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- PDB-5lj5: Overall structure of the yeast spliceosome immediately after bran... -

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Basic information

Entry
Database: PDB / ID: 5lj5
TitleOverall structure of the yeast spliceosome immediately after branching.
Components
  • (Pre-mRNA-processing ...) x 2
  • (Pre-mRNA-splicing factor ...) x 14
  • (Small nuclear ribonucleoprotein ...) x 6
  • (U2 small nuclear ribonucleoprotein ...) x 2
  • CWC15
  • CWC22
  • Exon 1 (5' exon) of UBC4 pre-mRNA
  • Intron of UBC4 pre-mRNA
  • Pre-mRNA-splicing helicase BRR2
  • Protein CWC16
  • Small nuclear ribonucleoprotein-associated protein B
  • U2 snRNA (small nuclear RNA)
  • U5 snRNA (small nuclear RNA)
  • U6 snRNA (small nuclear RNA)
  • unknown
KeywordsSPLICING / spliceosome / snRNP / pre-mRNA splicing / trans-esterification / lariat intermediate / complex C
Function / homology
Function and homology information


RNA exon ligation / snRNA metabolic process / snRNA modification / post-spliceosomal complex / U2-type post-mRNA release spliceosomal complex / cellular bud site selection / post-mRNA release spliceosomal complex / 3'-5' RNA helicase activity / generation of catalytic spliceosome for first transesterification step / cis assembly of pre-catalytic spliceosome ...RNA exon ligation / snRNA metabolic process / snRNA modification / post-spliceosomal complex / U2-type post-mRNA release spliceosomal complex / cellular bud site selection / post-mRNA release spliceosomal complex / 3'-5' RNA helicase activity / generation of catalytic spliceosome for first transesterification step / cis assembly of pre-catalytic spliceosome / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) / U4/U6 snRNP / 7-methylguanosine cap hypermethylation / ATP-dependent activity, acting on RNA / pICln-Sm protein complex / U2-type catalytic step 1 spliceosome / pre-mRNA binding / snRNP binding / splicing factor binding / SMN-Sm protein complex / small nuclear ribonucleoprotein complex / spliceosomal tri-snRNP complex / commitment complex / mRNA cis splicing, via spliceosome / U2-type spliceosomal complex / U2-type prespliceosome assembly / U2-type catalytic step 2 spliceosome / U2 snRNP / U1 snRNP / U4 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 / mRNA 5'-splice site recognition / spliceosomal complex assembly / mRNA 3'-splice site recognition / Gap-filling DNA repair synthesis and ligation in TC-NER / DNA replication origin binding / spliceosomal tri-snRNP complex assembly / Dual incision in TC-NER / Prp19 complex / U5 snRNA binding / protein K63-linked ubiquitination / U5 snRNP / DNA replication initiation / 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 / RNA splicing / positive regulation of RNA splicing / spliceosomal complex / mRNA splicing, via spliceosome / RING-type E3 ubiquitin transferase / metallopeptidase activity / ubiquitin-protein transferase activity / ubiquitin protein ligase activity / nucleic acid binding / RNA helicase activity / RNA helicase / DNA repair / GTPase activity / mRNA binding / chromatin binding / chromatin / GTP binding / ATP hydrolysis activity / mitochondrion / DNA binding / RNA binding / zinc ion binding / ATP binding / metal ion binding / identical protein binding / nucleus / cytoplasm / cytosol
Similarity search - Function
CBF1-interacting co-repressor CIR, N-terminal domain / Pre-mRNA splicing factor / : / N-terminal domain of CBF1 interacting co-repressor CIR / Pre-mRNA splicing factor / N-terminal domain of CBF1 interacting co-repressor CIR / Sec63 N-terminal domain-like domain / Splicing factor Yju2 / Saf4/Yju2 protein / Saf4/Yju2 protein ...CBF1-interacting co-repressor CIR, N-terminal domain / Pre-mRNA splicing factor / : / N-terminal domain of CBF1 interacting co-repressor CIR / Pre-mRNA splicing factor / N-terminal domain of CBF1 interacting co-repressor CIR / Sec63 N-terminal domain-like domain / Splicing factor Yju2 / Saf4/Yju2 protein / Saf4/Yju2 protein / Sec63 N-terminal domain-like fold / HAT (Half-A-TPR) repeat / Pre-mRNA-splicing factor Isy1 / Pre-mRNA-splicing factor Isy1 superfamily / Isy1-like splicing family / cwf21 / Slt11, RNA recognition motif / Pre-mRNA-splicing factor Cwc2, RNA recognition motif / Torus domain / Torus domain / RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) / Helix hairpin bin domain superfamily / : / mRNA splicing factor Cwf21 domain / cwf21 domain / Pre-mRNA-splicing factor 19 / Pre-mRNA-processing factor 19 / : / Prp19/Pso4-like / : / Pre-mRNA-splicing factor SYF1 middle HAT repeat / G10 protein signature 2. / BUD31/G10-related, conserved site / G10 protein signature 1. / : / : / STL11, N-terminal / : / Pre-mRNA-splicing factor Syf1/CRNKL1 C-terminal HAT repeat / SKI-interacting protein SKIP, SNW domain / SKI-interacting protein, SKIP / SKIP/SNW domain / : / Pre-mRNA-splicing