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- PDB-7qtt: Structural organization of a late activated human spliceosome (Ba... -

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Basic information

Entry
Database: PDB / ID: 7qtt
TitleStructural organization of a late activated human spliceosome (Baqr, core region)
Components
  • (Peptidyl-prolyl cis-trans isomerase-like ...) x 2
  • (Pre-mRNA-splicing factor ...) x 2
  • (Small nuclear ribonucleoprotein ...) x 6
  • (Splicing factor 3A subunit ...) x 2
  • (Splicing factor 3B subunit ...) x 5
  • 116 kDa U5 small nuclear ribonucleoprotein component
  • BUD13 homolog
  • Cell division cycle 5-like protein
  • Crooked neck-like protein 1
  • G-patch domain and KOW motifs-containing protein
  • MINX
  • PHD finger-like domain-containing protein 5A
  • Pleiotropic regulator 1
  • Pre-mRNA-processing-splicing factor 8
  • Protein BUD31 homolog
  • Putative pre-mRNA-splicing factor ATP-dependent RNA helicase DHX16
  • RING finger protein 113A
  • RING-type E3 ubiquitin-protein ligase PPIL2
  • SNW domain-containing protein 1
  • Serine/arginine repetitive matrix protein 2
  • Small nuclear ribonucleoprotein-associated proteins B and B'
  • Spliceosome-associated protein CWC15 homolog
  • U2snRNA
  • U5 snRNA
  • U6snRNA
KeywordsSPLICING / Spliceosome / helicase / Prp2 / Aquarius / catalytic activation
Function / homology
Function and homology information


RES complex / negative regulation of chemokine-mediated signaling pathway / snoRNA splicing / U11/U12 snRNP / regulation of retinoic acid receptor signaling pathway / post-mRNA release spliceosomal complex / U2 snRNP binding / U7 snRNA binding / histone pre-mRNA DCP binding / U7 snRNP ...RES complex / negative regulation of chemokine-mediated signaling pathway / snoRNA splicing / U11/U12 snRNP / regulation of retinoic acid receptor signaling pathway / post-mRNA release spliceosomal complex / U2 snRNP binding / U7 snRNA binding / histone pre-mRNA DCP binding / U7 snRNP / histone pre-mRNA 3'end processing complex / regulation of vitamin D receptor signaling pathway / SLBP independent Processing of Histone Pre-mRNAs / SLBP Dependent Processing of Replication-Dependent Histone Pre-mRNAs / B-WICH complex / nuclear retinoic acid receptor binding / embryonic brain development / protein methylation / U12-type spliceosomal complex / 7-methylguanosine cap hypermethylation / U1 snRNP binding / methylosome / RNA splicing, via transesterification reactions / pICln-Sm protein complex / C2H2 zinc finger domain binding / U2-type catalytic step 1 spliceosome / pre-mRNA binding / regulation of mRNA splicing, via spliceosome / snRNP binding / positive regulation of mRNA splicing, via spliceosome / splicing factor binding / SMN-Sm protein complex / small nuclear ribonucleoprotein complex / spliceosomal tri-snRNP complex / host-mediated activation of viral transcription / P granule / telomerase holoenzyme complex / U2-type precatalytic spliceosome / positive regulation of vitamin D receptor signaling pathway / commitment complex / mRNA cis splicing, via spliceosome / telomerase RNA binding / U2-type spliceosomal complex / nuclear vitamin D receptor binding / U2-type prespliceosome assembly / Notch binding / U2-type catalytic step 2 spliceosome / Regulation of gene expression in late stage (branching morphogenesis) pancreatic bud precursor cells / RUNX3 regulates NOTCH signaling / positive regulation of neurogenesis / NOTCH4 Intracellular Domain Regulates Transcription / SAGA complex / RNA Polymerase II Transcription Termination / U2 snRNP / U1 snRNP / U4 snRNP / NOTCH3 Intracellular Domain Regulates Transcription / Basigin interactions / U2-type prespliceosome / positive regulation of transcription by RNA polymerase III / WD40-repeat domain binding / ubiquitin-ubiquitin ligase activity / protein peptidyl-prolyl isomerization / K63-linked polyubiquitin modification-dependent protein binding / nuclear androgen receptor binding / precatalytic spliceosome / pattern recognition receptor activity / Notch-HLH transcription pathway / Formation of paraxial mesoderm / SMAD binding / positive regulation of transforming growth factor beta receptor signaling pathway / regulation of RNA splicing / spliceosomal complex assembly / mRNA Splicing - Minor Pathway / mRNA 3'-splice site recognition / positive regulation of transcription by RNA polymerase I / spliceosomal tri-snRNP complex assembly / Prp19 complex / U5 snRNA binding / U5 snRNP / intrinsic apoptotic signaling pathway in response to DNA damage by p53 class mediator / protein localization to nucleus / U2 snRNA binding / positive regulation of G1/S transition of mitotic cell cycle / U6 snRNA binding / pre-mRNA intronic binding / spliceosomal snRNP assembly / U1 snRNA binding / Cajal body / regulation of DNA repair / retinoic acid receptor signaling pathway / U4/U6 x U5 tri-snRNP complex / cellular response to retinoic acid / catalytic step 2 spliceosome / antiviral innate immune response / mRNA Splicing - Major Pathway / RNA splicing / DNA damage checkpoint signaling / positive regulation of RNA splicing / positive regulation of protein export from nucleus
Similarity search - Function
G-patch domain and KOW motifs-containing protein, KOW 1 / G-patch domain and KOW motifs-containing protein, KOW 2 / GPKOW C-terminal domain / PPIL4-like, cyclophilin domain / Cyclophilin-RNA interacting protein / Peptidyl-prolyl cis-trans isomerase like 2, U-box domain / Spp2/MOS2, G-patch domain / Pre-mRNA-splicing factor Spp2-like / G-patch domain / Pre-mRNA-splicing factor CWC24-like ...G-patch domain and KOW motifs-containing protein, KOW 1 / G-patch domain and KOW motifs-containing protein, KOW 2 / GPKOW C-terminal domain / PPIL4-like, cyclophilin domain / Cyclophilin-RNA interacting protein / Peptidyl-prolyl cis-trans isomerase like 2, U-box domain / Spp2/MOS2, G-patch domain / Pre-mRNA-splicing factor Spp2-like / G-patch domain / Pre-mRNA-splicing factor CWC24-like / Bud13 / : / Pre-mRNA-splicing factor of RES complex / : / SF3B4, RNA recognition motif 2 / Splicing factor SF3a60 binding domain / Splicing factor SF3a60 binding domain / Cactus-binding C-terminus of cactin protein / : / Replication stress response SDE2 C-terminal / : / : / SF3A2 domain / : / Pre-mRNA-splicing factor SF3a complex subunit 2 (Prp11) / mRNA splicing factor Cwf21 domain / cwf21 domain / Splicing factor SF3a60 /Prp9 subunit, C-terminal / SF3A3 domain / SF3a60/Prp9 C-terminal / Pre-mRNA-splicing factor SF3A3, of SF3a complex, Prp9 / G-patch domain profile. / SF3B4, RNA recognition motif 1 / : / G-patch domain / glycine rich nucleic binding domain / Zinc finger C-x8-C-x5-C-x3-H type (and similar) / : / : / Domain of unknown function DUF382 / Domain of unknown function (DUF382) / G10 protein signature 2. / Splicing factor 3B, subunit 5 / Cyclophilin-type peptidyl-prolyl cis-trans isomerase, cyclophilin A-like / BUD31/G10-related, conserved site / G10 protein signature 1. / Splicing factor 3B subunit 1 / Splicing factor 3B subunit 1 / Zinc-finger of C2H2 type / : / STL11, N-terminal / : / Pre-mRNA-splicing factor Syf1/CRNKL1 C-terminal HAT repeat / SKI-interacting protein SKIP, SNW domain / PSP, proline-rich / SKI-interacting protein, SKIP / SKIP/SNW domain / PSP / proline-rich domain in spliceosome associated proteins / Pre-mRNA-splicing factor Cwf15/Cwc15 / Cwf15/Cwc15 cell cycle control protein / WD repeat Prp46/PLRG1-like / Pre-mRNA-splicing factor Cwc2/Slt11 / Matrin/U1-C, C2H2-type zinc finger / Zinc finger matrin-type profile. / G10 protein / Pre-mRNA-splicing factor BUD31 / PHF5-like / Pre-mRNA splicing factor component Cdc5p/Cef1, C-terminal / : / : / PHF5-like protein / pre-mRNA splicing factor component / Splicing factor 3B subunit 5/RDS3 complex subunit 10 / Splicing factor 3B subunit 10 (SF3b10) / Initiation factor eIF-4 gamma, MA3 / MA3 domain / MI domain profile. / Domain in DAP-5, eIF4G, MA-3 and other proteins. / Splicing factor 3B subunit 1-like / MIF4G domain / Small ribonucleoprotein associated, SmB/SmN / Myb-like DNA-binding domain / Middle domain of eukaryotic initiation factor 4G (eIF4G) / MIF4G-like, type 3 / Spt5 C-terminal domain / Spt5 C-terminal nonapeptide repeat binding Spt4 / Small nuclear ribonucleoprotein D1 / U-box domain profile. / Zinc finger, CCCH-type superfamily / Modified RING finger domain / zinc finger / : / U-box domain / PPP2R1A-like HEAT repeat / SAP motif profile. / 116kDa U5 small nuclear ribonucleoprotein component, N-terminal / 116kDa U5 small nuclear ribonucleoprotein component, C-terminal / Snu114, GTP-binding domain / 116 kDa U5 small nuclear ribonucleoprotein component N-terminus
