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- EMDB-19830: Human pseudouridine synthase 3 (PUS3 R116A mutant) and two tRNA-Gln -

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Basic information

Entry
Database: EMDB / ID: EMD-19830
TitleHuman pseudouridine synthase 3 (PUS3 R116A mutant) and two tRNA-Gln
Map data
Sample
  • Complex: Homodimer of human PUS3 and two tRNA-Gln
    • Complex: Pseudouridine synthase 3 (PUS3)
      • Protein or peptide: tRNA pseudouridine(38/39) synthase
    • Complex: tRNA-Gln
      • RNA: tRNA-Gln
  • Ligand: MAGNESIUM ION
KeywordsRNA modification / pseudouridylation / tRNA / homodimer / RNA BINDING PROTEIN
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 / cytosol / cytoplasm
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
tRNA pseudouridine(38/39) synthase
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.66 Å
AuthorsLin T-Y / Jezowski J / Glatt S
Funding support Poland, European Union, Switzerland, 3 items
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 12, 2024-
Header (metadata) releaseJul 10, 2024-
Map releaseJul 10, 2024-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19830.png

Downloads & links

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Map

FileDownload / File: emd_19830.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 320 pix.
= 270.592 Å		0.85 Å/pix.
x 320 pix.
= 270.592 Å		0.85 Å/pix.
x 320 pix.
= 270.592 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8456 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.36
Minimum - Maximum-0.63815033 - 1.5463488
Average (Standard dev.)0.0010510543 (±0.036034677)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 270.592 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_19830_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_19830_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Homodimer of human PUS3 and two tRNA-Gln

EntireName: Homodimer of human PUS3 and two tRNA-Gln
Components
  • Complex: Homodimer of human PUS3 and two tRNA-Gln
    • Complex: Pseudouridine synthase 3 (PUS3)
      • Protein or peptide: tRNA pseudouridine(38/39) synthase
    • Complex: tRNA-Gln
      • RNA: tRNA-Gln
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Homodimer of human PUS3 and two tRNA-Gln

SupramoleculeName: Homodimer of human PUS3 and two tRNA-Gln / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Molecular weightTheoretical: 160 KDa

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Supramolecule #2: Pseudouridine synthase 3 (PUS3)

SupramoleculeName: Pseudouridine synthase 3 (PUS3) / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: tRNA-Gln

SupramoleculeName: tRNA-Gln / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: tRNA pseudouridine(38/39) synthase

MacromoleculeName: tRNA pseudouridine(38/39) synthase / type: protein_or_peptide / ID: 1 / Details: R116A single mutation / Number of copies: 2 / Enantiomer: LEVO / EC number: tRNA pseudouridine38/39 synthase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 55.639102 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MAYNDTDRNQ TEKLLKRVRE LEQEVQRLKK EQAKNKEDSN IRENSAGAGK TKRAFDFSAH GRRHVALRIA YMGWGYQGFA SQENTNNTI EEKLFEALTK TRLVESRQTS NYHRCGATDK GVSAFGQVIS LDLRSQFPRG RDSEDFNVKE EANAAAEEIR Y THILNRVL ...String:
MAYNDTDRNQ TEKLLKRVRE LEQEVQRLKK EQAKNKEDSN IRENSAGAGK TKRAFDFSAH GRRHVALRIA YMGWGYQGFA SQENTNNTI EEKLFEALTK TRLVESRQTS NYHRCGATDK GVSAFGQVIS LDLRSQFPRG RDSEDFNVKE EANAAAEEIR Y THILNRVL PPDIRILAWA PVEPSFSARF SCLERTYRYF FPRADLDIVT MDYAAQKYVG THDFRNLCKM DVANGVINFQ RT ILSAQVQ LVGQSPGEGR WQEPFQLCQF EVTGQAFLYH QVRCMMAILF LIGQGMEKPE IIDELLNIEK NPQKPQYSMA VEF PLVLYD CKFENVKWIY DQEAQEFNIT HLQQLWANHA VKTHMLYSML QGLDTVPVPC GIGPKMDGMT EWGNVKPSVI KQTS AFVEG VKMRTYKPLM DRPKCQGLES RIQHFVRRGR IEHPHLFHEE ETKAKRDCND TLEEENTNLE TPTKRVCVDT EIKSI I

UniProtKB: tRNA pseudouridine(38/39) synthase

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Macromolecule #2: tRNA-Gln

MacromoleculeName: tRNA-Gln / type: rna / ID: 2 / Number of copies: 2
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.047236 KDa
SequenceString:
GGCCCCAUGG UGUAAUGGUU AGCACUCUGG ACUUUGAAUC CAGCGAUCCG AGUUCAAAUC UCGGUGGGAC CUCCA

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 6 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.3 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R2/1 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 8393 / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3452930
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.66 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.1) / Number images used: 569272
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.1)
Final 3D classificationNumber classes: 4 / Avg.num./class: 280000 / Software - Name: cryoSPARC (ver. 4.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-9enb:
Human pseudouridine synthase 3 (PUS3 R116A mutant) and two tRNA-Gln

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