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- PDB-6exn: Post-catalytic P complex spliceosome with 3' splice site docked -

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Basic information

Entry
Database: PDB / ID: 6exn
TitlePost-catalytic P complex spliceosome with 3' splice site docked
Components
  • (Pre-mRNA-processing factor ...) x 2
  • (Pre-mRNA-splicing factor ...) x 18
  • (Small nuclear ribonucleoprotein ...) x 6
  • (U2 small nuclear ribonucleoprotein ...) x 2
  • Intron lariat: UBC4 RNA
  • Ligated exons: UBC4 mRNA
  • Pre-mRNA-processing protein 45
  • Small nuclear ribonucleoprotein-associated protein B
  • U2 snRNA
  • U5 snRNA
  • U6 snRNA
  • Unassigned structure
KeywordsSPLICING / Spliceosome / P-complex / Exon ligation
Function / homology
Function and homology information


U2-type post-spliceosomal complex / spliceosomal complex disassembly / mRNA branch site recognition / U2-type post-mRNA release spliceosomal complex / cellular bud site selection / pre-mRNA 3'-splice site binding / post-mRNA release spliceosomal complex / nuclear mRNA surveillance / generation of catalytic spliceosome for first transesterification step / cis assembly of pre-catalytic spliceosome ...U2-type post-spliceosomal complex / spliceosomal complex disassembly / mRNA branch site recognition / U2-type post-mRNA release spliceosomal complex / cellular bud site selection / pre-mRNA 3'-splice site binding / post-mRNA release spliceosomal complex / nuclear mRNA surveillance / generation of catalytic spliceosome for first transesterification step / cis assembly of pre-catalytic spliceosome / splicing factor binding / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) / U4/U6 snRNP / 7-methylguanosine cap hypermethylation / pICln-Sm protein complex / U2-type catalytic step 1 spliceosome / pre-mRNA binding / snRNP binding / small nuclear ribonucleoprotein complex / SMN-Sm protein complex / spliceosomal tri-snRNP complex / U2-type spliceosomal complex / commitment complex / mRNA cis splicing, via spliceosome / U2-type prespliceosome assembly / U2-type catalytic step 2 spliceosome / U4 snRNP / U2 snRNP / U1 snRNP / poly(U) RNA binding / U2-type prespliceosome / precatalytic spliceosome / generation of catalytic spliceosome for second transesterification step / Formation of TC-NER Pre-Incision Complex / spliceosomal complex assembly / mRNA 5'-splice site recognition / Gap-filling DNA repair synthesis and ligation in TC-NER / mRNA 3'-splice site recognition / DNA replication origin binding / Dual incision in TC-NER / spliceosomal tri-snRNP complex assembly / Prp19 complex / protein K63-linked ubiquitination / U5 snRNA binding / DNA replication initiation / U5 snRNP / U2 snRNA binding / U6 snRNA binding / pre-mRNA intronic binding / spliceosomal snRNP assembly / U1 snRNA binding / U4/U6 x U5 tri-snRNP complex / positive regulation of cell cycle / catalytic step 2 spliceosome / nuclear periphery / RNA splicing / positive regulation of RNA splicing / spliceosomal complex / RING-type E3 ubiquitin transferase / mRNA splicing, via spliceosome / metallopeptidase activity / ubiquitin-protein transferase activity / ubiquitin protein ligase activity / RNA helicase activity / RNA helicase / DNA repair / GTPase activity / mRNA binding / chromatin binding / GTP binding / chromatin / ATP hydrolysis activity / mitochondrion / DNA binding / RNA binding / zinc ion binding / ATP binding / metal ion binding / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
RNA Binding Protein, Prp18; Chain A / Functional domain of the splicing factor Prp18 / Prp18 / Pre-mRNA-splicing factor 18 / Prp18 domain / Splicing factor Yju2 / Saf4/Yju2 protein / Saf4/Yju2 protein / Pre-mRNA-splicing factor SLU7 domain / Pre-mRNA-splicing factor SLU7 ...RNA Binding Protein, Prp18; Chain A / Functional domain of the splicing factor Prp18 / Prp18 / Pre-mRNA-splicing factor 18 / Prp18 domain / Splicing factor Yju2 / Saf4/Yju2 protein / Saf4/Yju2 protein / Pre-mRNA-splicing factor SLU7 domain / Pre-mRNA-splicing factor SLU7 / Pre-mRNA splicing Prp18-interacting factor / DHX8/ Prp22, DEXH-box helicase domain / : / : / HAT (Half-A-TPR) repeat / cwf21 / Slt11, RNA recognition motif / Pre-mRNA-splicing factor Cwc2, RNA recognition motif / Torus domain / Torus domain / mRNA splicing factor SYF2 / SYF2 splicing factor / RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) / : / : / mRNA splicing factor Cwf21 domain / cwf21 domain / Pre-mRNA-processing factor 17 / : / : / Pre-mRNA-splicing factor SYF1 middle HAT repeat / Pre-mRNA-splicing factor 19 / Pre-mRNA-processing factor 19 / Prp19/Pso4-like / G10 protein signature 2. / BUD31/G10-related, conserved site / G10 protein signature 1. / : / : / Pre-mRNA-splicing factor Syf1/CRNKL1 C-terminal HAT repeat / : / STL11, N-terminal / : / Pre-mRNA-splicing factor Syf1/CNRKL1 N-terminal HAT repeat / 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 / WD repeat Prp46/PLRG1-like / Pre-mRNA-splicing factor Cwc2/Slt11 / G10 protein / Pre-mRNA-splicing factor BUD31 / Pre-mRNA splicing factor component Cdc5p/Cef1, C-terminal / : / : / pre-mRNA splicing factor component / Initiation factor eIF-4 gamma, MA3 / MA3 domain / MI domain profile. / Domain in DAP-5, eIF4G, MA-3 and other proteins. / U2 small nuclear ribonucleoprotein A' / Middle domain of eukaryotic initiation factor 4G (eIF4G) / MIF4G-like, type 3 / U-box domain profile. / U2A'/phosphoprotein 32 family A, C-terminal / occurring C-terminal to leucine-rich repeats / Modified RING finger domain / U-box domain / Leucine-rich repeat / 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 / Pre-mRNA-splicing factor Syf1-like / SH3 type barrels. - #100 / : / Helicase associated domain (HA2), ratchet-like / DEAD-box helicase, OB fold / Oligonucleotide/oligosaccharide-binding (OB)-fold / Helicase associated domain (HA2), winged-helix / Helicase-associated domain / Helicase associated domain (HA2) Add an annotation / Small nuclear ribonucleoprotein Sm D3 / Small nuclear ribonucleoprotein Sm D2 / Small nuclear ribonucleoprotein E / Small nuclear ribonucleoprotein G / Small nuclear ribonucleoprotein F / Sm-like protein Lsm7/SmG / Like-Sm (LSM) domain containing protein, LSm4/SmD1/SmD3 / Sm-like protein Lsm6/SmF / Myb-type HTH DNA-binding domain profile. / Zinc finger, CCCH-type / Zinc finger C3H1-type profile. / LSM domain / LSM domain, eukaryotic/archaea-type / snRNP Sm proteins / HAT (Half-A-TPR) repeat / HAT (Half-A-TPR) repeats / Myb domain
Similarity search - Domain/homology
GUANOSINE-5'-TRIPHOSPHATE / INOSITOL HEXAKISPHOSPHATE / : / : / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / : / Pre-mRNA-splicing factor ATP-dependent RNA helicase PRP22 ...GUANOSINE-5'-TRIPHOSPHATE / INOSITOL HEXAKISPHOSPHATE / : / : / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / : / Pre-mRNA-splicing factor ATP-dependent RNA helicase PRP22 / Pre-mRNA-splicing factor BUD31 / Pre-mRNA-processing protein 45 / Splicing factor YJU2 / Pre-mRNA-processing factor 19 / Pre-mRNA-splicing factor 8 / Pre-mRNA-splicing factor 18 / Pre-mRNA-splicing factor SNU114 / Pre-mRNA-splicing factor SLT11 / Small nuclear ribonucleoprotein-associated protein B / Small nuclear ribonucleoprotein G / U2 small nuclear ribonucleoprotein B'' / Pre-mRNA-processing factor 17 / Small nuclear ribonucleoprotein Sm D3 / Pre-mRNA-splicing factor SYF2 / Pre-mRNA-splicing factor CWC22 / Small nuclear ribonucleoprotein F / Small nuclear ribonucleoprotein Sm D1 / Pre-mRNA-splicing factor SLU7 / Pre-mRNA-splicing factor CWC21 / Pre-mRNA-splicing factor CEF1 / Pre-mRNA-splicing factor CWC15 / Pre-mRNA-splicing factor SYF1 / Pre-mRNA-splicing factor SNT309 / Small nuclear ribonucleoprotein Sm D2 / U2 small nuclear ribonucleoprotein A' / Pre-mRNA-splicing factor CWC2 / Pre-mRNA-splicing factor CLF1 / Small nuclear ribonucleoprotein E / Pre-mRNA-splicing factor PRP46
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288c (yeast)
Saccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsWilkinson, M.E. / Fica, S.M. / Galej, W.P. / Norman, C.M. / Newman, A.J. / Nagai, K.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
European Research CouncilSPLICE3D United Kingdom
Medical Research Council (United Kingdom)MC_U105184330 United Kingdom
CitationJournal: Science / Year: 2017
Title: Postcatalytic spliceosome structure reveals mechanism of 3'-splice site selection.
Authors: Max E Wilkinson / Sebastian M Fica / Wojciech P Galej / Christine M Norman / Andrew J Newman / Kiyoshi Nagai /
Abstract: Introns are removed from eukaryotic messenger RNA precursors by the spliceosome in two transesterification reactions-branching and exon ligation. The mechanism of 3'-splice site recognition during ...Introns are removed from eukaryotic messenger RNA precursors by the spliceosome in two transesterification reactions-branching and exon ligation. The mechanism of 3'-splice site recognition during exon ligation has remained unclear. Here we present the 3.7-angstrom cryo-electron microscopy structure of the yeast P-complex spliceosome immediately after exon ligation. The 3'-splice site AG dinucleotide is recognized through non-Watson-Crick pairing with the 5' splice site and the branch-point adenosine. After the branching reaction, protein factors work together to remodel the spliceosome and stabilize a conformation competent for 3'-splice site docking, thereby promoting exon ligation. The structure accounts for the strict conservation of the GU and AG dinucleotides at the 5' and 3' ends of introns and provides insight into the catalytic mechanism of exon ligation.
History
DepositionNov 8, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 17, 2018Provider: repository / Type: Initial release
Revision 1.1Jan 31, 2018Group: Structure summary / Category: audit_author / Item: _audit_author.identifier_ORCID
Revision 1.2Oct 24, 2018Group: Advisory / Data collection / Derived calculations
Category: pdbx_unobs_or_zero_occ_atoms / pdbx_validate_close_contact / struct_conn
Revision 1.3Nov 21, 2018Group: Advisory / Data collection / Derived calculations
Category: pdbx_validate_close_contact / struct_conn / struct_conn_type
Item: _struct_conn_type.id
Revision 1.4Dec 11, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 2.0Oct 7, 2020Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Non-polymer description / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / entity / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / struct_conn / struct_conn_type / struct_site
Item: _atom_site.Cartn_x / _atom_site.Cartn_y ..._atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.pdbx_formal_charge / _atom_site.type_symbol / _chem_comp.id / _chem_comp.name / _chem_comp.pdbx_synonyms / _entity.pdbx_description / _pdbx_entity_nonpoly.comp_id / _pdbx_entity_nonpoly.name / _pdbx_nonpoly_scheme.mon_id / _pdbx_nonpoly_scheme.pdb_mon_id / _struct_conn.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_conn_type.id / _struct_site.details / _struct_site.pdbx_auth_comp_id

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Structure visualization

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Assembly

Deposited unit
2: U2 snRNA
5: U5 snRNA
6: U6 snRNA
A: Pre-mRNA-splicing factor Prp8
C: Pre-mRNA-splicing factor SNU114
D: Pre-mRNA-splicing factor CWC16
E: Ligated exons: UBC4 mRNA
H: Pre-mRNA-splicing factor CWC22
I: Intron lariat: UBC4 RNA
J: Pre-mRNA-splicing factor PRP46
K: Pre-mRNA-processing protein 45
L: Pre-mRNA-splicing factor BUD31
M: Pre-mRNA-splicing factor CWC2
N: Pre-mRNA-splicing factor SLT11
O: Pre-mRNA-splicing factor CEF1
P: Pre-mRNA-splicing factor CWC15
R: Pre-mRNA-splicing factor CWC21
S: Pre-mRNA-splicing factor CLF1
T: Pre-mRNA-splicing factor SYF1
V: Pre-mRNA-splicing factor ATP-dependent RNA helicase PRP22
W: U2 small nuclear ribonucleoprotein A'
X: Unassigned structure
Y: U2 small nuclear ribonucleoprotein B''
a: Pre-mRNA-splicing factor Prp18
b: Small nuclear ribonucleoprotein-associated protein B
c: Pre-mRNA-splicing factor SLU7
d: Small nuclear ribonucleoprotein Sm D3
e: Small nuclear ribonucleoprotein E
f: Small nuclear ribonucleoprotein F
g: Small nuclear ribonucleoprotein G
h: Small nuclear ribonucleoprotein Sm D1
j: Small nuclear ribonucleoprotein Sm D2
k: Small nuclear ribonucleoprotein-associated protein B
l: Small nuclear ribonucleoprotein Sm D1
m: Small nuclear ribonucleoprotein Sm D2
n: Small nuclear ribonucleoprotein Sm D3
o: Pre-mRNA-processing factor Prp17
p: Small nuclear ribonucleoprotein E
q: Small nuclear ribonucleoprotein F
r: Small nuclear ribonucleoprotein G
s: Pre-mRNA-splicing factor SNT309
t: Pre-mRNA-processing factor Prp19
u: Pre-mRNA-processing factor Prp19
v: Pre-mRNA-processing factor Prp19
w: Pre-mRNA-processing factor Prp19
y: Pre-mRNA-splicing factor SYF2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)2,244,12756
Polymers2,242,46146
Non-polymers1,66510
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area200780 Å2
ΔGint-1080 kcal/mol
Surface area402940 Å2
MethodPISA

