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- EMDB-19967: Cryo-electron microscopy structure of the Chaetomium thermofilum ... -

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Basic information

Entry
Database: EMDB / ID: EMD-19967
TitleCryo-electron microscopy structure of the Chaetomium thermofilum Uba4-Urm1 complex
Map data
Sample
  • Complex: Dimeric complex of Uba4 with Urm1
    • Protein or peptide: Adenylyltransferase and sulfurtransferase UBA4
    • Protein or peptide: Ubiquitin-related modifier 1
KeywordstRNA thiolation pathway / E1-activating enzyme / urmylation / sulfur transfer / persulfidation / TRANSLATION
Function / homology
Function and homology information


molybdopterin synthase sulfurtransferase / URM1 activating enzyme activity / molybdopterin-synthase sulfurtransferase activity / molybdopterin-synthase adenylyltransferase / molybdopterin-synthase adenylyltransferase activity / tRNA thio-modification / protein urmylation / tRNA wobble position uridine thiolation / tRNA wobble uridine modification / thiosulfate-cyanide sulfurtransferase activity ...molybdopterin synthase sulfurtransferase / URM1 activating enzyme activity / molybdopterin-synthase sulfurtransferase activity / molybdopterin-synthase adenylyltransferase / molybdopterin-synthase adenylyltransferase activity / tRNA thio-modification / protein urmylation / tRNA wobble position uridine thiolation / tRNA wobble uridine modification / thiosulfate-cyanide sulfurtransferase activity / Mo-molybdopterin cofactor biosynthetic process / ATP binding / metal ion binding / cytosol
Similarity search - Function
Adenylyltransferase and sulfurtransferase MOCS3/Uba4 / Ubiquitin-related modifier 1 / Urm1 (Ubiquitin related modifier) / Molybdopterin synthase/thiamin biosynthesis sulphur carrier, beta-grasp / ThiF/MoeB/HesA family / THIF-type NAD/FAD binding fold / ThiF family / Rhodanese Homology Domain / Ubiquitin-activating enzyme / Rhodanese-like domain ...Adenylyltransferase and sulfurtransferase MOCS3/Uba4 / Ubiquitin-related modifier 1 / Urm1 (Ubiquitin related modifier) / Molybdopterin synthase/thiamin biosynthesis sulphur carrier, beta-grasp / ThiF/MoeB/HesA family / THIF-type NAD/FAD binding fold / ThiF family / Rhodanese Homology Domain / Ubiquitin-activating enzyme / Rhodanese-like domain / Rhodanese domain profile. / Rhodanese-like domain superfamily / Rhodanese-like domain / Beta-grasp domain superfamily
Similarity search - Domain/homology
Adenylyltransferase and sulfurtransferase uba4 / Ubiquitin-related modifier 1
Similarity search - Component
Biological speciesThermochaetoides thermophila (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.9 Å
AuthorsKwasna D / Sokolowski M / Jaciuk M / Glatt S
Funding supportEuropean Union, Poland, 2 items
OrganizationGrant numberCountry
European Research Council (ERC)101001394European Union
Foundation for Polish ScienceFirstTEAM/2016-1/2 Poland
CitationJournal: Nucleic Acids Res / Year: 2024
Title: Molecular basis for thiocarboxylation and release of Urm1 by its E1-activating enzyme Uba4.
Authors: Mikołaj Sokołowski / Dominika Kwasna / Keerthiraju E Ravichandran / Cristian Eggers / Rościsław Krutyhołowa / Magdalena Kaczmarczyk / Bozena Skupien-Rabian / Marcin Jaciuk / Marta ...Authors: Mikołaj Sokołowski / Dominika Kwasna / Keerthiraju E Ravichandran / Cristian Eggers / Rościsław Krutyhołowa / Magdalena Kaczmarczyk / Bozena Skupien-Rabian / Marcin Jaciuk / Marta Walczak / Priyanka Dahate / Marta Pabis / Małgorzata Jemioła-Rzemińska / Urszula Jankowska / Sebastian A Leidel / Sebastian Glatt /
Abstract: Ubiquitin-related modifier 1 (Urm1) is a highly conserved member of the ubiquitin-like (UBL) family of proteins. Urm1 is a key component of the eukaryotic transfer RNA (tRNA) thiolation cascade, ...Ubiquitin-related modifier 1 (Urm1) is a highly conserved member of the ubiquitin-like (UBL) family of proteins. Urm1 is a key component of the eukaryotic transfer RNA (tRNA) thiolation cascade, responsible for introducing sulfur at wobble uridine (U34) in several eukaryotic tRNAs. Urm1 must be thiocarboxylated (Urm1-SH) by its E1 activating enzyme UBL protein activator 4 (Uba4). Uba4 first adenylates and then thiocarboxylates the C-terminus of Urm1 using its adenyl-transferase (AD) and rhodanese (RHD) domains. However, the detailed mechanisms of Uba4, the interplay between the two domains, and the release of Urm1 remain elusive. Here, we report a cryo-EM-based structural model of the Uba4/Urm1 complex that reveals the position of its RHD domains after Urm1 binding, and by analyzing the in vitro and in vivo consequence of mutations at the interface, we show its importance for the thiocarboxylation of Urm1. Our results confirm that the formation of the Uba4-Urm1 thioester and thiocarboxylation of Urm1's C-terminus depend on conserved cysteine residues of Uba4 and that the complex avoids unwanted side-reactions of the adenylate by forming a thioester intermediate. We show how the Urm1-SH product can be released and how Urm1 interacts with upstream (Tum1) and downstream (Ncs6) components of the pathway. Our work provides a detailed mechanistic description of the reaction steps that are needed to produce Urm1-SH, which is required to thiolate tRNAs and persulfidate proteins.
History
DepositionMar 27, 2024-
Header (metadata) releaseDec 25, 2024-
Map releaseDec 25, 2024-
UpdateJan 8, 2025-
Current statusJan 8, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19967.png

Downloads & links

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Map

FileDownload / File: emd_19967.map.gz / Format: CCP4 / Size: 46.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 230 pix.
= 197.8 Å		0.86 Å/pix.
x 230 pix.
= 197.8 Å		0.86 Å/pix.
x 230 pix.
= 197.8 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.014
Minimum - Maximum-0.09982629 - 0.1868402
Average (Standard dev.)0.00064499077 (±0.01020588)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions230230230
Spacing230230230
CellA=B=C: 197.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_19967_msk_1.map
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Mask #2

Fileemd_19967_msk_2.map
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Additional map: 7 A low-pass filtered map

Fileemd_19967_additional_1.map
Annotation7 A low-pass filtered map
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Half map: #2

Fileemd_19967_half_map_1.map
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Half map: #1

Fileemd_19967_half_map_2.map
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Sample components

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Entire : Dimeric complex of Uba4 with Urm1

EntireName: Dimeric complex of Uba4 with Urm1
Components
  • Complex: Dimeric complex of Uba4 with Urm1
    • Protein or peptide: Adenylyltransferase and sulfurtransferase UBA4
    • Protein or peptide: Ubiquitin-related modifier 1

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Supramolecule #1: Dimeric complex of Uba4 with Urm1

SupramoleculeName: Dimeric complex of Uba4 with Urm1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Thermochaetoides thermophila (fungus)
Molecular weightTheoretical: 120.7 KDa

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Macromolecule #1: Adenylyltransferase and sulfurtransferase UBA4

MacromoleculeName: Adenylyltransferase and sulfurtransferase UBA4 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
EC number: Transferases; Transferring sulfur-containing groups; Sulfurtransferases
Source (natural)Organism: Thermochaetoides thermophila (fungus)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSTQKSLSK EEIERYSRQM IVPGMGKEGQ LRLMNAKVLI IGAGGLGCPA AQYLAGAGVG TIGIVDGDSV ETSNLHRQVA HATKRVGMLK VDSLITHLIE INPLPVYVPY RFDLTPQNAA QIIKPWDVIL DCTDNPATRY LISDVCVLLG KPLVSAASVQ KSGQLIVLNC ...String:
MGSTQKSLSK EEIERYSRQM IVPGMGKEGQ LRLMNAKVLI IGAGGLGCPA AQYLAGAGVG TIGIVDGDSV ETSNLHRQVA HATKRVGMLK VDSLITHLIE INPLPVYVPY RFDLTPQNAA QIIKPWDVIL DCTDNPATRY LISDVCVLLG KPLVSAASVQ KSGQLIVLNC PPTPQGVVNK KAAPCYRCCF KKPPPPSAQT SKGEAGIMGP VVGMMGVAQA GEAIKILVSQ LHMPPKEGEE VSPEKNLVQP TLLIYTYDLN SAIGPYSFRA LKMGGRKKDC FACGENSTLT LDGIKSGNPN YVQFCGNMTQ STNLAPEDRI TATAYNEKRR NGELGEHILL DTREKEHFSF GSIPGAVNVP FSKFLVKASS IKREGNSPAE LLPMQPASDE APIVVVCRRG QDSQEVVEKL KELGLDNGGK RKIMDIVGGM KAWRDEVDPD FPFI

UniProtKB: Adenylyltransferase and sulfurtransferase uba4

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Macromolecule #2: Ubiquitin-related modifier 1

MacromoleculeName: Ubiquitin-related modifier 1 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Thermochaetoides thermophila (fungus)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSSKAKLEEI PITVDFSGGL EMLFDNQRRH SISLPAKDTE GKPVTIAFLI DYICKKLMKD PRTDLFVLDN HIRPGILVLI NDADWELEGE EAYEIQPNDN ILFVSTLHGG

UniProtKB: Ubiquitin-related modifier 1

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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
Component:
ConcentrationFormulaName
20.0 mMHEPESHEPES
150.0 mMNaClsodium chloride
1.0 mMDTTDithiothreitol
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Details: 8 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: 5 s blot time, 15 s wait time, 0 s drain time, and a blot force of 5..
DetailsUba4 and Urm1 were fused with isopeptide bond

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number real images: 5150 / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.1 µm / Nominal defocus min: 0.9 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3532190
Startup modelType of model: OTHER / Details: Ab-initio
Final reconstructionAlgorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 5.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.14.2) / Number images used: 80383
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.14.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.14.2)
Final 3D classificationNumber classes: 2 / Avg.num./class: 80000 / Software - Name: cryoSPARC (ver. 2.14.2)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model

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