstruction||Algorithm: 3D Autorefine / Software: Relion / CTF correction: CTFFind3 / Resolution: 4.5 Å / Resolution method: FSC 0.143, gold-standard|
-Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)
EMDB accession codes are about to change! (news from PDBe EMDB page)
- The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force. (see PDBe EMDB page)
- The EM Navigator/Yorodumi systems omit the EMD- prefix.
Related info.: Q: What is "EMD"? / ID/Accession-code notation in Yorodumi/EM Navigator
-Jul 12, 2017. Major update of PDB
Major update of PDB
- wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary. This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated. See below links for details.
- In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software). Now, EM Navigator and Yorodumi are based on the updated data.
+Jun 16, 2017. Omokage search with filter
Omokage search with filter
- Result of Omokage search can be filtered by keywords and the database types
Related info.: Omokage search
+Sep 15, 2016. EM Navigator & Yorodumi renewed
EM Navigator & Yorodumi renewed
- New versions of EM Navigator and Yorodumi started
Related info.: Changes in new EM Navigator and Yorodumi
+Aug 31, 2016. New EM Navigator & Yorodumi
New EM Navigator & Yorodumi
- In 15th Sep 2016, the development versions of EM Navigator and Yorodumi will replace the official versions.
- Current version will continue as 'legacy version' for some time.
Related info.: Changes in new EM Navigator and Yorodumi / EM Navigator / Yorodumi
Thousand views of thousand structures
- Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
- This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
Related info.: EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi