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- PDB-7abf: Human pre-Bact-1 spliceosome core structure -

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Entry
Database: PDB / ID: 7abf
TitleHuman pre-Bact-1 spliceosome core structure
Components
  • 116 kDa U5 small nuclear ribonucleoprotein component
  • MINX M3 RNA
  • Microfibrillar-associated protein 1
  • Pleiotropic regulator 1
  • Pre-mRNA-processing-splicing factor 8
  • Pre-mRNA-splicing factor 38A
  • Protein BUD31 homolog
  • SNW domain-containing protein 1
  • Spliceosome-associated protein CWC15 homolog
  • Transcription elongation regulator 1
  • U5 small nuclear RNA
  • U6 small nuclear RNA
  • Ubiquitin-like protein 5
  • WW domain-binding protein 11
  • Zinc finger matrin-type protein 2
KeywordsSPLICING / Complex / spliceosome / catalytic activation
Function / homology
Function and homology information


microfibril / regulation of retinoic acid receptor signaling pathway / WW domain binding / regulation of vitamin D receptor signaling pathway / transcription elongation factor activity / nuclear retinoic acid receptor binding / Prp19 complex / positive regulation of androgen receptor activity / U2-type catalytic step 1 spliceosome / RNA splicing, via transesterification reactions ...microfibril / regulation of retinoic acid receptor signaling pathway / WW domain binding / regulation of vitamin D receptor signaling pathway / transcription elongation factor activity / nuclear retinoic acid receptor binding / Prp19 complex / positive regulation of androgen receptor activity / U2-type catalytic step 1 spliceosome / RNA splicing, via transesterification reactions / mRNA cis splicing, via spliceosome / U2-type spliceosomal complex / positive regulation by host of viral transcription / U2-type precatalytic spliceosome / positive regulation of vitamin D receptor signaling pathway / U2-type catalytic step 2 spliceosome / nuclear vitamin D receptor binding / ubiquitin-like protein conjugating enzyme binding / RNA polymerase binding / Notch binding / Regulation of gene expression in late stage (branching morphogenesis) pancreatic bud precursor cells / RUNX3 regulates NOTCH signaling / positive regulation of mRNA splicing, via spliceosome / NOTCH4 Intracellular Domain Regulates Transcription / positive regulation of protein targeting to mitochondrion / NOTCH3 Intracellular Domain Regulates Transcription / positive regulation of neurogenesis / K63-linked polyubiquitin modification-dependent protein binding / nuclear androgen receptor binding / precatalytic spliceosome / Notch-HLH transcription pathway / Formation of paraxial mesoderm / mRNA Splicing - Minor Pathway / negative regulation of transcription elongation by RNA polymerase II / positive regulation of transforming growth factor beta receptor signaling pathway / SMAD binding / protein localization to nucleus / spliceosomal tri-snRNP complex assembly / intrinsic apoptotic signaling pathway in response to DNA damage by p53 class mediator / retinoic acid receptor signaling pathway / U5 snRNA binding / positive regulation of G1/S transition of mitotic cell cycle / U5 snRNP / U2 snRNA binding / Cajal body / U6 snRNA binding / pre-mRNA intronic binding / cellular response to retinoic acid / U1 snRNA binding / U4/U6 x U5 tri-snRNP complex / catalytic step 2 spliceosome / mRNA Splicing - Major Pathway / RNA splicing / nuclear receptor coactivator activity / response to cocaine / nuclear receptor binding / positive regulation of transcription elongation by RNA polymerase II / Downregulation of SMAD2/3:SMAD4 transcriptional activity / transcription coregulator activity / spliceosomal complex / protein modification process / NOTCH1 Intracellular Domain Regulates Transcription / fibrillar center / mRNA processing / Pre-NOTCH Transcription and Translation / Constitutive Signaling by NOTCH1 PEST Domain Mutants / Constitutive Signaling by NOTCH1 HD+PEST Domain Mutants / nuclear matrix / mRNA splicing, via spliceosome / rRNA processing / protein tag activity / transcription corepressor activity / cellular response to xenobiotic stimulus / cellular response to tumor necrosis factor / single-stranded DNA binding / nuclear membrane / cellular response to lipopolysaccharide / RNA polymerase II-specific DNA-binding transcription factor binding / transcription coactivator activity / nuclear body / nuclear speck / intracellular membrane-bounded organelle / GTPase activity / negative regulation of DNA-templated transcription / centrosome / chromatin / regulation of transcription by RNA polymerase II / GTP binding / enzyme binding / negative regulation of transcription by RNA polymerase II / positive regulation of transcription by RNA polymerase II / mitochondrion / DNA binding / RNA binding / zinc ion binding / nucleoplasm / membrane / identical protein binding / nucleus / cytosol
Similarity search - Function