factor Syf1/CNRKL1 N-terminal HAT repeat / 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 / U2 small nuclear ribonucleoprotein A' / U2A'/phosphoprotein 32 family A, C-terminal / occurring C-terminal to leucine-rich repeats / Brr2, N-terminal helicase PWI domain / : / N-terminal helicase PWI domain / Pre-mRNA-splicing helicase BRR2 plug domain / U-box domain profile. / Winged Helix-turn-helix domain / Sec63 Brl domain / : / 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 / Sec63 domain / Sec63 Brl domain / : / Small nuclear ribonucleoprotein Sm D3 / Helicase associated domain (HA2), ratchet-like / DEAD-box helicase, OB fold / Oligonucleotide/oligosaccharide-binding (OB)-fold / Helicase associated domain (HA2), winged-helix / Small nuclear ribonucleoprotein Sm D2 / Small nuclear ribonucleoprotein E / Helicase-associated domain / Small nuclear ribonucleoprotein G / Helicase associated domain (HA2) Add an annotation / 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 / : / Sm domain profile.
Similarity search - Domain/homology
GUANOSINE-5'-TRIPHOSPHATE / : / : / : / : / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / : ...GUANOSINE-5'-TRIPHOSPHATE / : / : / : / : / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / : / : / Pre-mRNA-splicing factor ATP-dependent RNA helicase PRP16 / Pre-mRNA-splicing factor ISY1 / Pre-mRNA-splicing factor BUD31 / Pre-mRNA-processing protein 45 / Splicing factor YJU2 / Pre-mRNA-processing factor 19 / Pre-mRNA-splicing helicase BRR2 / Pre-mRNA-splicing factor 8 / 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'' / Small nuclear ribonucleoprotein Sm D3 / Pre-mRNA-splicing factor CWC25 / Small nuclear ribonucleoprotein F / Small nuclear ribonucleoprotein Sm D1 / Pre-mRNA-splicing factor CWC21 / Pre-mRNA-splicing factor CEF1 / 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 (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 10 Å
AuthorsGalej, W.P. / Wilkinson, M.F. / Fica, S.M. / Oubridge, C. / Newman, A.J. / Nagai, K.
CitationJournal: Nature / Year: 2016
Title: Cryo-EM structure of the spliceosome immediately after branching.
Authors: Wojciech P Galej / Max E Wilkinson / Sebastian M Fica / Chris Oubridge / Andrew J Newman / Kiyoshi Nagai /
Abstract: Precursor mRNA (pre-mRNA) splicing proceeds by two consecutive transesterification reactions via a lariat-intron intermediate. Here we present the 3.8 Å cryo-electron microscopy structure of the ...Precursor mRNA (pre-mRNA) splicing proceeds by two consecutive transesterification reactions via a lariat-intron intermediate. Here we present the 3.8 Å cryo-electron microscopy structure of the spliceosome immediately after lariat formation. The 5'-splice site is cleaved but remains close to the catalytic Mg site in the U2/U6 small nuclear RNA (snRNA) triplex, and the 5'-phosphate of the intron nucleotide G(+1) is linked to the branch adenosine 2'OH. The 5'-exon is held between the Prp8 amino-terminal and linker domains, and base-pairs with U5 snRNA loop 1. Non-Watson-Crick interactions between the branch helix and 5'-splice site dock the branch adenosine into the active site, while intron nucleotides +3 to +6 base-pair with the U6 snRNA ACAGAGA sequence. Isy1 and the step-one factors Yju2 and Cwc25 stabilize docking of the branch helix. The intron downstream of the branch site emerges between the Prp8 reverse transcriptase and linker domains and extends towards the Prp16 helicase, suggesting a plausible mechanism of remodelling before exon ligation.
History
DepositionJul 17, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 31, 2016Provider: repository / Type: Initial release
Revision 1.1Sep 21, 2016Group: Database references
Revision 1.2Aug 30, 2017Group: Data collection / Derived calculations / Category: em_imaging_optics / em_software / struct_conn
Item: _em_imaging_optics.energyfilter_name / _em_software.name
Revision 1.3Oct 3, 2018Group: Advisory / Data collection / Refinement description
Category: pdbx_unobs_or_zero_occ_atoms / refine ...pdbx_unobs_or_zero_occ_atoms / refine / refine_hist / refine_ls_restr / refine_ls_shell
Item: _refine.pdbx_refine_id / _refine_hist.pdbx_refine_id ..._refine.pdbx_refine_id / _refine_hist.pdbx_refine_id / _refine_ls_restr.pdbx_refine_id / _refine_ls_shell.pdbx_refine_id
Revision 1.4Oct 24, 2018Group: Advisory / Data collection / Derived calculations
Category: pdbx_unobs_or_zero_occ_atoms / pdbx_validate_close_contact ...pdbx_unobs_or_zero_occ_atoms / pdbx_validate_close_contact / struct_conn / struct_conn_type
Revision 1.5Dec 11, 2019Group: Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB
Revision 1.6Nov 13, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature / refine / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update / _refine.ls_d_res_high / _refine.ls_d_res_low / _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

