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- PDB-8h2h: Cryo-EM structure of a Group II Intron Complexed with its Reverse... -

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Basic information

Entry
Database: PDB / ID: 8h2h
TitleCryo-EM structure of a Group II Intron Complexed with its Reverse Transcriptase
Components
  • Group II intron-encoded protein LtrA
  • LtrB
  • RNA (5'-R(P*CP*AP*CP*AP*UP*CP*CP*AP*UP*AP*AP*C)-3')
KeywordsRNA BINDING PROTEIN/RNA / intron / RNA BINDING PROTEIN-RNA COMPLEX
Function / homology
Function and homology information


intron homing / mRNA processing / RNA-directed DNA polymerase / RNA-directed DNA polymerase activity / endonuclease activity / Hydrolases; Acting on ester bonds
Similarity search - Function
Domain X / : / Type II intron maturase / AI2M/AI1M-like, HNH endonuclease / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase domain / Reverse transcriptase (RT) catalytic domain profile. / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
RNA / RNA (> 10) / RNA (> 100) / Group II intron-encoded protein LtrA
Similarity search - Component
Biological speciesLactococcus lactis (lactic acid bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsLiu, N. / Dong, X.L. / Qu, G.S. / Wang, J. / Wang, H.W. / Belfort, M.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2022
Title: Functionalized graphene grids with various charges for single-particle cryo-EM.
Authors: Ye Lu / Nan Liu / Yongbo Liu / Liming Zheng / Junhao Yang / Jia Wang / Xia Jia / Qinru Zi / Hailin Peng / Yu Rao / Hong-Wei Wang /
Abstract: A major hurdle for single particle cryo-EM in structural determination lies in the specimen preparation impaired by the air-water interface (AWI) and preferential particle-orientation problems. In ...A major hurdle for single particle cryo-EM in structural determination lies in the specimen preparation impaired by the air-water interface (AWI) and preferential particle-orientation problems. In this work, we develop functionalized graphene grids with various charges via a dediazoniation reaction for cryo-EM specimen preparation. The graphene grids are paraffin-assistant fabricated, which appear with less contaminations compared with those produced by polymer transfer method. By applying onto three different types of macromolecules, we demonstrate that the high-yield charged graphene grids bring macromolecules away from the AWI and enable adjustable particle-orientation distribution for more robust single particle cryo-EM structural determination.
History
DepositionOct 6, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 23, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: LtrB
B: RNA (5'-R(P*CP*AP*CP*AP*UP*CP*CP*AP*UP*AP*AP*C)-3')
D: Group II intron-encoded protein LtrA


Theoretical massNumber of molelcules
Total (without water)365,3903
Polymers365,3903
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: RNA chain LtrB


Mass: 291363.969 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactococcus lactis (lactic acid bacteria)
Production host: Lactococcus lactis (lactic acid bacteria)
#2: RNA chain RNA (5'-R(P*CP*AP*CP*AP*UP*CP*CP*AP*UP*AP*AP*C)-3')


Mass: 3739.312 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactococcus lactis (lactic acid bacteria)
Production host: Lactococcus lactis (lactic acid bacteria)
#3: Protein Group II intron-encoded protein LtrA


Mass: 70286.664 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactococcus lactis (lactic acid bacteria)
Gene: ltrA, matR / Production host: Lactococcus lactis (lactic acid bacteria)
References: UniProt: P0A3U0, RNA-directed DNA polymerase, Hydrolases; Acting on ester bonds

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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: a Group II Intron Complexed with its Reverse Transcriptase
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Lactococcus lactis (lactic acid bacteria)
Source (recombinant)Organism: Lactococcus lactis (lactic acid bacteria)
Buffer solutionpH: 8
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: 2500 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 399660 / Symmetry type: POINT

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