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- EMDB-13690: Structure of U5 snRNP assembly and recycling factor TSSC4 in comp... -

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Basic information

Entry
Database: EMDB / ID: EMD-13690
TitleStructure of U5 snRNP assembly and recycling factor TSSC4 in complex with BRR2 and Jab1 domain of PRPF8
Map data
Sample
  • Complex: SNRNP200-TSSC4-PRP8F complex
    • Complex: SNRNP200
      • Protein or peptide: U5 small nuclear ribonucleoprotein 200 kDa helicase
    • Complex: PRPF8-Jab1/MPN
      • Protein or peptide: Pre-mRNA-processing-splicing factor 8
    • Complex: TSSC4
      • Protein or peptide: Protein TSSC4
Function / homology
Function and homology information


cis assembly of pre-catalytic spliceosome / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) / molecular sequestering activity / U2-type catalytic step 1 spliceosome / RNA splicing, via transesterification reactions / U2-type precatalytic spliceosome / U2-type catalytic step 2 spliceosome / K63-linked polyubiquitin modification-dependent protein binding / mRNA Splicing - Minor Pathway / spliceosomal tri-snRNP complex assembly ...cis assembly of pre-catalytic spliceosome / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) / molecular sequestering activity / U2-type catalytic step 1 spliceosome / RNA splicing, via transesterification reactions / U2-type precatalytic spliceosome / U2-type catalytic step 2 spliceosome / K63-linked polyubiquitin modification-dependent protein binding / mRNA Splicing - Minor Pathway / spliceosomal tri-snRNP complex assembly / U5 snRNA binding / U5 snRNP / U2 snRNA binding / spliceosomal snRNP assembly / U6 snRNA binding / pre-mRNA intronic binding / U1 snRNA binding / U4/U6 x U5 tri-snRNP complex / catalytic step 2 spliceosome / mRNA Splicing - Major Pathway / RNA splicing / helicase activity / spliceosomal complex / mRNA splicing, via spliceosome / mRNA processing / osteoblast differentiation / cellular response to tumor necrosis factor / cellular response to lipopolysaccharide / RNA helicase activity / RNA helicase / nuclear speck / protein-containing complex binding / ATP hydrolysis activity / RNA binding / nucleoplasm / ATP binding / membrane / identical protein binding / nucleus / cytoplasm
Similarity search - Function
Tumour suppressing sub-chromosomal transferable candidate 4 / Tumour suppressing sub-chromosomal transferable candidate 4 / Brr2, N-terminal helicase PWI domain / : / N-terminal helicase PWI domain / Pre-mRNA-splicing helicase BRR2 plug domain / Sec63 Brl domain / Sec63 domain / Sec63 Brl domain / JAB1/Mov34/MPN/PAD-1 ubiquitin protease ...Tumour suppressing sub-chromosomal transferable candidate 4 / Tumour suppressing sub-chromosomal transferable candidate 4 / Brr2, N-terminal helicase PWI domain / : / N-terminal helicase PWI domain / Pre-mRNA-splicing helicase BRR2 plug domain / Sec63 Brl domain / Sec63 domain / Sec63 Brl domain / JAB1/Mov34/MPN/PAD-1 ubiquitin protease / PROCT domain / Prp8 RNase domain IV, fingers region / PROCT (NUC072) domain / PRO8NT domain / PROCN domain / Pre-mRNA-processing-splicing factor 8, U6-snRNA-binding / Pre-mRNA-processing-splicing factor 8, U5-snRNA-binding / RNA recognition motif, spliceosomal PrP8 / PRP8 domain IV core / Pre-mRNA-processing-splicing factor 8, U5-snRNA-binding domain superfamily / Prp8 RNase domain IV, palm region / PRO8NT (NUC069), PrP8 N-terminal domain / PROCN (NUC071) domain / U6-snRNA interacting domain of PrP8 / U5-snRNA binding site 2 of PrP8 / RNA recognition motif of the spliceosomal PrP8 / PRP8 domain IV core / Pre-mRNA-processing-splicing factor 8 / JAB/MPN domain / JAB1/MPN/MOV34 metalloenzyme domain / MPN domain / MPN domain profile. / DEAD/DEAH box helicase / DEAD/DEAH box helicase domain / C2 domain superfamily / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Immunoglobulin E-set / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / Winged helix DNA-binding domain superfamily / Ribonuclease H-like superfamily / Winged helix-like DNA-binding domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
U5 small nuclear ribonucleoprotein 200 kDa helicase / Pre-mRNA-processing-splicing factor 8 / U5 small nuclear ribonucleoprotein TSSC4
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.05 Å
AuthorsBergfort A / Kuropka B / Ilik IA / Freund C / Aktas T / Hilal T / Weber G / Wahl MC
Funding support Germany, 1 items
OrganizationGrant numberCountry
German Research Foundation (DFG)TRR186/2 Germany
CitationJournal: Nucleic Acids Res / Year: 2022
Title: The intrinsically disordered TSSC4 protein acts as a helicase inhibitor, placeholder and multi-interaction coordinator during snRNP assembly and recycling.
Authors: Alexandra Bergfort / Tarek Hilal / Benno Kuropka / İbrahim Avşar Ilik / Gert Weber / Tuğçe Aktaş / Christian Freund / Markus C Wahl /
Abstract: Biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) and their recycling after splicing require numerous assembly/recycling factors whose modes of action are often poorly understood. ...Biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) and their recycling after splicing require numerous assembly/recycling factors whose modes of action are often poorly understood. The intrinsically disordered TSSC4 protein has been identified as a nuclear-localized U5 snRNP and U4/U6-U5 tri-snRNP assembly/recycling factor, but how TSSC4's intrinsic disorder supports TSSC4 functions remains unknown. Using diverse interaction assays and cryogenic electron microscopy-based structural analysis, we show that TSSC4 employs four conserved, non-contiguous regions to bind the PRPF8 Jab1/MPN domain and the SNRNP200 helicase at functionally important sites. It thereby inhibits SNRNP200 helicase activity, spatially aligns the proteins, coordinates formation of a U5 sub-module and transiently blocks premature interaction of SNRNP200 with at least three other spliceosomal factors. Guided by the structure, we designed a TSSC4 variant that lacks stable binding to the PRPF8 Jab1/MPN domain or SNRNP200 in vitro. Comparative immunoprecipitation/mass spectrometry from HEK293 nuclear extract revealed distinct interaction profiles of wild type TSSC4 and the variant deficient in PRPF8/SNRNP200 binding with snRNP proteins, other spliceosomal proteins as well as snRNP assembly/recycling factors and chaperones. Our findings elucidate molecular strategies employed by an intrinsically disordered protein to promote snRNP assembly, and suggest multiple TSSC4-dependent stages during snRNP assembly/recycling.
History
DepositionOct 7, 2021-
Header (metadata) releaseJan 26, 2022-
Map releaseJan 26, 2022-
UpdateAug 10, 2022-
Current statusAug 10, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7px3
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13690.png

Downloads & links

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Map

FileDownload / File: emd_13690.map.gz / Format: CCP4 / Size: 129.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.88 Å/pix.
x 324 pix.
= 283.824 Å		0.88 Å/pix.
x 324 pix.
= 283.824 Å		0.88 Å/pix.
x 324 pix.
= 283.824 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.876 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3 / Movie #1: 0.5
Minimum - Maximum0.0 - 2.9160206
Average (Standard dev.)0.00810555 (±0.06897149)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions324324324
Spacing324324324
CellA=B=C: 283.824 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8760.8760.876
M x/y/z324324324
origin x/y/z0.0000.0000.000
length x/y/z283.824283.824283.824
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS324324324
D min/max/mean0.0002.9160.008

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Supplemental data

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Additional map: #1

Fileemd_13690_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : SNRNP200-TSSC4-PRP8F complex

EntireName: SNRNP200-TSSC4-PRP8F complex
Components
  • Complex: SNRNP200-TSSC4-PRP8F complex
    • Complex: SNRNP200
      • Protein or peptide: U5 small nuclear ribonucleoprotein 200 kDa helicase
    • Complex: PRPF8-Jab1/MPN
      • Protein or peptide: Pre-mRNA-processing-splicing factor 8
    • Complex: TSSC4
      • Protein or peptide: Protein TSSC4