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
U: U5 snRNA (small nuclear RNA)
E: Exon 1 (5' exon) of UBC4 pre-mRNA
I: Intron of UBC4 pre-mRNA
Z: U2 snRNA (small nuclear RNA)
V: U6 snRNA (small nuclear RNA)
A: Pre-mRNA-splicing factor 8
B: Pre-mRNA-splicing helicase BRR2
D: Protein CWC16
F: Pre-mRNA-splicing factor CWC25
C: Pre-mRNA-splicing factor SNU114
G: Pre-mRNA-splicing factor ISY1
H: CWC22
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: CWC15
R: Pre-mRNA-splicing factor CWC21
S: Pre-mRNA-splicing factor CLF1
T: Pre-mRNA-splicing factor SYF1
b: Small nuclear ribonucleoprotein-associated protein B
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
W: U2 small nuclear ribonucleoprotein A'
Y: U2 small nuclear ribonucleoprotein B''
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
p: Small nuclear ribonucleoprotein E
q: Small nuclear ribonucleoprotein F
r: Small nuclear ribonucleoprotein G
Q: Pre-mRNA-splicing factor ATP-dependent RNA helicase PRP16
t: Pre-mRNA-processing factor 19
u: Pre-mRNA-processing factor 19
v: Pre-mRNA-processing factor 19
w: Pre-mRNA-processing factor 19
s: Pre-mRNA-splicing factor SNT309
x: unknown
hetero molecules


Theoretical massNumber of molelcules
Total (without water)2,376,12255
Polymers2,375,09245
Non-polymers1,03010
Water00
1


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

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Components

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RNA chain , 5 types, 5 molecules UEIZV

#1: RNA chain U5 snRNA (small nuclear RNA)


Mass: 57444.875 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: GenBank: 1039023403
#2: RNA chain Exon 1 (5' exon) of UBC4 pre-mRNA


Mass: 5170.152 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast)
#3: RNA chain Intron of UBC4 pre-mRNA


