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Yorodumi- PDB-9enf: Human pseudouridine synthase 3 (PUS3 D118A mutant) and two pre-tR... -
+Open data
-Basic information
Entry | Database: PDB / ID: 9enf | ||||||||||||
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Title | Human pseudouridine synthase 3 (PUS3 D118A mutant) and two pre-tRNA-Arg | ||||||||||||
Components |
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Keywords | RNA BINDING PROTEIN / RNA modification / pseudouridylation / tRNA / homodimer | ||||||||||||
Function / homology | Function and homology information tRNA pseudouridine38/39 synthase / tRNA pseudouridine(38/39) synthase activity / mRNA pseudouridine synthesis / tRNA pseudouridine synthesis / pseudouridine synthase activity / tRNA modification in the nucleus and cytosol / tRNA modification / RNA binding / nucleus / cytosol / cytoplasm Similarity search - Function | ||||||||||||
Biological species | Homo sapiens (human) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.97 Å | ||||||||||||
Authors | Lin, T.-Y. / Jezowski, J. / Glatt, S. | ||||||||||||
Funding support | Poland, European Union, Switzerland, 3items
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Citation | Journal: Mol Cell / Year: 2024 Title: The molecular basis of tRNA selectivity by human pseudouridine synthase 3. Authors: Ting-Yu Lin / Leon Kleemann / Jakub Jeżowski / Dominika Dobosz / Michał Rawski / Paulina Indyka / Grzegorz Ważny / Rahul Mehta / Andrzej Chramiec-Głąbik / Łukasz Koziej / Tristan Ranff ...Authors: Ting-Yu Lin / Leon Kleemann / Jakub Jeżowski / Dominika Dobosz / Michał Rawski / Paulina Indyka / Grzegorz Ważny / Rahul Mehta / Andrzej Chramiec-Głąbik / Łukasz Koziej / Tristan Ranff / Christian Fufezan / Mateusz Wawro / Jakub Kochan / Joanna Bereta / Sebastian A Leidel / Sebastian Glatt / Abstract: Pseudouridine (Ψ), the isomer of uridine, is ubiquitously found in RNA, including tRNA, rRNA, and mRNA. Human pseudouridine synthase 3 (PUS3) catalyzes pseudouridylation of position 38/39 in tRNAs. ...Pseudouridine (Ψ), the isomer of uridine, is ubiquitously found in RNA, including tRNA, rRNA, and mRNA. Human pseudouridine synthase 3 (PUS3) catalyzes pseudouridylation of position 38/39 in tRNAs. However, the molecular mechanisms by which it recognizes its RNA targets and achieves site specificity remain elusive. Here, we determine single-particle cryo-EM structures of PUS3 in its apo form and bound to three tRNAs, showing how the symmetric PUS3 homodimer recognizes tRNAs and positions the target uridine next to its active site. Structure-guided and patient-derived mutations validate our structural findings in complementary biochemical assays. Furthermore, we deleted PUS1 and PUS3 in HEK293 cells and mapped transcriptome-wide Ψ sites by Pseudo-seq. Although PUS1-dependent sites were detectable in tRNA and mRNA, we found no evidence that human PUS3 modifies mRNAs. Our work provides the molecular basis for PUS3-mediated tRNA modification in humans and explains how its tRNA modification activity is linked to intellectual disabilities. | ||||||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 9enf.cif.gz | 198.2 KB | Display | PDBx/mmCIF format |
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PDB format | pdb9enf.ent.gz | 148.1 KB | Display | PDB format |
PDBx/mmJSON format | 9enf.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 9enf_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
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Full document | 9enf_full_validation.pdf.gz | 1.2 MB | Display | |
Data in XML | 9enf_validation.xml.gz | 36 KB | Display | |
Data in CIF | 9enf_validation.cif.gz | 53.6 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/en/9enf ftp://data.pdbj.org/pub/pdb/validation_reports/en/9enf | HTTPS FTP |
-Related structure data
Related structure data | 19833MC 8okdC 9enbC 9encC 9eneC 9f9qC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 55681.207 Da / Num. of mol.: 2 / Mutation: D118A Source method: isolated from a genetically manipulated source Details: D118A single mutation / Source: (gene. exp.) Homo sapiens (human) / Gene: PUS3, FKSG32 / Production host: Spodoptera frugiperda (fall armyworm) References: UniProt: Q9BZE2, tRNA pseudouridine38/39 synthase #2: RNA chain | Mass: 30298.900 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component |
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Molecular weight | Value: 0.18 MDa / Experimental value: NO | ||||||||||||||||||||||||
Source (natural) |
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Source (recombinant) |
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Buffer solution | pH: 7.5 | ||||||||||||||||||||||||
Specimen | Conc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||
Specimen support | Grid material: COPPER / Grid type: Quantifoil R2/1 | ||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: TFS KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 900 nm / Cs: 2.7 mm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8673 |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 9139918 | ||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 381809 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: OTHER / Space: REAL | ||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Source name: AlphaFold / Type: in silico model |