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- PDB-8q7n: cryo-EM structure of the human spliceosomal B complex protomer (t... -

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Entry
Database: PDB / ID: 8q7n
Titlecryo-EM structure of the human spliceosomal B complex protomer (tri-snRNP core region)
Components
  • (U4/U6 small nuclear ribonucleoprotein ...) x 3
  • 116 kDa U5 small nuclear ribonucleoprotein component
  • MINX pre-mRNA
  • Microfibrillar-associated protein 1
  • NHP2-like protein 1, N-terminally processed
  • Pre-mRNA-processing factor 6
  • Pre-mRNA-processing-splicing factor 8
  • Pre-mRNA-splicing factor 38A
  • Protein BUD31 homolog
  • Splicing factor 3A subunit 1
  • Thioredoxin-like protein 4A
  • Transcription elongation regulator 1
  • U4 snRNA
  • U4/U6.U5 tri-snRNP-associated protein 1
  • U5 snRNA
  • U6 snRNA
  • Ubiquitin-like protein 5
  • WW domain-binding protein 4
  • Zinc finger matrin-type protein 2
KeywordsSPLICING / spliceosome / pre-catalytic spliceosome / spliceosomal B complex
Function / homology
Function and homology information


microfibril / spliceosomal snRNP complex / ribonucleoprotein complex localization / U4atac snRNP / positive regulation of cytotoxic T cell differentiation / maturation of 5S rRNA / snRNP binding / RNA localization / U4atac snRNA binding / box C/D sno(s)RNA binding ...microfibril / spliceosomal snRNP complex / ribonucleoprotein complex localization / U4atac snRNP / positive regulation of cytotoxic T cell differentiation / maturation of 5S rRNA / snRNP binding / RNA localization / U4atac snRNA binding / box C/D sno(s)RNA binding / positive regulation of primary miRNA processing / dense fibrillar component / U4/U6 snRNP / transcription elongation factor activity / positive regulation of androgen receptor activity / RNA splicing, via transesterification reactions / U2-type catalytic step 1 spliceosome / U4 snRNA binding / box C/D methylation guide snoRNP complex / spliceosomal tri-snRNP complex / proline-rich region binding / U2-type precatalytic spliceosome / U2-type spliceosomal complex / mRNA cis splicing, via spliceosome / ubiquitin-like protein conjugating enzyme binding / U2-type prespliceosome assembly / RNA polymerase binding / U2-type catalytic step 2 spliceosome / rRNA modification in the nucleus and cytosol / box C/D snoRNP assembly / U4 snRNP / U2 snRNP / U3 snoRNA binding / positive regulation of protein targeting to mitochondrion / U2-type prespliceosome / K63-linked polyubiquitin modification-dependent protein binding / precatalytic spliceosome / positive regulation of miRNA metabolic process / spliceosomal complex assembly / negative regulation of transcription elongation by RNA polymerase II / mRNA Splicing - Minor Pathway / mRNA 3'-splice site recognition / MLL1 complex / single fertilization / spliceosomal tri-snRNP complex assembly / Major pathway of rRNA processing in the nucleolus and cytosol / U5 snRNA binding / U5 snRNP / RNA processing / U2 snRNA binding / ribonucleoprotein complex binding / U6 snRNA binding / spliceosomal snRNP assembly / Cajal body / pre-mRNA intronic binding / U1 snRNA binding / U4/U6 x U5 tri-snRNP complex / catalytic step 2 spliceosome / mRNA Splicing - Major Pathway / RNA splicing / nuclear receptor coactivator activity / maturation of LSU-rRNA / response to cocaine / maturation of SSU-rRNA / nuclear receptor binding / positive regulation of transcription elongation by RNA polymerase II / small-subunit processome / transcription coregulator activity / spliceosomal complex / protein modification process / mRNA splicing, via spliceosome / mRNA processing / protein tag activity / transcription corepressor activity / cellular response to xenobiotic stimulus / protein-macromolecule adaptor activity / cellular response to tumor necrosis factor / ribosomal small subunit biogenesis / ATPase binding / cellular response to lipopolysaccharide / RNA polymerase II-specific DNA-binding transcription factor binding / cytosolic large ribosomal subunit / transcription coactivator activity / nuclear speck / cell division / intracellular membrane-bounded organelle / GTPase activity / centrosome / chromatin / GTP binding / nucleolus / Golgi apparatus / negative regulation of transcription by RNA polymerase II / positive regulation of transcription by RNA polymerase II / protein-containing complex / DNA binding / RNA binding / zinc ion binding / nucleoplasm / identical protein binding
Similarity search - Function
WW domain-binding protein 4 / 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 ...WW domain-binding protein 4 / 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 / U1-C, C2H2-type zinc finger / U1 zinc finger / PRP4-like superfamily / pre-mRNA processing factor 4 (PRP4) like / FF domain / SNU66/SART1 family / HIND motif / SART-1 family / HIND motif / FF domain / FF domain superfamily / FF domain profile. / Contains two conserved F residues / PWI domain superfamily / PWI domain / PWI domain profile. / Pre-mRNA processing factor 4 (PRP4)-like / Splicing Factor Motif, present in Prp18 and Pr04 / Pre-mRNA-splicing factor 3 / U4/U6 small nuclear ribonucleoprotein Prp3 / pre-mRNA processing factor 3 domain / Prp31 C-terminal / U4/U6 small nuclear ribonucleoprotein Prp31 / Prp31 C terminal domain / Small nuclear ribonucleoprotein Prp3, C-terminal domain / Splicing factor 3A subunit 1, ubiquitin domain / Small nuclear ribonucleoprotein Prp3, C-terminal domain / PWI domain / PWI, domain in splicing factors / Dim1 family / Mitosis protein DIM1 / Mitosis protein DIM1 / PRP1 splicing factor, N-terminal / PRP1 splicing factor, N-terminal / Splicing factor 3A subunit 1 / Splicing factor 3A subunit 1, conserved domain / Pre-mRNA splicing factor PRP21 like protein / BUD31/G10-related, conserved site / G10 protein signature 1. / G10 protein signature 2. / SWAP/Surp / SWAP/Surp superfamily / Surp module / SURP motif repeat profile. / Suppressor-of-White-APricot splicing regulator / G10 protein / Pre-mRNA-splicing factor BUD31 / Matrin/U1-C, C2H2-type zinc finger / Zinc finger matrin-type profile. / Zinc-finger double-stranded RNA-binding / Zinc finger, double-stranded RNA binding / H/ACA ribonucleoprotein complex, subunit Nhp2-like / NOSIC / NOSIC (NUC001) domain / Nop domain / Nop domain superfamily / Nop, C-terminal domain / snoRNA binding domain, fibrillarin / Nop domain profile. / Pre-mRNA-splicing factor Syf1-like / Tetratricopeptide repeat / 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 / Tetratricopeptide repeat / HAT (Half-A-TPR) repeat / HAT (Half-A-TPR) repeats / 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 / WW/rsp5/WWP domain signature. / Elongation factor EFG, domain V-like / Elongation factor G C-terminus / WW domain superfamily / WW/rsp5/WWP domain profile. / Domain with 2 conserved Trp (W) residues / WW domain / EF-G domain III/V-like / PROCT domain / Prp8 RNase domain IV, fingers region / PROCT (NUC072) domain
Similarity search - Domain/homology
: / : / RNA / RNA (> 10) / RNA (> 100) / Transcription elongation regulator 1 / U4/U6 small nuclear ribonucleoprotein Prp4 / U4/U6.U5 tri-snRNP-associated protein 1 / U4/U6 small nuclear ribonucleoprotein Prp3 / WW domain-binding protein 4 ...: / : / RNA / RNA (> 10) / RNA (> 100) / Transcription elongation regulator 1 / U4/U6 small nuclear ribonucleoprotein Prp4 / U4/U6.U5 tri-snRNP-associated protein 1 / U4/U6 small nuclear ribonucleoprotein Prp3 / WW domain-binding protein 4 / Pre-mRNA-processing factor 6 / Protein BUD31 homolog / Microfibrillar-associated protein 1 / NHP2-like protein 1 / Thioredoxin-like protein 4A / 116 kDa U5 small nuclear ribonucleoprotein component / Splicing factor 3A subunit 1 / Pre-mRNA-processing-splicing factor 8 / Pre-mRNA-splicing factor 38A / U4/U6 small nuclear ribonucleoprotein Prp31 / Zinc finger matrin-type protein 2 / Ubiquitin-like protein 5
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsZhang, Z. / Kumar, V. / Dybkov, O. / Will, C.L. / Urlaub, H. / Stark, H. / Luehrmann, R.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation (DFG) Germany
CitationJournal: EMBO J / Year: 2024
Title: Cryo-EM analyses of dimerized spliceosomes provide new insights into the functions of B complex proteins.
Authors: Zhenwei Zhang / Vinay Kumar / Olexandr Dybkov / Cindy L Will / Henning Urlaub / Holger Stark / Reinhard Lührmann /
Abstract: The B complex is a key intermediate stage of spliceosome assembly. To improve the structural resolution of monomeric, human spliceosomal B (hB) complexes and thereby generate a more comprehensive hB ...The B complex is a key intermediate stage of spliceosome assembly. To improve the structural resolution of monomeric, human spliceosomal B (hB) complexes and thereby generate a more comprehensive hB molecular model, we determined the cryo-EM structure of B complex dimers formed in the presence of ATP S. The enhanced resolution of these complexes allows a finer molecular dissection of how the 5' splice site (5'ss) is recognized in hB, and new insights into molecular interactions of FBP21, SNU23 and PRP38 with the U6/5'ss helix and with each other. It also reveals that SMU1 and RED are present as a heterotetrameric complex and are located at the interface of the B dimer protomers. We further show that MFAP1 and UBL5 form a 5' exon binding channel in hB, and elucidate the molecular contacts stabilizing the 5' exon at this stage. Our studies thus yield more accurate models of protein and RNA components of hB complexes. They further allow the localization of additional proteins and protein domains (such as SF3B6, BUD31 and TCERG1) whose position was not previously known, thereby uncovering new functions for B-specific and other hB proteins during pre-mRNA splicing.
History
DepositionAug 16, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 24, 2024Provider: repository / Type: Initial release
Revision 1.1Aug 7, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
5: U5 snRNA
6: U6 snRNA
7: Splicing factor 3A subunit 1
C: 116 kDa U5 small nuclear ribonucleoprotein component
D: Thioredoxin-like protein 4A
I: Pre-mRNA-splicing factor 38A
K: Microfibrillar-associated protein 1
M: NHP2-like protein 1, N-terminally processed
Q: Protein BUD31 homolog
X: WW domain-binding protein 4
Z: MINX pre-mRNA
r: Zinc finger matrin-type protein 2
s: Ubiquitin-like protein 5
L: U4/U6 small nuclear ribonucleoprotein Prp31
F: U4/U6 small nuclear ribonucleoprotein Prp4
N: Pre-mRNA-processing factor 6
A: Pre-mRNA-processing-splicing factor 8
S: U4/U6.U5 tri-snRNP-associated protein 1
T: Transcription elongation regulator 1
4: U4 snRNA
J: U4/U6 small nuclear ribonucleoprotein Prp3