Mass: 24109.115 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: GenBank: 4718
#4: RNA chain U2 snRNA (small nuclear RNA)


Mass: 376267.406 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: GenBank: 536627
#5: RNA chain U6 snRNA (small nuclear RNA)


Mass: 35883.176 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: GenBank: 1039022925

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Pre-mRNA-splicing factor ... , 14 types, 14 molecules AFCGJLMNORSTQs

#6: Protein Pre-mRNA-splicing factor 8


Mass: 279867.469 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P33334
#9: Protein Pre-mRNA-splicing factor CWC25 / Complexed with CEF1 protein 25


Mass: 20412.477 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P53854
#10: 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 (brewer's yeast) / References: UniProt: P36048
#11: Protein Pre-mRNA-splicing factor ISY1 / Interactor of SYF1 / PRP19-associated complex protein 30


Mass: 28073.836 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P21374
#13: 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: 50771.289 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q12417
#15: 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 (brewer's yeast) / References: UniProt: P25337
#16: 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 (brewer's yeast) / References: UniProt: Q12046
#17: 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 (brewer's yeast) / References: UniProt: P38241
#18: Protein Pre-mRNA-splicing factor CEF1 / PRP nineteen-associated complex protein 85 / PRP19-associated complex protein 85


Mass: 67837.773 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q03654
#20: 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 (brewer's yeast) / References: UniProt: Q03375
#21: Protein Pre-mRNA-splicing factor CLF1 / Crooked neck-like factor 1 / PRP19-associated complex protein 77 / Synthetic lethal with CDC40 protein 3


Mass: 82555.859 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q12309
#22: Protein Pre-mRNA-splicing factor SYF1 / PRP19-associated complex protein 90 / Synthetic lethal with CDC40 protein 1


Mass: 100344.016 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q04048
#32: Protein Pre-mRNA-splicing factor ATP-dependent RNA helicase PRP16


Mass: 121815.234 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P15938, RNA helicase
#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 (brewer's yeast) / References: UniProt: Q06091

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Protein , 6 types, 7 molecules BDHPbkx

#7: Protein Pre-mRNA-splicing helicase BRR2 / Protein Snu246


Mass: 246470.266 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P32639, RNA helicase
#8: Protein Protein CWC16


Mass: 32371.086 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P28320
#12: Protein CWC22


Mass: 69118.984 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A162ERE7
#19: Protein CWC15


Mass: 19970.195 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A162GIZ3
#23: 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 (brewer's yeast) / References: UniProt: P40018
#35: Protein unknown


Mass: 16017.708 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast)

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Pre-mRNA-processing ... , 2 types, 5 molecules Ktuvw

#14: 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 (brewer's yeast) / References: UniProt: P28004
#33: Protein
Pre-mRNA-processing factor 19


Mass: 56629.777 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast)
References: UniProt: P32523, Ligases; Forming carbon-nitrogen bonds; Acid-amino-acid ligases (peptide synthases)

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

#24: 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 (brewer's yeast) / References: UniProt: P43321
#25: 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 (brewer's yeast) / References: UniProt: Q12330
#26: 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 (brewer's yeast) / References: UniProt: P54999
#27: 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 (brewer's yeast) / References: UniProt: P40204
#28: 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 (brewer's yeast) / References: UniProt: Q02260
#29: 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 (brewer's yeast) / References: UniProt: Q06217

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

#30: 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 (brewer's yeast) / References: UniProt: Q08963
#31: 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 (brewer's yeast) / References: UniProt: P40567

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Non-polymers , 3 types, 10 molecules

#36: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#37: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: Zn
#38: 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

