|Entry||Database: PDB / ID: 5xwy|
|Title||Electron cryo-microscopy structure of LbuCas13a-crRNA binary complex|
|Keywords||RNA BINDING PROTEIN/RNA / Cas13a / CRISPR / RNA BINDING PROTEIN-RNA COMPLEX|
|Function / homology||defense response to virus / endonuclease activity / Acting on Ester Bonds / RNA binding / CRISPR-associated endoribonuclease Cas13a|
Function and homology information
|Specimen source||Leptotrichia buccalis (bacteria)|
synthetic construct (others)
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.2 Å resolution|
|Authors||Zhang, X. / Wang, Y. / Ma, J. / Liu, L. / Li, X. / Li, Z. / You, L. / Wang, J. / Wang, M.|
|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.
SummaryFull reportAbout validation report
|Date||Deposition: Jun 30, 2017 / Release: Sep 13, 2017|
|Structure viewer||Molecule: |
Downloads & links
A: A type VI-A CRISPR-Cas RNA-guided RNA ribonuclease, Cas13a
B: RNA (59-MER)
|#1: Protein/peptide|| |
Mass: 138555.156 Da / Num. of mol.: 1 / Mutation: R1048A, H1053A
Source: (gene. exp.) 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
Gene: Lebu_1799 / Production host: Escherichia coli (E. coli) / References: UniProt: C7NBY4
|#2: RNA chain|| |
Mass: 18869.348 Da / Num. of mol.: 1 / Source: (synth.) synthetic construct (others)
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / Reconstruction method: single particle reconstruction|
|Component||Name: LbuCas13a-crRNA binary complex / Type: COMPLEX / Entity ID: 1,||Source (natural)||Organism: Leptotrichia buccalis (bacteria) / Strain: ATCC 14201||Source (recombinant)||Organism: Escherichia coli (E. coli)||Buffer solution||pH: 7.5||Specimen||Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES||Vitrification||Cryogen name: ETHANE|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Microscope model: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy|
|Image recording||Electron dose: 60 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)|
|Software||Name: PHENIX / Version: dev_2411: / Classification: refinement|
|CTF correction||Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|3D reconstruction||Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 238758 / Symmetry type: POINT|
|Refine LS restraints|
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