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- PDB-5xwy: Electron cryo-microscopy structure of LbuCas13a-crRNA binary complex -

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Basic information

Entry
Database: PDB / ID: 5xwy
TitleElectron cryo-microscopy structure of LbuCas13a-crRNA binary complex
Components
  • A type VI-A CRISPR-Cas RNA-guided RNA ribonuclease, Cas13a
  • RNA (59-MER)
KeywordsRNA BINDING PROTEIN/RNA / Cas13a / CRISPR / RNA BINDING PROTEIN-RNA COMPLEX
Function / homologydefense response to virus / endonuclease activity / Acting on Ester Bonds / RNA binding / CRISPR-associated endoribonuclease Cas13a
Function and homology information
Specimen sourceLeptotrichia buccalis (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.2 Å resolution
AuthorsZhang, X. / Wang, Y. / Ma, J. / Liu, L. / Li, X. / Li, Z. / You, L. / Wang, J. / Wang, M.
CitationJournal: 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
SummaryFull reportAbout validation report
DateDeposition: Jun 30, 2017 / Release: Sep 13, 2017

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Structure visualization

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Assembly

Deposited unit
A: A type VI-A CRISPR-Cas RNA-guided RNA ribonuclease, Cas13a
B: RNA (59-MER)


Theoretical massNumber of molelcules
Total (without water)157,4252
Polyers157,4252
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area (Å2)11180
ΔGint (kcal/M)-71
Surface area (Å2)56350

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Components

#1: Protein/peptide A type VI-A CRISPR-Cas RNA-guided RNA ribonuclease, Cas13a


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 RNA (59-MER)


Mass: 18869.348 Da / Num. of mol.: 1 / Source: (synth.) synthetic construct (others)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: LbuCas13a-crRNA binary complex / Type: COMPLEX / Entity ID: 1, 2 / Source: RECOMBINANT
Source (natural)Organism: Leptotrichia buccalis (bacteria) / Strain: ATCC 14201
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: dev_2411: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 238758 / Symmetry type: POINT
Refine
Refine IDHighest resolution
13.20
ELECTRON MICROSCOPY
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01010528
ELECTRON MICROSCOPYf_angle_d1.20114384
ELECTRON MICROSCOPYf_dihedral_angle_d11.7718615
ELECTRON MICROSCOPYf_chiral_restr0.0631619
ELECTRON MICROSCOPYf_plane_restr0.0051642

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