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Supramolecule #1: SNRNP200-TSSC4-PRP8F complex

SupramoleculeName: SNRNP200-TSSC4-PRP8F complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: SNRNP200

SupramoleculeName: SNRNP200 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)

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Supramolecule #3: PRPF8-Jab1/MPN

SupramoleculeName: PRPF8-Jab1/MPN / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)

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Supramolecule #4: TSSC4

SupramoleculeName: TSSC4 / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Macromolecule #1: U5 small nuclear ribonucleoprotein 200 kDa helicase

MacromoleculeName: U5 small nuclear ribonucleoprotein 200 kDa helicase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA helicase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 199.666656 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: GAEFMDLDQG GEALAPRQVL DLEDLVFTQG SHFMANKRCQ LPDGSFRRQR KGYEEVHVPA LKPKPFGSEE QLLPVEKLPK YAQAGFEGF KTLNRIQSKL YRAALETDEN LLLCAPTGAG KTNVALMCML REIGKHINMD GTINVDDFKI IYIAPMRSLV Q EMVGSFGK ...String:
GAEFMDLDQG GEALAPRQVL DLEDLVFTQG SHFMANKRCQ LPDGSFRRQR KGYEEVHVPA LKPKPFGSEE QLLPVEKLPK YAQAGFEGF KTLNRIQSKL YRAALETDEN LLLCAPTGAG KTNVALMCML REIGKHINMD GTINVDDFKI IYIAPMRSLV Q EMVGSFGK RLATYGITVA ELTGDHQLCK EEISATQIIV CTPEKWDIIT RKGGERTYTQ LVRLIILDEI HLLHDDRGPV LE ALVARAI RNIEMTQEDV RLIGLSATLP NYEDVATFLR VDPAKGLFYF DNSFRPVPLE QTYVGITEKK AIKRFQIMNE IVY EKIMEH AGKNQVLVFV HSRKETGKTA RAIRDMCLEK DTLGLFLREG SASTEVLRTE AEQCKNLELK DLLPYGFAIH HAGM TRVDR TLVEDLFADK HIQVLVSTAT LAWGVNLPAH TVIIKGTQVY SPEKGRWTEL GALDILQMLG RAGRPQYDTK GEGIL ITSH GELQYYLSLL NQQLPIESQM VSKLPDMLNA EIVLGNVQNA KDAVNWLGYA YLYIRMLRSP TLYGISHDDL KGDPLL DQR RLDLVHTAAL MLDKNNLVKY DKKTGNFQVT ELGRIASHYY ITNDTVQTYN QLLKPTLSEI ELFRVFSLSS EFKNITV RE EEKLELQKLL ERVPIPVKES IEEPSAKINV LLQAFISQLK LEGFALMADM VYVTQSAGRL MRAIFEIVLN RGWAQLTD K TLNLCKMIDK RMWQSMCPLR QFRKLPEEVV KKIEKKNFPF ERLYDLNHNE IGELIRMPKM GKTIHKYVHL FPKLELSVH LQPITRSTLK VELTITPDFQ WDEKVHGSSE AFWILVEDVD SEVILHHEYF LLKAKYAQDE HLITFFVPVF EPLPPQYFIR VVSDRWLSC ETQLPVSFRH LILPEKYPPP TELLDLQPLP VSALRNSAFE SLYQDKFPFF NPIQTQVFNT VYNSDDNVFV G APTGSGKT ICAEFAILRM LLQSSEGRCV YITPMEALAE QVYMDWYEKF QDRLNKKVVL LTGETSTDLK LLGKGNIIIS TP EKWDILS RRWKQRKNVQ NINLFVVDEV HLIGGENGPV LEVICSRMRY ISSQIERPIR IVALSSSLSN AKDVAHWLGC SAT STFNFH PNVRPVPLEL HIQGFNISHT QTRLLSMAKP VYHAITKHSP KKPVIVFVPS RKQTRLTAID ILTTCAADIQ RQRF LHCTE KDLIPYLEKL SDSTLKETLL NGVGYLHEGL SPMERRLVEQ LFSSGAIQVV VASRSLCWGM NVAAHLVIIM DTQYY NGKI HAYVDYPIYD VLQMVGHANR PLQDDEGRCV IMCQGSKKDF FKKFLYEPLP VESHLDHCMH DHFNAEIVTK TIENKQ DAV DYLTWTFLYR RMTQNPNYYN LQGISHRHLS DHLSELVEQT LSDLEQSKCI SIEDEMDVAP LNLGMIAAYY YINYTTI EL FSMSLNAKTK VRGLIEIISN AAEYENIPIR HHEDNLLRQL AQKVPHKLNN PKFNDPHVKT NLLLQAHLSR MQLSAELQ S DTEEILSKAI RLIQACVDVL SSNGWLSPAL AAMELAQMVT QAMWSKDSYL KQLPHFTSEH IKRCTDKGVE SVFDIMEME DEERNALLQL TDSQIADVAR FCNRYPNIEL SYEVVDKDSI RSGGPVVVLV QLEREEEVTG PVIAPLFPQK REEGWWVVIG DAKSNSLIS IKRLTLQQKA KVKLDFVAPA TGAHNYTLYF MSDAYMGCDQ EYKFSVDVKE AETDSDSD

