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- PDB-8e27: RNA-free Human Dis3L2 -

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Basic information

Entry
Database: PDB / ID: 8.0E+27
TitleRNA-free Human Dis3L2
ComponentsDIS3-like exonuclease 2
KeywordsRNA BINDING PROTEIN/RNA / 3'-5' exonuclease / RNA-free exonuclease / human exonuclease / RNA BIND PROTEIN-RNA complex / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


polyuridylation-dependent mRNA catabolic process / miRNA catabolic process / mitotic sister chromatid separation / : / Z-decay: degradation of maternal mRNAs by zygotically expressed factors / poly(U) RNA binding / stem cell population maintenance / : / mRNA catabolic process / RNA nuclease activity ...polyuridylation-dependent mRNA catabolic process / miRNA catabolic process / mitotic sister chromatid separation / : / Z-decay: degradation of maternal mRNAs by zygotically expressed factors / poly(U) RNA binding / stem cell population maintenance / : / mRNA catabolic process / RNA nuclease activity / P-body / mitotic cell cycle / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / 3'-5'-RNA exonuclease activity / negative regulation of cell population proliferation / cell division / magnesium ion binding / cytoplasm / cytosol
Similarity search - Function
DIS3-like exonuclease 2 / DIS3-like exonuclease 2, C-terminal / DIS3-like exonuclease 2 C terminal / Rrp44-like cold shock domain / Rrp44-like cold shock domain / Dis3-like cold-shock domain 2 / Dis3-like cold-shock domain 2 (CSD2) / Ribonuclease II/R, conserved site / Ribonuclease II family signature. / Ribonuclease II/R ...DIS3-like exonuclease 2 / DIS3-like exonuclease 2, C-terminal / DIS3-like exonuclease 2 C terminal / Rrp44-like cold shock domain / Rrp44-like cold shock domain / Dis3-like cold-shock domain 2 / Dis3-like cold-shock domain 2 (CSD2) / Ribonuclease II/R, conserved site / Ribonuclease II family signature. / Ribonuclease II/R / RNB domain / RNB / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
DIS3-like exonuclease 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsMeze, K. / Thomas, D.R. / Joshua-Tor, L.
Funding support United States, 2items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM114147 United States
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: A shape-shifting nuclease unravels structured RNA.
Authors: Katarina Meze / Armend Axhemi / Dennis R Thomas / Ahmet Doymaz / Leemor Joshua-Tor /
Abstract: RNA turnover pathways ensure appropriate gene expression levels by eliminating unwanted transcripts. Dis3-like 2 (Dis3L2) is a 3'-5' exoribonuclease that plays a critical role in human development. ...RNA turnover pathways ensure appropriate gene expression levels by eliminating unwanted transcripts. Dis3-like 2 (Dis3L2) is a 3'-5' exoribonuclease that plays a critical role in human development. Dis3L2 independently degrades structured substrates, including coding and noncoding 3' uridylated RNAs. While the basis for Dis3L2's substrate recognition has been well characterized, the mechanism of structured RNA degradation by this family of enzymes is unknown. We characterized the discrete steps of the degradation cycle by determining cryogenic electron microscopy structures representing snapshots along the RNA turnover pathway and measuring kinetic parameters for RNA processing. We discovered a dramatic conformational change that is triggered by double-stranded RNA (dsRNA), repositioning two cold shock domains by 70 Å. This movement exposes a trihelix linker region, which acts as a wedge to separate the two RNA strands. Furthermore, we show that the trihelix linker is critical for dsRNA, but not single-stranded RNA, degradation. These findings reveal the conformational plasticity of Dis3L2 and detail a mechanism of structured RNA degradation.
History
DepositionAug 14, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 1, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 8, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Mar 29, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Jun 12, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / em_3d_fitting_list
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DIS3-like exonuclease 2


Theoretical massNumber of molelcules
Total (without water)96,5431
Polymers96,5431
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry, The purified product was confirmed by mass spectrometry and gel filtration. The structure is highly similar to that of the mouse Dis3L2 (PDB: 4PMW)
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein DIS3-like exonuclease 2 / hDIS3L2


Mass: 96543.320 Da / Num. of mol.: 1 / Mutation: D391N
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DIS3L2, FAM6A / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: Q8IYB7, Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters

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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: Inactive RNA-free HsDis3L2 (D391N) / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.1 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHEPES1
2150 mMsodium chlorideNaCl1
35 mMDithiothreitolDTT1
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 295 K

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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 magnification: 160000 X / Nominal defocus max: 2600 nm / Nominal defocus min: 700 nm / Calibrated defocus min: 500 nm / Calibrated defocus max: 2800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 80 K / Temperature (min): 80 K
Image recordingAverage exposure time: 5 sec. / Electron dose: 51.75 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4095
Image scansSampling size: 5 µm / Width: 3840 / Height: 3712 / Movie frames/image: 25 / Used frames/image: 1-25

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Processing

SoftwareName: PHENIX / Version: 1.18_3855: / Classification: refinement
EM software
IDNameVersionCategory
1Warp1.0.8particle selection
2EPU2.5image acquisition
4Warp1.0.8CTF correction
7Cootmodel fitting
9cryoSPARC3.0.0initial Euler assignment
10cryoSPARC3.0.0final Euler assignment
11cryoSPARCv3.0.0classification
12cryoSPARC3.0.03D reconstruction
13PHENIX1.18-3855-000model refinement
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 990287
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 162793 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: Chain A of PDB 4PMW (MmDis3L2) was fit into the map via rigid body fitting. Residues were then mutated to match the human protein and manual building done to correct for differences. Next ...Details: Chain A of PDB 4PMW (MmDis3L2) was fit into the map via rigid body fitting. Residues were then mutated to match the human protein and manual building done to correct for differences. Next rounds of refinement and building were done in PHENIX and Coot, respectively, until the final structure was obtained.
Atomic model buildingPDB-ID: 4PMW

4pmw


Pdb chain-ID: A / Accession code: 4PMW / Source name: PDB / Type: experimental model

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