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- PDB-8okd: Human pseudouridine synthase 3 and tRNA-Gln -

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Basic information

Entry
Database: PDB / ID: 8okd
TitleHuman pseudouridine synthase 3 and tRNA-Gln
Components
  • human tRNA-Gln (70-MER)
  • tRNA pseudouridine(38/39) synthase
KeywordsRNA BINDING PROTEIN / RNA modification / pseudouridylation / tRNA / homodimer
Function / homology
Function and homology information


tRNA pseudouridine38/39 synthase / tRNA pseudouridine(38/39) synthase activity / tRNA pseudouridine synthesis / mRNA pseudouridine synthesis / pseudouridine synthase activity / tRNA modification in the nucleus and cytosol / tRNA modification / RNA binding / nucleus / cytoplasm / cytosol
Similarity search - Function
Pseudouridine synthase Pus3-like / Pseudouridine synthase I, TruA / Pseudouridine synthase I, TruA, C-terminal / Pseudouridine synthase I, TruA, alpha/beta domain / tRNA pseudouridine synthase / Pseudouridine synthase TruA/RsuA/RluB/E/F, N-terminal / Pseudouridine synthase, catalytic domain superfamily
Similarity search - Domain/homology
RNA / RNA (> 10) / tRNA pseudouridine(38/39) synthase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsLin, T.-Y. / Glatt, S.
Funding support Poland, European Union, Switzerland, 3items
OrganizationGrant numberCountry
Polish National Science Centre2019/35/D/NZ1/02397 Poland
European Research Council (ERC)101001394European Union
Swiss National Science Foundation310030_184947 Switzerland
CitationJournal: 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
DepositionMar 28, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 10, 2024Provider: repository / Type: Initial release
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Revision 1.2Jul 9, 2025Group: Data collection / Structure summary / Category: em_admin / em_software / pdbx_entry_details / Item: _em_admin.last_update / _em_software.name
Revision 1.1Jul 9, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: tRNA pseudouridine(38/39) synthase
B: tRNA pseudouridine(38/39) synthase
C: human tRNA-Gln (70-MER)
X: human tRNA-Gln (70-MER)


Theoretical massNumber of molelcules
Total (without water)159,5034
Polymers159,5034
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area10280 Å2
ΔGint-91 kcal/mol
Surface area49000 Å2

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Components

#1: Protein tRNA pseudouridine(38/39) synthase / tRNA pseudouridine synthase 3 / tRNA pseudouridylate synthase 3 / tRNA-uridine isomerase 3


Mass: 55704.199 Da / Num. of mol.: 2 / Mutation: D118A
Source method: isolated from a genetically manipulated source
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 human tRNA-Gln (70-MER)


Mass: 24047.236 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
Has protein modificationN

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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

Component
IDNameTypeEntity IDParent-IDSource
1homodimer of PUS3 and tRNA-Gln complexCOMPLEXall0MULTIPLE SOURCES
2Pseudouridine synthase 3 (PUS3)COMPLEX#11RECOMBINANT
3tRNA-GlnCOMPLEX#21RECOMBINANT
Molecular weightValue: 0.11 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Spodoptera frugiperda (fall armyworm)7108
33synthetic construct (others)32630
Buffer solutionpH: 7.5
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 8 mA / Grid material: COPPER / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 900 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8320

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARC4.1particle selection
2EPUimage acquisition
4cryoSPARC4.1CTF correction
9cryoSPARC4.1initial Euler assignment
10cryoSPARC4.1final Euler assignment
11cryoSPARC4.1classification
12cryoSPARC4.13D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 482246
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 147307 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0038212
ELECTRON MICROSCOPYf_angle_d0.49411778
ELECTRON MICROSCOPYf_dihedral_angle_d12.9442312
ELECTRON MICROSCOPYf_chiral_restr0.0341406
ELECTRON MICROSCOPYf_plane_restr0.0031002

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