Theoretical massNumber of molelcules
Total (without water)1,427,47921
Polymers1,427,47921
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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RNA chain , 4 types, 4 molecules 56Z4

#1: RNA chain U5 snRNA


Mass: 37254.855 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: GenBank: 20330981
#2: RNA chain U6 snRNA


Mass: 34098.270 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: GenBank: NR_004394.1
#11: RNA chain MINX pre-mRNA


Mass: 111300.453 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human)
#20: RNA chain U4 snRNA


Mass: 46528.465 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human)

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Protein , 14 types, 14 molecules 7CDIKMQXrsNAST

#3: Protein Splicing factor 3A subunit 1 / SF3a120 / Spliceosome-associated protein 114 / SAP 114


Mass: 88991.094 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q15459
#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) / References: UniProt: Q15029
#5: Protein Thioredoxin-like protein 4A / DIM1 protein homolog / Spliceosomal U5 snRNP-specific 15 kDa protein / Thioredoxin-like U5 snRNP ...DIM1 protein homolog / Spliceosomal U5 snRNP-specific 15 kDa protein / Thioredoxin-like U5 snRNP protein U5-15kD


Mass: 16807.346 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P83876
#6: 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) / References: UniProt: Q8NAV1
#7: Protein Microfibrillar-associated protein 1 / Spliceosome B complex protein MFAP1


Mass: 52050.527 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P55081
#8: Protein NHP2-like protein 1, N-terminally processed


Mass: 14191.524 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P55769
#9: 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) / References: UniProt: P41223
#10: Protein WW domain-binding protein 4 / WBP-4 / Formin-binding protein 21 / WW domain-containing-binding protein 4


Mass: 42575.801 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: O75554
#12: 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) / References: UniProt: Q96NC0
#13: 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) / References: UniProt: Q9BZL1
#16: Protein Pre-mRNA-processing factor 6 / Androgen receptor N-terminal domain-transactivating protein 1 / ANT-1 / PRP6 homolog / U5 snRNP- ...Androgen receptor N-terminal domain-transactivating protein 1 / ANT-1 / PRP6 homolog / U5 snRNP-associated 102 kDa protein / U5-102 kDa protein


Mass: 107092.242 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: O94906
#17: 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) / References: UniProt: Q6P2Q9
#18: Protein U4/U6.U5 tri-snRNP-associated protein 1 / SNU66 homolog / hSnu66 / Squamous cell carcinoma antigen recognized by T-cells 1 / SART-1 / hSART-1 ...SNU66 homolog / hSnu66 / Squamous cell carcinoma antigen recognized by T-cells 1 / SART-1 / hSART-1 / U4/U6.U5 tri-snRNP-associated 110 kDa protein


Mass: 90414.117 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: O43290
#19: Protein Transcription elongation regulator 1 / TATA box-binding protein-associated factor 2S / Transcription factor CA150


Mass: 124083.000 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: O14776

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U4/U6 small nuclear ribonucleoprotein ... , 3 types, 3 molecules LFJ

#14: Protein U4/U6 small nuclear ribonucleoprotein Prp31 / Pre-mRNA-processing factor 31 / Serologically defined breast cancer antigen NY-BR-99 / U4/U6 snRNP ...Pre-mRNA-processing factor 31 / Serologically defined breast cancer antigen NY-BR-99 / U4/U6 snRNP 61 kDa protein / Protein 61K / hPrp31


Mass: 55528.969 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q8WWY3
#15: Protein U4/U6 small nuclear ribonucleoprotein Prp4 / PRP4 homolog / hPrp4 / U4/U6 snRNP 60 kDa protein / WD splicing factor Prp4


Mass: 58536.105 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: O43172
#21: Protein U4/U6 small nuclear ribonucleoprotein Prp3 / Pre-mRNA-splicing factor 3 / hPrp3 / U4/U6 snRNP 90 kDa protein


Mass: 77669.188 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: O43395

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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

ComponentName: human pre-catalytic spliceosome / Type: COMPLEX / Entity ID: #1-#11, #13-#20, #12, #21 / Source: NATURAL
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 7.9
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 48 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 251564 / Symmetry type: POINT

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