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- PDB-8k0p: The Anoxybacillus pushchinoensis ORF-less Group IIC Intron HYER1 ... -

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Basic information

Entry
Database: PDB / ID: 8k0p
TitleThe Anoxybacillus pushchinoensis ORF-less Group IIC Intron HYER1 at symmetric apo state
ComponentsRNA (542-MER)
KeywordsRNA / ORF-less Group IIC intron / ribozymes
Function / homologyRNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesAnoxybacillus pushchinoensis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.99 Å
AuthorsZhu, H.Z. / Liu, J.J.G.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Science / Year: 2024
Title: Hydrolytic endonucleolytic ribozyme (HYER) is programmable for sequence-specific DNA cleavage.
Authors: Zi-Xian Liu / Shouyue Zhang / Han-Zhou Zhu / Zhi-Hang Chen / Yun Yang / Long-Qi Li / Yuan Lei / Yun Liu / Dan-Yuan Li / Ao Sun / Cheng-Ping Li / Shun-Qing Tan / Gao-Li Wang / Jie-Yi Shen / ...Authors: Zi-Xian Liu / Shouyue Zhang / Han-Zhou Zhu / Zhi-Hang Chen / Yun Yang / Long-Qi Li / Yuan Lei / Yun Liu / Dan-Yuan Li / Ao Sun / Cheng-Ping Li / Shun-Qing Tan / Gao-Li Wang / Jie-Yi Shen / Shuai Jin / Caixia Gao / Jun-Jie Gogo Liu /
Abstract: Ribozymes are catalytic RNAs with diverse functions including self-splicing and polymerization. This work aims to discover natural ribozymes that behave as hydrolytic and sequence-specific DNA ...Ribozymes are catalytic RNAs with diverse functions including self-splicing and polymerization. This work aims to discover natural ribozymes that behave as hydrolytic and sequence-specific DNA endonucleases, which could be repurposed as DNA manipulation tools. Focused on bacterial group II-C introns, we found that many systems without intron-encoded protein propagate multiple copies in their resident genomes. These introns, named HYdrolytic Endonucleolytic Ribozymes (HYERs), cleaved RNA, single-stranded DNA, bubbled double-stranded DNA (dsDNA), and plasmids in vitro. HYER1 generated dsDNA breaks in the mammalian genome. Cryo-electron microscopy analysis revealed a homodimer structure for HYER1, where each monomer contains a Mg-dependent hydrolysis pocket and captures DNA complementary to the target recognition site (TRS). Rational designs including TRS extension, recruiting sequence insertion, and heterodimerization yielded engineered HYERs showing improved specificity and flexibility for DNA manipulation.
History
DepositionJul 10, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 15, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: RNA (542-MER)
C: RNA (542-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)410,3676
Polymers410,2702
Non-polymers974
Water00
1


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

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Components

#1: RNA chain RNA (542-MER)


Mass: 205134.922 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Anoxybacillus pushchinoensis (bacteria)
Production host: Escherichia coli (E. coli)
#2: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN

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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: The Anoxybacillus pushchinoensis ORF-less Group IIC Intron HYER1 at symmetric apo state
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Anoxybacillus pushchinoensis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
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 FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 2.99 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1276808 / Symmetry type: POINT

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