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Macromolecule #2: Pre-mRNA-processing-splicing factor 8

MacromoleculeName: Pre-mRNA-processing-splicing factor 8 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 30.133229 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: GPLGSMTQTF SSKTEWRVRA ISAANLHLRT NHIYVSSDDI KETGYTYILP KNVLKKFICI SDLRAQIAGY LYGVSPPDNP QVKEIRCIV MVPQWGTHQT VHLPGQLPQH EYLKEMEPLG WIHTQPNESP QLSPQDVTTH AKIMADNPSW DGEKTIIITC S FTPGSCTL ...String:
GPLGSMTQTF SSKTEWRVRA ISAANLHLRT NHIYVSSDDI KETGYTYILP KNVLKKFICI SDLRAQIAGY LYGVSPPDNP QVKEIRCIV MVPQWGTHQT VHLPGQLPQH EYLKEMEPLG WIHTQPNESP QLSPQDVTTH AKIMADNPSW DGEKTIIITC S FTPGSCTL TAYKLTPSGY EWGRQNTDKG NNPKGYLPSH YERVQMLLSD RFLGFFMVPA QSSWNYNFMG VRHDPNMKYE LQ LANPKEF YHEVHRPSHF LNFALL

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Macromolecule #3: Protein TSSC4

MacromoleculeName: Protein TSSC4 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 34.366465 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAEAGTGEPS PSVEGEHGTE YDTLPSDTVS LSDSDSDLSL PGGAEVEALS PMGLPGEEDS GPDEPPSPPS GLLPATVQPF HLRGMSSTF SQRSRDIFDC LEGAARRAPS SVAHTSMSDN GGFKRPLAPS GRSPVEGLGR AHRSPASPRV PPVPDYVAHP E RWTKYSLE ...String:
MAEAGTGEPS PSVEGEHGTE YDTLPSDTVS LSDSDSDLSL PGGAEVEALS PMGLPGEEDS GPDEPPSPPS GLLPATVQPF HLRGMSSTF SQRSRDIFDC LEGAARRAPS SVAHTSMSDN GGFKRPLAPS GRSPVEGLGR AHRSPASPRV PPVPDYVAHP E RWTKYSLE DVTEVSEQSN QATALAFLGS QSLAAPTDCV SSFNQDPSSC GEGRVIFTKP VRGVEARHER KRVLGKVGEP GR GGLGNPA TDRGEGPVEL AHLAGPGSPE AEEWGSHHGG LQEVEALSGS VHSGSVPGLP PVETVGFHGS RKRSRDHFRN KSS SPEDPG AEV

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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

Concentration5 mg/mL
BufferpH: 7.6
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 308 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 120000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 3349 / Average exposure time: 30.59 sec. / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1025529
CTF correctionSoftware - Name: cryoSPARC (ver. 2.9)
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 2.9)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.9)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.05 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3) / Number images used: 387973
FSC plot (resolution estimation)

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