WW domain binding protein 11 / WW domain binding protein 11 / Ubiquitin-like modifier Hub1/Ubl5 / Pre-mRNA-splicing factor 38, C-terminal / Pre-mRNA-splicing factor 38-associated hydrophilic C-term / Micro-fibrillar-associated protein 1, C-terminal / Microfibrillar-associated protein 1 / Microfibril-associated/Pre-mRNA processing / U4/U6.U5 small nuclear ribonucleoprotein component Snu23 / Pre-mRNA-splicing factor 38 ...WW domain binding protein 11 / WW domain binding protein 11 / Ubiquitin-like modifier Hub1/Ubl5 / Pre-mRNA-splicing factor 38, C-terminal / Pre-mRNA-splicing factor 38-associated hydrophilic C-term / Micro-fibrillar-associated protein 1, C-terminal / Microfibrillar-associated protein 1 / Microfibril-associated/Pre-mRNA processing / U4/U6.U5 small nuclear ribonucleoprotein component Snu23 / Pre-mRNA-splicing factor 38 / Transcription elongation regulator 1-like / PRP38 family / FF domain / FF domain / FF domain superfamily / FF domain profile. / Contains two conserved F residues / WD repeat Prp46/PLRG1-like / BUD31/G10-related, conserved site / G10 protein signature 1. / G10 protein signature 2. / SKI-interacting protein SKIP, SNW domain / SKI-interacting protein, SKIP / SKIP/SNW domain / Pre-mRNA-splicing factor Cwf15/Cwc15 / Cwf15/Cwc15 cell cycle control protein / G10 protein / Pre-mRNA-splicing factor BUD31 / Zinc-finger double-stranded RNA-binding / Zinc finger, double-stranded RNA binding / Snu114, GTP-binding domain / 116kDa U5 small nuclear ribonucleoprotein component, N-terminal / 116kDa U5 small nuclear ribonucleoprotein component, C-terminal / 116 kDa U5 small nuclear ribonucleoprotein component N-terminus / Matrin/U1-C-like, C2H2-type zinc finger / U1-like zinc finger / Elongation Factor G, domain II / Elongation Factor G, domain III / JAB1/Mov34/MPN/PAD-1 ubiquitin protease / Translation elongation factor EFG/EF2, domain IV / Elongation factor G, domain IV / Elongation factor G, domain IV / Elongation factor G C-terminus / WW domain / Elongation factor EFG, domain V-like / Elongation factor G C-terminus / WW/rsp5/WWP domain signature. / EF-G domain III/V-like / WW domain superfamily / WW/rsp5/WWP domain profile. / Domain with 2 conserved Trp (W) residues / WW domain / 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 / Translation elongation factor EFTu-like, domain 2 / Elongation factor Tu domain 2 / JAB/MPN domain / JAB1/MPN/MOV34 metalloenzyme domain / MPN domain / MPN domain profile. / Zinc finger C2H2 superfamily / Translational (tr)-type GTP-binding domain / Elongation factor Tu GTP binding domain / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / Ubiquitin family / Small GTP-binding protein domain / Ubiquitin-like domain / Translation protein, beta-barrel domain superfamily / Ribosomal protein S5 domain 2-type fold, subgroup / G-protein beta WD-40 repeat / Ubiquitin-like domain superfamily / Ribonuclease H-like superfamily / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Ribosomal protein S5 domain 2-type fold / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
GUANOSINE-5'-TRIPHOSPHATE / INOSITOL HEXAKISPHOSPHATE / : / RNA / RNA (> 10) / RNA (> 100) / Transcription elongation regulator 1 / Pleiotropic regulator 1 / Protein BUD31 homolog / Microfibrillar-associated protein 1 ...GUANOSINE-5'-TRIPHOSPHATE / INOSITOL HEXAKISPHOSPHATE / : / RNA / RNA (> 10) / RNA (> 100) / Transcription elongation regulator 1 / Pleiotropic regulator 1 / Protein BUD31 homolog / Microfibrillar-associated protein 1 / SNW domain-containing protein 1 / 116 kDa U5 small nuclear ribonucleoprotein component / Pre-mRNA-processing-splicing factor 8 / Pre-mRNA-splicing factor 38A / Zinc finger matrin-type protein 2 / Ubiquitin-like protein 5 / Spliceosome-associated protein CWC15 homolog / WW domain-binding protein 11
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsTownsend, C. / Kastner, B. / Leelaram, M.N. / Bertram, K. / Stark, H. / Luehrmann, R.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation (DFG)860 Germany
CitationJournal: Science / Year: 2020
Title: Mechanism of protein-guided folding of the active site U2/U6 RNA during spliceosome activation.
Authors: Cole Townsend / Majety N Leelaram / Dmitry E Agafonov / Olexandr Dybkov / Cindy L Will / Karl Bertram / Henning Urlaub / Berthold Kastner / Holger Stark / Reinhard Lührmann /
Abstract: Spliceosome activation involves extensive protein and RNA rearrangements that lead to formation of a catalytically active U2/U6 RNA structure. At present, little is known about the assembly pathway ...Spliceosome activation involves extensive protein and RNA rearrangements that lead to formation of a catalytically active U2/U6 RNA structure. At present, little is known about the assembly pathway of the latter and the mechanism whereby proteins aid its proper folding. Here, we report the cryo-electron microscopy structures of two human, activated spliceosome precursors (that is, pre-B complexes) at core resolutions of 3.9 and 4.2 angstroms. These structures elucidate the order of the numerous protein exchanges that occur during activation, the mutually exclusive interactions that ensure the correct order of ribonucleoprotein rearrangements needed to form the U2/U6 catalytic RNA, and the stepwise folding pathway of the latter. Structural comparisons with mature B complexes reveal the molecular mechanism whereby a conformational change in the scaffold protein PRP8 facilitates final three-dimensional folding of the U2/U6 catalytic RNA.
History
DepositionSep 7, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 9, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 30, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
Q: Protein BUD31 homolog
I: Pre-mRNA-splicing factor 38A
A: Pre-mRNA-processing-splicing factor 8
r: 116 kDa U5 small nuclear ribonucleoprotein component
N: Zinc finger matrin-type protein 2
q: Ubiquitin-like protein 5
R: Spliceosome-associated protein CWC15 homolog
5: U5 small nuclear RNA
6: U6 small nuclear RNA
X: WW domain-binding protein 11
v: SNW domain-containing protein 1
G: Pleiotropic regulator 1
Z: MINX M3 RNA
K: Microfibrillar-associated protein 1
A4: Transcription elongation regulator 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,005,86918
Polymers1,004,66115
Non-polymers1,2083
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: cross-linking
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 12 types, 12 molecules QIArNqRXvGKA4