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Components

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RNA chain , 5 types, 5 molecules 256EI

#1: RNA chain U2 snRNA


Mass: 376267.406 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288c (yeast) / References: TPG: 329136699
#2: RNA chain U5 snRNA


Mass: 57444.875 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288c (yeast) / References: GenBank: 1039024057
#3: RNA chain U6 snRNA


Mass: 35883.176 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288c (yeast) / References: GenBank: 1039023280
#7: RNA chain Ligated exons: UBC4 mRNA


Mass: 12523.501 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Production host: Transcription vector pUC18-pES2 (others)
#9: RNA chain Intron lariat: UBC4 RNA


Mass: 30200.730 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288c (yeast) / Production host: Transcription vector pUC18-pES2 (others) / References: TPG: 329136699

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Pre-mRNA-splicing factor ... , 18 types, 18 molecules ACDHJLMNOPRSTVacsy

#4: Protein Pre-mRNA-splicing factor Prp8


Mass: 279867.469 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P33334
#5: Protein Pre-mRNA-splicing factor SNU114 / 114 kDa U5 small nuclear ribonucleoprotein component / Growth inhibitory protein 10


Mass: 114174.008 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P36048
#6: Protein Pre-mRNA-splicing factor CWC16


Mass: 32371.086 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P28320
#8: Protein Pre-mRNA-splicing factor CWC22 / Complexed with CEF1 protein 22


Mass: 67386.062 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / Strain: ATCC 204508 / S288c / References: UniProt: P53333
#10: Protein Pre-mRNA-splicing factor PRP46 / Complexed with CEF1 protein 1 / PRP nineteen-associated complex protein 50 / PRP19-associated ...Complexed with CEF1 protein 1 / PRP nineteen-associated complex protein 50 / PRP19-associated complex protein 50 / Pre-mRNA-processing protein 46


Mass: 50801.316 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: Q12417
#12: Protein Pre-mRNA-splicing factor BUD31 / Bud site selection protein 31 / Complexed with CEF1 protein 14


Mass: 18484.502 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P25337
#13: Protein Pre-mRNA-splicing factor CWC2 / Complexed with CEF1 protein 2 / PRP19-associated complex protein 40 / Synthetic lethal with CLF1 protein 3


Mass: 38486.562 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: Q12046
#14: Protein Pre-mRNA-splicing factor SLT11 / Extracellular mutant protein 2 / Synthetic lethality with U2 protein 11


Mass: 40988.590 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P38241
#15: Protein Pre-mRNA-splicing factor CEF1 / PRP nineteen-associated complex protein 85 / PRP19-associated complex protein 85


Mass: 67829.812 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: Q03654
#16: Protein Pre-mRNA-splicing factor CWC15 / Complexed with CEF1 protein 15


Mass: 19970.195 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / Strain: ATCC 204508 / S288c / References: UniProt: Q03772
#17: Protein Pre-mRNA-splicing factor CWC21 / Complexed with CEF1 protein 21


Mass: 15793.596 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: Q03375
#18: Protein Pre-mRNA-splicing factor CLF1 / Crooked neck-like factor 1 / PRP19-associated complex protein 77 / Synthetic lethal with CDC40 protein 3


Mass: 82560.922 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: Q12309
#19: Protein Pre-mRNA-splicing factor SYF1 / PRP19-associated complex protein 90 / Synthetic lethal with CDC40 protein 1


Mass: 100356.070 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: Q04048
#20: Protein Pre-mRNA-splicing factor ATP-dependent RNA helicase PRP22


Mass: 130187.359 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P24384, RNA helicase
#24: Protein Pre-mRNA-splicing factor Prp18


