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- EMDB-16917: Human apo pseudouridine synthase 3 (PUS3) -

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Basic information

Entry
Database: EMDB / ID: EMD-16917
TitleHuman apo pseudouridine synthase 3 (PUS3)
Map datahuman full-legnth apo PUS3
Sample
  • Complex: homodimer of PUS3
    • Protein or peptide: pseudouridine syntase 3
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 / 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
tRNA pseudouridine(38/39) synthase
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.5 Å
AuthorsLin T-Y / Glatt S / Koziej L
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 27, 2023-
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_16917.png

Downloads & links

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Map

FileDownload / File: emd_16917.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationhuman full-legnth apo PUS3
Projections & slices

Image control

Size
Brightness
Contrast
Others
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

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0573
Minimum - Maximum-0.058290984 - 0.17048551
Average (Standard dev.)0.00036348976 (±0.007825954)
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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Mask #1

Fileemd_16917_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: human full-legnth apo PUS3 half map A

Fileemd_16917_half_map_1.map
Annotationhuman full-legnth apo PUS3 half map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: human full-legnth apo PUS3 half map B

Fileemd_16917_half_map_2.map
Annotationhuman full-legnth apo PUS3 half map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : homodimer of PUS3

EntireName: homodimer of PUS3
Components
  • Complex: homodimer of PUS3
    • Protein or peptide: pseudouridine syntase 3

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Supramolecule #1: homodimer of PUS3

SupramoleculeName: homodimer of PUS3 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 550 KDa

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Macromolecule #1: pseudouridine syntase 3

MacromoleculeName: pseudouridine syntase 3 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: tRNA pseudouridine38/39 synthase
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: AMADNDTDRN QTEKLLKRVR ELEQEVQRLK KEQAKNKEDS NIRENSAGAG KTKRAFDFSA HGRRHVALRI AYMGWGYQGF ASQENTNNTI EEKLFEALTK TRLVESRQTS NYHRCGRTAK GVSAFGQVIS LDLRSQFPRG RDSEDFNVKE EANAAAEEIR YTHILNRVLP ...String:
AMADNDTDRN QTEKLLKRVR ELEQEVQRLK KEQAKNKEDS NIRENSAGAG KTKRAFDFSA HGRRHVALRI AYMGWGYQGF ASQENTNNTI EEKLFEALTK TRLVESRQTS NYHRCGRTAK GVSAFGQVIS LDLRSQFPRG RDSEDFNVKE EANAAAEEIR YTHILNRVLP PDIRILAWAP VEPSFSARFS CLERTYRYFF PRADLDIVTM DYAAQKYVGT HDFRNLCKMD VANGVINFQR TILSAQVQLV GQSPGEGRWQ EPFQLCQFEV TGQAFLYHQV RCMMAILFLI GQGMEKPEII DELLNIEKNP QKPQYSMAVE FPLVLYDCKF ENVKWIYDQE AQEFNITHLQ QLWANHAVKT HMLYSMLQGL DTVPVPCGIG PKMDGMTEWG NVKPSVIKQT SAFVEGVKMR TYKPLMDRPK CQGLESRIQH FVRRGRIEHP HLFHEEETKA KRDCNDTLEE ENTNLETPTK RVCVDTEIKS II

UniProtKB: tRNA pseudouridine(38/39) synthase

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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: C-flat-2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Details: 8 mA
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 (6k x 4k) / Number grids imaged: 1 / Number real images: 7353 / 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: 3.8000000000000003 µm / Nominal defocus min: 3.0 µm
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: 334282
Startup modelType of model: INSILICO MODEL / In silico model: alphafold2
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 6.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.1) / Number images used: 63173
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 4.1)
Final 3D classificationNumber classes: 3 / Avg.num./class: 65000 / Software - Name: cryoSPARC (ver. 4.1)
FSC plot (resolution estimation)

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