#1: Protein Protein BUD31 homolog / Protein EDG-2 / Protein G10 homolog


Mass: 17032.850 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela / References: UniProt: P41223
#2: Protein Pre-mRNA-splicing factor 38A


Mass: 37563.863 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela / References: UniProt: Q8NAV1
#3: Protein Pre-mRNA-processing-splicing factor 8 / 220 kDa U5 snRNP-specific protein / PRP8 homolog / Splicing factor Prp8 / p220


Mass: 273974.250 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela / References: UniProt: Q6P2Q9
#4: Protein 116 kDa U5 small nuclear ribonucleoprotein component / Elongation factor Tu GTP-binding domain-containing protein 2 / SNU114 homolog / hSNU114 / U5 snRNP- ...Elongation factor Tu GTP-binding domain-containing protein 2 / SNU114 homolog / hSNU114 / U5 snRNP-specific protein / 116 kDa / U5-116 kDa


Mass: 109560.625 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela / References: UniProt: Q15029
#5: Protein Zinc finger matrin-type protein 2


Mass: 23664.047 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela / References: UniProt: Q96NC0
#6: Protein Ubiquitin-like protein 5


Mass: 8560.945 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela / References: UniProt: Q9BZL1
#7: Protein Spliceosome-associated protein CWC15 homolog


Mass: 26674.447 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela / References: UniProt: Q9P013
#10: Protein WW domain-binding protein 11 / WBP-11 / Npw38-binding protein / NpwBP / SH3 domain-binding protein SNP70 / Splicing factor that ...WBP-11 / Npw38-binding protein / NpwBP / SH3 domain-binding protein SNP70 / Splicing factor that interacts with PQBP-1 and PP1


Mass: 70098.641 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela / References: UniProt: Q9Y2W2
#11: Protein SNW domain-containing protein 1 / Nuclear protein SkiP / Nuclear receptor coactivator NCoA-62 / Ski-interacting protein


Mass: 61610.703 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela / References: UniProt: Q13573
#12: Protein Pleiotropic regulator 1


Mass: 57280.758 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela / References: UniProt: O43660
#14: Protein Microfibrillar-associated protein 1


Mass: 52050.527 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela / References: UniProt: P55081
#15: Protein Transcription elongation regulator 1 / / TATA box-binding protein-associated factor 2S / Transcription factor CA150


Mass: 121870.320 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela / References: UniProt: O14776

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RNA chain , 3 types, 3 molecules 56Z

#8: RNA chain U5 small nuclear RNA


Mass: 36908.668 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela / References: GenBank: 36515
#9: RNA chain U6 small nuclear RNA


Mass: 34098.270 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela
#13: RNA chain MINX M3 RNA


Mass: 73712.359 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 3 molecules

#16: Chemical ChemComp-IHP / INOSITOL HEXAKISPHOSPHATE / MYO-INOSITOL HEXAKISPHOSPHATE / INOSITOL 1,2,3,4,5,6-HEXAKISPHOSPHATE / Phytic acid


Mass: 660.035 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H18O24P6
#17: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE / Guanosine triphosphate


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#18: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1pre-Bact-1 spliceosomal complexCOMPLEX#1-#150MULTIPLE SOURCES
2pre-Bact-1 spliceosomal complexCOMPLEX#1-#12, #14-#151NATURAL
3MINX M3 RNACOMPLEX#131RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23synthetic construct (others)32630
Source (recombinant)Organism: synthetic construct (others)
Buffer solutionpH: 7.9
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R3.5/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingAverage exposure time: 1 sec. / Electron dose: 2.25 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k)
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
7UCSF Chimeramodel fitting
8Cootmodel fitting
13RELION33D reconstruction
20PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 84539 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL

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