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- EMDB-19834: Human pseudouridine synthase 3 (PUS3 R116A mutant) -

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Basic information

Entry
Database: EMDB / ID: EMD-19834
TitleHuman pseudouridine synthase 3 (PUS3 R116A mutant)
Map data
Sample
  • Complex: Homodimer of human PUS3 (R116A mutant)
    • Protein or peptide: Pseudouridine synthase 3 homodimer
KeywordsRNA modification / pseudouridylation / tRNA / homodimer / RNA BINDING PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.49 Å
AuthorsLin T-Y / 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-
UpdateJul 24, 2024-
Current statusJul 24, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19834.png

Downloads & links

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Map

FileDownload / File: emd_19834.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 256 pix.
= 220.16 Å		0.86 Å/pix.
x 256 pix.
= 220.16 Å		0.86 Å/pix.
x 256 pix.
= 220.16 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.08770094 - 0.21645698
Average (Standard dev.)0.00018239992 (±0.008178629)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 220.16 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_19834_half_map_1.map
Projections & Slices
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Half map: #2

Fileemd_19834_half_map_2.map
Projections & Slices
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Sample components

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Entire : Homodimer of human PUS3 (R116A mutant)

EntireName: Homodimer of human PUS3 (R116A mutant)
Components
  • Complex: Homodimer of human PUS3 (R116A mutant)
    • Protein or peptide: Pseudouridine synthase 3 homodimer

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Supramolecule #1: Homodimer of human PUS3 (R116A mutant)

SupramoleculeName: Homodimer of human PUS3 (R116A mutant) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 110 KDa

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Macromolecule #1: Pseudouridine synthase 3 homodimer

MacromoleculeName: Pseudouridine synthase 3 homodimer / type: protein_or_peptide / ID: 1 / Details: R116A single mutation / Enantiomer: LEVO / EC number: tRNA pseudouridine38/39 synthase
Source (natural)Organism: Homo sapiens (human)
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

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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: 7980 / 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: 1918835
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.49 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.1) / Number images used: 377322
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: 3 / Avg.num./class: 3333333 / Software - Name: cryoSPARC (ver. 4.1)
FSC plot (resolution estimation)

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