Similarity search - Domain/homology
GUANOSINE-5'-TRIPHOSPHATE / INOSITOL HEXAKISPHOSPHATE / : / : / RNA / RNA (> 10) / RNA (> 100) / E3 ubiquitin-protein ligase RNF113A / Pleiotropic regulator 1 / Pre-mRNA-splicing factor ATP-dependent RNA helicase DHX16 ...GUANOSINE-5'-TRIPHOSPHATE / INOSITOL HEXAKISPHOSPHATE / : / : / RNA / RNA (> 10) / RNA (> 100) / E3 ubiquitin-protein ligase RNF113A / Pleiotropic regulator 1 / Pre-mRNA-splicing factor ATP-dependent RNA helicase DHX16 / Splicing factor 3B subunit 1 / Small nuclear ribonucleoprotein-associated proteins B and B' / Protein BUD31 homolog / Small nuclear ribonucleoprotein E / Small nuclear ribonucleoprotein F / Small nuclear ribonucleoprotein G / Small nuclear ribonucleoprotein Sm D1 / Small nuclear ribonucleoprotein Sm D2 / Small nuclear ribonucleoprotein Sm D3 / Splicing factor 3A subunit 3 / RING-type E3 ubiquitin-protein ligase PPIL2 / Splicing factor 3B subunit 2 / SNW domain-containing protein 1 / 116 kDa U5 small nuclear ribonucleoprotein component / Splicing factor 3B subunit 3 / Splicing factor 3B subunit 4 / Splicing factor 3A subunit 2 / Pre-mRNA-processing-splicing factor 8 / PHD finger-like domain-containing protein 5A / Peptidyl-prolyl cis-trans isomerase-like 4 / G-patch domain and KOW motifs-containing protein / Cell division cycle 5-like protein / BUD13 homolog / Splicing factor 3B subunit 5 / Crooked neck-like protein 1 / Pre-mRNA-splicing factor CWC22 homolog / Pre-mRNA-splicing factor RBM22 / Spliceosome-associated protein CWC15 homolog / Serine/arginine repetitive matrix protein 2 / Peptidyl-prolyl cis-trans isomerase-like 1
Similarity search - Component
Biological speciesHomo sapiens (human)
unidentified adenovirus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsCretu, C. / Pena, V.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation (DFG)241796087 Germany
CitationJournal: Nature / Year: 2023
Title: Structural basis of catalytic activation in human splicing.
Authors: Jana Schmitzová / Constantin Cretu / Christian Dienemann / Henning Urlaub / Vladimir Pena /
Abstract: Pre-mRNA splicing follows a pathway driven by ATP-dependent RNA helicases. A crucial event of the splicing pathway is the catalytic activation, which takes place at the transition between the ...Pre-mRNA splicing follows a pathway driven by ATP-dependent RNA helicases. A crucial event of the splicing pathway is the catalytic activation, which takes place at the transition between the activated B and the branching-competent B spliceosomes. Catalytic activation occurs through an ATP-dependent remodelling mediated by the helicase PRP2 (also known as DHX16). However, because PRP2 is observed only at the periphery of spliceosomes, its function has remained elusive. Here we show that catalytic activation occurs in two ATP-dependent stages driven by two helicases: PRP2 and Aquarius. The role of Aquarius in splicing has been enigmatic. Here the inactivation of Aquarius leads to the stalling of a spliceosome intermediate-the B complex-found halfway through the catalytic activation process. The cryogenic electron microscopy structure of B reveals how PRP2 and Aquarius remodel B and B, respectively. Notably, PRP2 translocates along the intron while it strips away the RES complex, opens the SF3B1 clamp and unfastens the branch helix. Translocation terminates six nucleotides downstream of the branch site through an assembly of PPIL4, SKIP and the amino-terminal domain of PRP2. Finally, Aquarius enables the dissociation of PRP2, plus the SF3A and SF3B complexes, which promotes the relocation of the branch duplex for catalysis. This work elucidates catalytic activation in human splicing, reveals how a DEAH helicase operates and provides a paradigm for how helicases can coordinate their activities.
History
DepositionJan 15, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 10, 2023Provider: repository / Type: Initial release
Revision 1.1May 24, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Jun 7, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Jun 14, 2023Group: Database references / Category: citation_author / Item: _citation_author.identifier_ORCID
Revision 1.4Jul 17, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Splicing factor 3B subunit 3
B: Splicing factor 3B subunit 5
C: Splicing factor 3B subunit 1
D: PHD finger-like domain-containing protein 5A
E: Splicing factor 3B subunit 2
F: Splicing factor 3B subunit 4
G: G-patch domain and KOW motifs-containing protein
I: Splicing factor 3A subunit 2
J: Splicing factor 3A subunit 3
L: BUD13 homolog
N: Putative pre-mRNA-splicing factor ATP-dependent RNA helicase DHX16
O: Pleiotropic regulator 1
P: Cell division cycle 5-like protein
Q: Spliceosome-associated protein CWC15 homolog
T: Protein BUD31 homolog
U: Pre-mRNA-splicing factor RBM22
V: Peptidyl-prolyl cis-trans isomerase-like 4
W: Pre-mRNA-splicing factor CWC22 homolog
X: Crooked neck-like protein 1
Y: SNW domain-containing protein 1
Z: RING finger protein 113A
a: Pre-mRNA-processing-splicing factor 8
b: 116 kDa U5 small nuclear ribonucleoprotein component
d: U6snRNA
e: U5 snRNA
f: U2snRNA
g: MINX
h: Small nuclear ribonucleoprotein F
i: Small nuclear ribonucleoprotein E
j: Small nuclear ribonucleoprotein Sm D3
k: Small nuclear ribonucleoprotein Sm D1
l: Small nuclear ribonucleoprotein Sm D2
m: Small nuclear ribonucleoprotein G
n: Small nuclear ribonucleoprotein-associated proteins B and B'
p: Peptidyl-prolyl cis-trans isomerase-like 1
q: Serine/arginine repetitive matrix protein 2
u: RING-type E3 ubiquitin-protein ligase PPIL2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)2,495,69258
Polymers2,493,55437
Non-polymers2,13821
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: homology
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Splicing factor 3B subunit ... , 5 types, 5 molecules ABCEF

