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- PDB-8wce: Cryo-EM structure of a protein-RNA complex -

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Basic information

Entry
Database: PDB / ID: 8wce
TitleCryo-EM structure of a protein-RNA complex
Components
  • RNA (31-MER)
  • RNA (66-MER)
  • Uncharacterized protein
KeywordsRNA BINDING PROTEIN/RNA / RNA / ribonuclease / RNA BINDING PROTEIN-RNA complex
Function / homologyRNA / RNA (> 10)
Function and homology information
Biological speciesunidentified (others)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.09 Å
AuthorsLi, Z.
Funding support China, 1items
OrganizationGrant numberCountry
Not fundedNA China
CitationJournal: Nucleic Acids Res / Year: 2024
Title: Molecular mechanism for target RNA recognition and cleavage of Cas13h.
Authors: Fugen Chen / Chendi Zhang / Jialin Xue / Feng Wang / Zhuang Li /
Abstract: RNA-targeting type VI CRISPR-Cas effectors are widely used in RNA applications. Cas13h is a recently identified subtype of Cas13 ribonuclease, with strong RNA cleavage activity and robust in vivo RNA ...RNA-targeting type VI CRISPR-Cas effectors are widely used in RNA applications. Cas13h is a recently identified subtype of Cas13 ribonuclease, with strong RNA cleavage activity and robust in vivo RNA knockdown efficiency. However, little is known regarding its biochemical properties and working mechanisms. Biochemical characterization of Cas13h1 indicated that it lacks in vitro pre-crRNA processing activity and adopts a central seed. The cleavage activity of Cas13h1 is enhanced by a R(G/A) 5'-PFS, and inhibited by tag:anti-tag RNA pairing. We determined the structures of Cas13h1-crRNA binary complex at 3.1 Å and Cas13h1-crRNA-target RNA ternary complex at 3.0 Å. The ternary complex adopts an elongated architecture, and encodes a nucleotide-binding pocket within Helical-2 domain to recognize the guanosine at the 5'-end of the target RNA. Base pairing between crRNA guide and target RNA disrupts Cas13h1-guide interactions, leading to dramatic movement of HEPN domains. Upon target RNA engagement, Cas13h1 adopts a complicated activation mechanism, including separation of HEPN catalytic residues and destabilization of the active site loop and NTD domain, to get activated. Collectively, these insights expand our understanding into Cas13 effectors.
History
DepositionSep 11, 2023Deposition site: PDBJ / Processing site: PDBC
Revision 1.0May 22, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Uncharacterized protein
G: RNA (66-MER)
T: RNA (31-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)162,2375
Polymers162,1883
Non-polymers492
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Uncharacterized protein


Mass: 130957.328 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) unidentified (others) / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: RNA chain RNA (66-MER)


Mass: 21257.666 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: RNA chain RNA (31-MER)


Mass: 9973.005 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: protein-RNA complex / Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: unidentified (others)
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
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 54 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 3.09 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 291461 / Symmetry type: POINT

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