|Entry||Database: EMDB / ID: 6777|
|Title||Electron cryo-microscopy structure of LbuCas13a-crRNA binary complex|
|Map data||Electron cryo-microscopy structure of LbuCas13a-crRNA binary complex|
|Sample||LbuCas13a-crRNA binary complex|
|Function / homology||defense response to virus / endonuclease activity / Acting on Ester Bonds / RNA binding / CRISPR-associated endoribonuclease Cas13a|
Function and homology information
|Source||Leptotrichia buccalis (bacteria)|
|Method||single particle reconstruction / cryo EM / 3.2 Å resolution|
|Authors||Zhang X / Wang Y|
|Citation||Journal: Cell / Year: 2017|
Title: The Molecular Architecture for RNA-Guided RNA Cleavage by Cas13a.
Authors: Liang Liu / Xueyan Li / Jun Ma / Zongqiang Li / Lilan You / Jiuyu Wang / Min Wang / Xinzheng Zhang / Yanli Wang
Abstract: Cas13a, a type VI-A CRISPR-Cas RNA-guided RNA ribonuclease, degrades invasive RNAs targeted by CRISPR RNA (crRNA) and has potential applications in RNA technology. To understand how Cas13a ...Cas13a, a type VI-A CRISPR-Cas RNA-guided RNA ribonuclease, degrades invasive RNAs targeted by CRISPR RNA (crRNA) and has potential applications in RNA technology. To understand how Cas13a is activated to cleave RNA, we have determined the crystal structure of Leptotrichia buccalis (Lbu) Cas13a bound to crRNA and its target RNA, as well as the cryo-EM structure of the LbuCas13a-crRNA complex. The crRNA-target RNA duplex binds in a positively charged central channel of the nuclease (NUC) lobe, and Cas13a protein and crRNA undergo a significant conformational change upon target RNA binding. The guide-target RNA duplex formation triggers HEPN1 domain to move toward HEPN2 domain, activating the HEPN catalytic site of Cas13a protein, which subsequently cleaves both single-stranded target and collateral RNAs in a non-specific manner. These findings reveal how Cas13a of type VI CRISPR-Cas systems defend against RNA phages and set the stage for its development as a tool for RNA manipulation.
|Validation Report||PDB-ID: 5xwy|
SummaryFull reportAbout validation report
|Date||Deposition: Jun 30, 2017 / Header (metadata) release: Sep 13, 2017 / Map release: Sep 13, 2017 / Last update: Sep 13, 2017|
|Structure viewer||EM map: |
Downloads & links
|File||emd_6777.map.gz (map file in CCP4 format, 32001 KB)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 0.82 Å|
CCP4 map header:
-Entire LbuCas13a-crRNA binary complex
|Entire||Name: LbuCas13a-crRNA binary complex / Number of components: 3|
-Component #1: protein, LbuCas13a-crRNA binary complex
|Protein||Name: LbuCas13a-crRNA binary complex / Recombinant expression: No|
|Source||Species: Leptotrichia buccalis (bacteria) / Strain: ATCC 14201|
|Source (engineered)||Expression System: Escherichia coli (E. coli)|
-Component #2: protein, A type VI-A CRISPR-Cas RNA-guided RNA ribonuclease, Cas13a
|Protein||Name: A type VI-A CRISPR-Cas RNA-guided RNA ribonuclease, Cas13a|
Recombinant expression: No
|Mass||Theoretical: 138.555156 kDa|
|Source (engineered)||Expression System: Leptotrichia buccalis (strain ATCC 14201 / DSM 1135 / JCM 12969 / NCTC 10249 / C-1013-b) (bacteria)|
Strain: ATCC 14201 / DSM 1135 / JCM 12969 / NCTC 10249 / C-1013-b
-Component #3: nucleic-acid, RNA (59-MER)
|Nucleic-acid||Name: RNA (59-MER) / Class: RNA / Structure: OTHER / Synthetic: No|
GGACCACCCC AAAAAUGAAG GGGACUAAAA CACAAAUCUA UCUGAAUAAA CUCUUCUUC
|Mass||Theoretical: 18.869348 kDa|
|Specimen||Specimen state: particle / Method: cryo EM|
|Sample solution||pH: 7.5|
|Vitrification||Cryogen name: ETHANE|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 60 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Imaging mode: BRIGHT FIELD|
|Specimen Holder||Model: OTHER|
|Camera||Detector: GATAN K2 (4k x 4k)|
|Processing||Method: single particle reconstruction / Number of projections: 238758|
|3D reconstruction||Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF|
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