#1: Protein Splicing factor 3B subunit 3 / Pre-mRNA-splicing factor SF3b 130 kDa subunit / SF3b130 / STAF130 / Spliceosome-associated protein ...Pre-mRNA-splicing factor SF3b 130 kDa subunit / SF3b130 / STAF130 / Spliceosome-associated protein 130 / SAP 130


Mass: 135718.844 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q15393
#2: Protein Splicing factor 3B subunit 5 / SF3b5 / Pre-mRNA-splicing factor SF3b 10 kDa subunit


Mass: 10149.369 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q9BWJ5
#3: Protein Splicing factor 3B subunit 1 / Pre-mRNA-splicing factor SF3b 155 kDa subunit / SF3b155 / Spliceosome-associated protein 155 / SAP 155


Mass: 146024.938 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: O75533
#5: Protein Splicing factor 3B subunit 2 / Pre-mRNA-splicing factor SF3b 145 kDa subunit / SF3b145 / Spliceosome-associated protein 145 / SAP 145


Mass: 100377.812 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q13435
#6: Protein Splicing factor 3B subunit 4 / Pre-mRNA-splicing factor SF3b 49 kDa subunit / Spliceosome-associated protein 49 / SAP 49


Mass: 44436.570 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q15427

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Protein , 16 types, 16 molecules DGLNOPQTXYZabnqu

#4: Protein PHD finger-like domain-containing protein 5A / PHD finger-like domain protein 5A / Splicing factor 3B-associated 14 kDa protein / SF3b14b