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Details

Has protein modificationY

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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: Spliceosome immediately after branching / Type: COMPLEX
Details: Splicing extract was prepared from Prp18-HA or Slu7-TAPS yeast strains. An in vitro transcribed yeast UBC4 pre-mRNA substrate (with 2 x MS2 bacteriophage coat protein-binding stem loops at ...Details: Splicing extract was prepared from Prp18-HA or Slu7-TAPS yeast strains. An in vitro transcribed yeast UBC4 pre-mRNA substrate (with 2 x MS2 bacteriophage coat protein-binding stem loops at the 5' end and with the 3'-splice site sequence UAGAG mutated to UACAC) was pre-bound to an MS2-maltose binding protein fusion protein. This substrate-protein complex was added to the splicing extract. The splicing reaction proceeded through the first step but the second step was blocked by the 3' splice site mutation. Substrate-bound spliceosomes from the splicing extract were purified on amylose resin and eluted with maltose. Subsequently the spliceosomes were captured on anti-HA-agarose (for Prp18-HA-tagged) or streptactin resin (for Slu7-TAPS tagged) and eluted with HA peptide or desthiobiotin, respectively. Purified spliceosomes were then dialysed against 20 mM HEPES KOH pH 7.8, 75 mM KCl, 0.25 mM EDTA
Entity ID: #1-#35 / Source: NATURAL
Molecular weightValue: 2 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHepes.KOH pH 7.81
275 mMpotassium chlorideKCl1
3250 micromolarEDTA1
41
51
SpecimenConc.: 0.3 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 microlitres sample were applied to the grid, left for 30 seconds and then blotted for 2.5-3.0 seconds before plunging.

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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: 81000 X / Calibrated magnification: 35714 X / Nominal defocus max: 4000 nm / Nominal defocus min: 500 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 0.8 sec. / Electron dose: 2 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of real images: 2213
Details: Total dose: 40 electrons/Angstrom^2 over 16 seconds. 20 movie frames collected at 1.25 frames per second.
EM imaging opticsEnergyfilter name: GIF Quantum
Image scansMovie frames/image: 20 / Used frames/image: 1-20

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Processing

SoftwareName: REFMAC / Version: 5.8.0124 / Classification: refinement
EM software
IDNameVersionCategoryDetails
3CTFFIND4CTF correction
6Coot0.8.3model fittingPaul Emsley's experimental version.
8REFMAC5.8model refinement
9RELION1.4initial Euler assignment
10RELION1.4final Euler assignment
11RELION1.4classification
12RELION1.43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 15872
3D reconstructionResolution: 10 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 15872 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: RECIPROCAL
Details: Used secondary structure restraints generated in ProSMART and LibG.
RefinementResolution: 10→10 Å / Cor.coef. Fo:Fc: 0.954 / SU B: 27.594 / SU ML: 0.398 / ESU R: 0.522
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.31901 --
obs0.31901 592776 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 180.1 Å2
Baniso -1Baniso -2Baniso -3
1-1.84 Å21.69 Å2-1.19 Å2
2---1.95 Å2-1.26 Å2
3---0.11 Å2
Refinement stepCycle: 1 / Total: 63166
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0070.01865428
ELECTRON MICROSCOPYr_bond_other_d0.0020.0253593
ELECTRON MICROSCOPYr_angle_refined_deg1.2521.83290631
ELECTRON MICROSCOPYr_angle_other_deg1.023122463
ELECTRON MICROSCOPYr_dihedral_angle_1_deg9.4125.2417501
ELECTRON MICROSCOPYr_dihedral_angle_2_deg36.16323.6681963
ELECTRON MICROSCOPYr_dihedral_angle_3_deg18.101157986
ELECTRON MICROSCOPYr_dihedral_angle_4_deg13.28515310
ELECTRON MICROSCOPYr_chiral_restr0.0890.20310628
ELECTRON MICROSCOPYr_gen_planes_refined0.0050.0268092
ELECTRON MICROSCOPYr_gen_planes_other0.0020.0214494
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it4.93120.95729635
ELECTRON MICROSCOPYr_mcbond_other4.9320.95729634
ELECTRON MICROSCOPYr_mcangle_it8.83931.34736846
ELECTRON MICROSCOPYr_mcangle_other8.83931.34836847
ELECTRON MICROSCOPYr_scbond_it5.32920.04235793
ELECTRON MICROSCOPYr_scbond_other5.32920.04235794
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other9.20830.54753786
ELECTRON MICROSCOPYr_long_range_B_refined19.477137799
ELECTRON MICROSCOPYr_long_range_B_other19.477137800
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.8→3.899 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork0.563 44142 -
Rfree-0 -
obs--100 %

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