Mass: 28414.391 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P33411
#26: Protein Pre-mRNA-splicing factor SLU7 / Synthetic lethal with U2 snRNA protein 17 / Synthetic lethal with U5 snRNA protein 7


Mass: 44722.875 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: Q02775
#34: Protein Pre-mRNA-splicing factor SNT309 / PRP19-associated complex protein 25 / Synergistic to PRP19 mutation protein 309


Mass: 20741.455 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: Q06091
#36: Protein Pre-mRNA-splicing factor SYF2 / PRP19 complex protein 31 / Synthetic lethal with CDC40 protein 2


Mass: 24850.719 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P53277

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Protein , 3 types, 4 molecules KXbk

#11: Protein Pre-mRNA-processing protein 45


Mass: 42548.727 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P28004
#22: Protein Unassigned structure


Mass: 8102.979 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288c (yeast)
#25: Protein Small nuclear ribonucleoprotein-associated protein B / snRNP-B / Sm protein B / SmB


Mass: 22426.990 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P40018

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U2 small nuclear ribonucleoprotein ... , 2 types, 2 molecules WY

#21: Protein U2 small nuclear ribonucleoprotein A' / U2 snRNP A' / Looks exceptionally like U2A protein 1


Mass: 27232.252 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: Q08963
#23: Protein U2 small nuclear ribonucleoprotein B'' / U2 snRNP B''


Mass: 12850.944 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P40567

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Small nuclear ribonucleoprotein ... , 6 types, 12 molecules dnepfqgrhljm

#27: Protein Small nuclear ribonucleoprotein Sm D3 / Sm-D3 / snRNP core protein D3


Mass: 11240.139 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P43321
#28: Protein Small nuclear ribonucleoprotein E / snRNP-E / Sm protein E / SmE


Mass: 10385.098 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: Q12330
#29: Protein Small nuclear ribonucleoprotein F / snRNP-F / Sm protein F / SmF


Mass: 9669.945 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P54999
#30: Protein Small nuclear ribonucleoprotein G / snRNP-G / Sm protein G / SmG


Mass: 8490.809 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P40204
#31: Protein Small nuclear ribonucleoprotein Sm D1 / Sm-D1 / snRNP core protein D1


Mass: 16296.798 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: Q02260
#32: Protein Small nuclear ribonucleoprotein Sm D2 / Sm-D2 / snRNP core protein D2


Mass: 12876.066 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: Q06217

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Pre-mRNA-processing factor ... , 2 types, 5 molecules otuvw

#33: Protein Pre-mRNA-processing factor Prp17 / Cell division control protein 40


Mass: 52128.762 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P40968
#35: Protein
Pre-mRNA-processing factor Prp19 / RING-type E3 ubiquitin transferase PRP19


Mass: 56629.777 Da / Num. of mol.: 4 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
References: UniProt: P32523, RING-type E3 ubiquitin transferase

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Non-polymers , 4 types, 10 molecules

#37: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#38: 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
#39: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


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


Mass: 65.409 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: Zn

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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
IDNameTypeDetailsEntity IDParent-IDSource
1P complex spliceosome with 3' exon dockedCOMPLEXP complex was assembled in vitro in splicing extract supplemented with dominant negative mutant Prp22 protein. P complex was purified via MS2-MBP fusion protein bound to 3 MS2 hairpin loops on the mRNA.#1-#360MULTIPLE SOURCES
2P complex spliceosomeCOMPLEX#1-#6, #8, #10-#361NATURAL
33' exonCOMPLEX#7, #91RECOMBINANT
Molecular weightValue: 2.2 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)559292
33Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)559292
Source (recombinant)Organism: Transcription vector pUC18-pES2 (others)
Buffer solutionpH: 7.9
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES.KOH pH 7.91
275 mMPotassium chlorideKCl1
30.25 mMEDTA1
SpecimenConc.: 1.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: 3 uL sample was applied to the grid, left for 30s, then blotted for 3s and immediately plunged 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Calibrated defocus min: 200 nm / Calibrated defocus max: 3000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 12 sec. / Electron dose: 47 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2295
Image scansMovie frames/image: 20 / Used frames/image: 1-20

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Processing

EM software
IDNameVersionCategoryDetails
1Gautomatchparticle selection
2EPUimage acquisition
4GctfCTF correction
9RELION2.1initial Euler assignment
10RELION2.1final Euler assignmentpatched version
12RELION2.13D reconstructionpatched version
13PHENIXdev-2919model refinementphenix.real_space_refine
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 206529
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 48617 / Symmetry type: POINT

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