Mass: 12427.524 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q7RTV0
#7: Protein G-patch domain and KOW motifs-containing protein / G-patch domain-containing protein 5 / Protein MOS2 homolog / Protein T54


Mass: 52299.711 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q92917
#10: Protein BUD13 homolog


Mass: 70669.211 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q9BRD0
#11: Protein Putative pre-mRNA-splicing factor ATP-dependent RNA helicase DHX16 / ATP-dependent RNA helicase #3 / DEAH-box protein 16


Mass: 119443.633 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: O60231, RNA helicase
#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) / References: UniProt: O43660
#13: Protein Cell division cycle 5-like protein / Cdc5-like protein / Pombe cdc5-related protein


Mass: 92406.883 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q99459
#14: 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) / References: UniProt: Q9P013
#15: 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
#19: Protein Crooked neck-like protein 1 / Crooked neck homolog / hCrn


Mass: 100610.008 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q9BZJ0
#20: 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) / References: UniProt: Q13573
#21: Protein RING finger protein 113A / Zinc finger protein 183


Mass: 38847.199 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: O15541
#22: 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
#23: 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
#34: Protein Small nuclear ribonucleoprotein-associated proteins B and B' / snRNP-B / Sm protein B/B' / SmB/B'


Mass: 24642.131 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P14678
#36: Protein Serine/arginine repetitive matrix protein 2 / 300 kDa nuclear matrix antigen / Serine/arginine-rich splicing factor-related nuclear matrix ...300 kDa nuclear matrix antigen / Serine/arginine-rich splicing factor-related nuclear matrix protein of 300 kDa / Ser/Arg-related nuclear matrix protein of 300 kDa / Splicing coactivator subunit SRm300 / Tax-responsive enhancer element-binding protein 803 / TaxREB803


Mass: 300255.312 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q9UQ35
#37: Protein RING-type E3 ubiquitin-protein ligase PPIL2 / CYC4 / Cyclophilin-60 / Cyclophilin-like protein Cyp-60 / Cyp60 / hCyP-60 / Probable inactive ...CYC4 / Cyclophilin-60 / Cyclophilin-like protein Cyp-60 / Cyp60 / hCyP-60 / Probable inactive peptidyl-prolyl cis-trans isomerase-like 2 / PPIase / Rotamase PPIL2


Mass: 58910.379 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human)
References: UniProt: Q13356, RING-type E3 ubiquitin transferase

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Splicing factor 3A subunit ... , 2 types, 2 molecules IJ

#8: Protein Splicing factor 3A subunit 2 / SF3a66 / Spliceosome-associated protein 62 / SAP 62


Mass: 49327.355 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q15428
#9: Protein Splicing factor 3A subunit 3 / SF3a60 / Spliceosome-associated protein 61 / SAP 61


Mass: 58934.844 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q12874

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Pre-mRNA-splicing factor ... , 2 types, 2 molecules UW

#16: Protein Pre-mRNA-splicing factor RBM22 / RNA-binding motif protein 22 / Zinc finger CCCH domain-containing protein 16


Mass: 46959.555 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q9NW64
#18: Protein Pre-mRNA-splicing factor CWC22 homolog / Nucampholin homolog / fSAPb


Mass: 105646.578 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q9HCG8

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Peptidyl-prolyl cis-trans isomerase-like ... , 2 types, 2 molecules Vp

#17: Protein Peptidyl-prolyl cis-trans isomerase-like 4 / PPIase / Cyclophilin-like protein PPIL4 / Rotamase PPIL4


Mass: 57324.176 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q8WUA2, peptidylprolyl isomerase
#35: Protein Peptidyl-prolyl cis-trans isomerase-like 1 / PPIase / Rotamase PPIL1


Mass: 18257.805 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q9Y3C6, peptidylprolyl isomerase

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RNA chain , 4 types, 4 molecules defg

#24: RNA chain U6snRNA


Mass: 34098.270 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human)
#25: 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
#26: RNA chain U2snRNA


Mass: 60186.445 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: GenBank: 36516
#27: RNA chain MINX


Mass: 102348.188 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: The MINX pre-mRNA substrate was obtained by in vitro transcription with T7 RNA polymerase. The full-length transcript contains three MS2 aptamer sequences at its 3' end for affinity purification.
Source: (synth.) unidentified adenovirus

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Small nuclear ribonucleoprotein ... , 6 types, 6 molecules hijklm

#28: Protein Small nuclear ribonucleoprotein F / snRNP-F / Sm protein F / SmF


Mass: 9734.171 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P62306
#29: Protein Small nuclear ribonucleoprotein E / snRNP-E / Sm protein E / SmE


Mass: 10817.601 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P62304
#30: Protein Small nuclear ribonucleoprotein Sm D3 / Sm-D3 / snRNP core protein D3


Mass: 13940.308 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P62318
#31: Protein Small nuclear ribonucleoprotein Sm D1 / Sm-D1 / Sm-D autoantigen / snRNP core protein D1


Mass: 13310.653 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P62314
#32: Protein Small nuclear ribonucleoprotein Sm D2 / Sm-D2 / snRNP core protein D2


Mass: 13551.928 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P62316
#33: Protein Small nuclear ribonucleoprotein G / snRNP-G / Sm protein G / SmG


Mass: 8508.084 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P62308

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Non-polymers , 4 types, 21 molecules

#38: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Zn
#39: Chemical ChemComp-IHP / INOSITOL HEXAKISPHOSPHATE / MYO-INOSITOL HEXAKISPHOSPHATE / INOSITOL 1,2,3,4,5,6-HEXAKISPHOSPHATE


Mass: 660.035 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H18O24P6 / Feature type: SUBJECT OF INVESTIGATION
#40: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


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


Mass: 24.305 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestY

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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: Late human activated spliceosome arrested with a dominant-negative mutant of the splicing helicase Aquarius (Aquarius K829A)
Type: COMPLEX
Details: The spliceosome complex was assembled in vitro in the HeLa nuclear extract on a model pre-mRNA substrate (MINX) tagged with three MS2 aptamer sequences for affinity purification.
Entity ID: #1-#37 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 7.9
Details: All solutions were sterile filtered using a 0.22um vacuum filtration unit.
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)HEPES1
2120 mMSodium ChlorideNaCl1
31.5 mMMagnesium AcetateMg(CH3COO)21
42 % (w/v)GlycerolC3H8O31
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The Baqr spliceosome was purified by affinity selection and gradient ultracentrifugation and crosslinked with 0.1% (v/v) glutaraldehyde in batch for cryo-EM grid preparation.
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: UltrAuFoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
Details: Volumes of 4 ul of the concentrated sample were applied to one side of glow-discharged UltrAuFoil 200 2/2 grids (Quantifoil) in a Vitrobot Mark IV (FEI) operating at 4 degrees Celsius and ...Details: Volumes of 4 ul of the concentrated sample were applied to one side of glow-discharged UltrAuFoil 200 2/2 grids (Quantifoil) in a Vitrobot Mark IV (FEI) operating at 4 degrees Celsius and 100% humidity. The grids were blotted for 2s with blotting force 5 and immediately frozen by plunging into liquid 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 FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 9 sec. / Electron dose: 45.47 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 10013
Details: Automated data acquisition for dataset 1 (untilted, 5229 micrographs) and dataset 2 (tilted, 25 degrees, 4784 micrographs) was performed with FEI EPU software package at a nominal ...Details: Automated data acquisition for dataset 1 (untilted, 5229 micrographs) and dataset 2 (tilted, 25 degrees, 4784 micrographs) was performed with FEI EPU software package at a nominal magnification of 130,000 (1.05 A per pixel). Micrographs for these two datasets, dose fractionated over 40 frames, were collected at a dose rate of 5.04 or 5.06 e/A2/s-1 over 9 s, resulting in a total dose of 45.38 and 45.55 e/A2, respectively.
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 30 eV
Image scansMovie frames/image: 40

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
1Warpparticle selection
2EPUimage acquisition
4WarpCTF correction
7Coot0.9.6model fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
19PHENIX1.19.2_4158model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 734691
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 146157 / Symmetry type: POINT
Atomic model buildingB value: 177.21 / Protocol: OTHER / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
16FF4

6ff4

16FF41PDBexperimental model
26FF7

6ff7

16FF72PDBexperimental model
35Z57

5z57

15Z573PDBexperimental model
46QDV

6qdv

16QDV4PDBexperimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 180.35 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00475501
ELECTRON MICROSCOPYf_angle_d0.833104676
ELECTRON MICROSCOPYf_dihedral_angle_d11.03814411
ELECTRON MICROSCOPYf_chiral_restr0.0512560
ELECTRON MICROSCOPYf_plane_restr0.00712556

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