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- PDB-5nrl: Structure of a pre-catalytic spliceosome -

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Entry
Database: PDB / ID: 5nrl
TitleStructure of a pre-catalytic spliceosome
Components
  • (Pre-mRNA-splicing factor ...) x 10
  • (Small nuclear ribonucleoprotein ...SnRNP) x 6
  • (U2 small nuclear ribonucleoprotein ...) x 2
  • (U4/U6 small nuclear ribonucleoprotein ...) x 2
  • (U6 snRNA-associated Sm-like protein ...) x 7
  • 13 kDa ribonucleoprotein-associated protein
  • 23 kDa U4/U6.U5 small nuclear ribonucleoprotein component
  • 66 kDa U4/U6.U5 small nuclear ribonucleoprotein component
  • Cold sensitive U2 snRNA suppressor 1
  • Pre-mRNA-processing factor 31
  • Pre-mRNA-splicing helicase BRR2
  • Protein HSH49
  • RDS3 complex subunit 10
  • Small nuclear ribonucleoprotein-associated protein B
  • Spliceosomal protein DIB1Spliceosome
  • U2 snRNAU2 spliceosomal RNA
  • U2 snRNP component HSH155
  • U4 snRNAU4 spliceosomal RNA
  • U5 snRNAU5 spliceosomal RNA
  • U6 snRNAU6 spliceosomal RNA
  • Unknown
  • Yeast UBC4 gene for ubiquitin-conjugating enzyme
KeywordsSPLICING / Spliceosome / Ribonucleoprotein
Function / homology
Function and homology information


maturation of 5S rRNA / response to xenobiotic stimulus / spliceosomal conformational changes to generate catalytic conformation / U4/U6 snRNP / snoRNA splicing / Lsm1-7-Pat1 complex / U6 snRNP / rRNA 2'-O-methylation / positive regulation of RNA binding / generation of catalytic spliceosome for first transesterification step ...maturation of 5S rRNA / response to xenobiotic stimulus / spliceosomal conformational changes to generate catalytic conformation / U4/U6 snRNP / snoRNA splicing / Lsm1-7-Pat1 complex / U6 snRNP / rRNA 2'-O-methylation / positive regulation of RNA binding / generation of catalytic spliceosome for first transesterification step / box C/D RNP complex / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) / splicing factor binding / U2-type spliceosomal complex / P-body assembly / pICln-Sm protein complex / U4 snRNP / nuclear-transcribed mRNA catabolic process / small nuclear ribonucleoprotein complex / spliceosomal tri-snRNP complex / SMN-Sm protein complex / U4 snRNA binding / commitment complex / U2 snRNP / U2-type catalytic step 2 spliceosome / mRNA cis splicing, via spliceosome / U12-type spliceosomal complex / U3 snoRNA binding / poly(A) binding / generation of catalytic spliceosome for second transesterification step / spliceosomal complex assembly / U2-type prespliceosome / spliceosomal snRNP assembly / sno(s)RNA-containing ribonucleoprotein complex / U1 snRNP / RNA metabolic process / mRNA 3'-splice site recognition / precatalytic spliceosome / mRNA 5'-splice site recognition / tRNA processing / spliceosomal tri-snRNP complex assembly / rRNA methylation / U5 snRNP / U5 snRNA binding / poly(U) RNA binding / U2 snRNA binding / U6 snRNA binding / pre-mRNA intronic binding / maturation of SSU-rRNA / U4/U6 x U5 tri-snRNP complex / U1 snRNA binding / small-subunit processome / isopeptidase activity / catalytic step 2 spliceosome / maturation of LSU-rRNA / spliceosomal complex / mRNA 3'-UTR binding / P-body / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / rRNA processing / metallopeptidase activity / mRNA splicing, via spliceosome / ribonucleoprotein complex / RNA helicase / RNA helicase activity / GTPase activity / mRNA binding / GTP binding / nucleolus / mitochondrion / RNA binding / zinc ion binding / nucleoplasm / ATP binding / identical protein binding / metal ion binding / nucleus / cytosol / cytoplasm
Splicing factor 3B subunit 10 (SF3b10) / 116kDa U5 small nuclear ribonucleoprotein component, N-terminal / U4/U6 small nuclear ribonucleoprotein Prp31 / Pre-mRNA-processing factor 6/Prp1/STA1 / Like-Sm (LSM) domain containing protein, LSm4/SmD1/SmD3 / Small nuclear ribonucleoprotein Sm D2 / P-loop containing nucleoside triphosphate hydrolase / Pre-mRNA-processing-splicing factor 8 / 50S ribosomal protein L30e-like / Pre-mRNA-splicing factor Prp9, N-terminal ...Splicing factor 3B subunit 10 (SF3b10) / 116kDa U5 small nuclear ribonucleoprotein component, N-terminal / U4/U6 small nuclear ribonucleoprotein Prp31 / Pre-mRNA-processing factor 6/Prp1/STA1 / Like-Sm (LSM) domain containing protein, LSm4/SmD1/SmD3 / Small nuclear ribonucleoprotein Sm D2 / P-loop containing nucleoside triphosphate hydrolase / Pre-mRNA-processing-splicing factor 8 / 50S ribosomal protein L30e-like / Pre-mRNA-splicing factor Prp9, N-terminal / SF3A3 domain / Splicing factor 3A subunit 3 / SF3A2 domain / Leucine-rich repeat domain superfamily / Small nuclear ribonucleoprotein E / Sm-like protein LSm5 / Small nuclear ribonucleoprotein G / Small nuclear ribonucleoprotein Sm D3 / Small nuclear ribonucleoprotein F / Sm-like protein Lsm4 / Sm-like protein Lsm8 / Sm-like protein Lsm3 / SF3B4, RNA recognition motif 1 / EF-G domain III/V-like / 116kDa U5 small nuclear ribonucleoprotein component, C-terminal / C2 domain superfamily / SWAP/Surp superfamily / U4/U6 small nuclear ribonucleoprotein Prp3 / Splicing factor SF3a60 subunit C-terminal / Nop domain superfamily / U6 snRNA-associated Sm-like protein LSm2 / PROCT domain / Tetratricopeptide repeat-containing domain / Zinc finger C2H2-type / Pre-mRNA-splicing factor 3 / Helicase superfamily 1/2, ATP-binding domain / Ribosomal protein S5 domain 2-type fold, subgroup / Immunoglobulin E-set / Pre-mRNA processing factor 4 (PRP4)-like / WD40/YVTN repeat-like-containing domain superfamily / Armadillo-type fold / Sm-like protein Lsm6/SmF / Sm-like protein Lsm7 / Zinc finger, double-stranded RNA binding / WD40-repeat-containing domain / Ribosomal protein L7Ae/L8/Nhp2 family / Cleavage/polyadenylation specificity factor, A subunit, N-terminal / Prp31 C-terminal / Pre-mRNA-processing-splicing factor 8, U6-snRNA-binding / Pre-mRNA-processing-splicing factor 8, U5-snRNA-binding / Tetratricopeptide repeat / WD40 repeat, conserved site / G-protein beta WD-40 repeat / Ribosomal protein S5 domain 2-type fold / PRP8 domain IV core / Splicing factor 3A subunit 1 / RNA-binding domain superfamily / Zinc finger C2H2 superfamily / Nucleotide-binding alpha-beta plait domain superfamily / Mono-functional DNA-alkylating methyl methanesulfonate N-term / SART-1 family / snoRNA binding domain, fibrillarin / Prp31 C terminal domain / Protein of unknown function (DUF1115) / pre-mRNA processing factor 3 (PRP3) / WD domain, G-beta repeat / PRP1 splicing factor, N-terminal / Ribosomal protein L7Ae/L30e/S12e/Gadd45 family / Zinc-finger of C2H2 type / PRP38 family / CPSF A subunit region / Domain of unknown function (DUF382) / 116 kDa U5 small nuclear ribonucleoprotein component N-terminus / PSP / RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) / PHF5-like protein / Domain of unknown function (DUF3449) / Zinc-finger double-stranded RNA-binding / Pre-mRNA-splicing factor SF3A3, of SF3a complex, Prp9 / Pre-mRNA-splicing factor PRP9 N-terminus / Pre-mRNA-splicing factor SF3a complex subunit 2 (Prp11) / Surp module / Pre-mRNA splicing factor PRP21 like protein / Leucine-rich repeat / RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) / Mitosis protein DIM1 / Elongation factor G, domain IV / Thioredoxin-like superfamily / LSM domain / WD40-repeat-containing domain superfamily / Winged helix DNA-binding domain superfamily / MPN domain / Splicing factor 3B subunit 1-like / U6 snRNA-associated Sm-like protein Lsm3 / U4/U6.U5 small nuclear ribonucleoprotein component Snu23 / Brr2, N-terminal helicase PWI domain / Nop, C-terminal domain / Pre-mRNA-processing-splicing factor 8, U5-snRNA-binding domain superfamily / Prp8 RNase domain IV, palm region / Prp8 RNase domain IV, fingers region
U6 snRNA-associated Sm-like protein LSm5 / Small nuclear ribonucleoprotein Sm D2 / U6 snRNA-associated Sm-like protein LSm4 / Small nuclear ribonucleoprotein-associated protein B / Pre-mRNA-splicing factor SPP381 / U6 snRNA-associated Sm-like protein LSm2 / Pre-mRNA-splicing factor SNU114 / Pre-mRNA-splicing helicase BRR2 / Pre-mRNA-splicing factor PRP21 / U4/U6 small nuclear ribonucleoprotein PRP4 ...U6 snRNA-associated Sm-like protein LSm5 / Small nuclear ribonucleoprotein Sm D2 / U6 snRNA-associated Sm-like protein LSm4 / Small nuclear ribonucleoprotein-associated protein B / Pre-mRNA-splicing factor SPP381 / U6 snRNA-associated Sm-like protein LSm2 / Pre-mRNA-splicing factor SNU114 / Pre-mRNA-splicing helicase BRR2 / Pre-mRNA-splicing factor PRP21 / U4/U6 small nuclear ribonucleoprotein PRP4 / Pre-mRNA-splicing factor PRP9 / Pre-mRNA-splicing factor 6 / RDS3 complex subunit 10 / U6 snRNA-associated Sm-like protein LSm6 / 13 kDa ribonucleoprotein-associated protein / Small nuclear ribonucleoprotein Sm D3 / Small nuclear ribonucleoprotein G / gb:4718: / Pre-mRNA-splicing factor RDS3 / Pre-mRNA-splicing factor PRP11 / U2 small nuclear ribonucleoprotein A' / Small nuclear ribonucleoprotein E / 23 kDa U4/U6.U5 small nuclear ribonucleoprotein component / 66 kDa U4/U6.U5 small nuclear ribonucleoprotein component / Protein HSH49 / gb:536627: / gb:1039024031: / gb:1039024040: / gb:1039023280: / Pre-mRNA-splicing factor RSE1 / U2 small nuclear ribonucleoprotein B'' / U4/U6 small nuclear ribonucleoprotein PRP3 / Cold sensitive U2 snRNA suppressor 1 / Small nuclear ribonucleoprotein Sm D1 / Pre-mRNA-splicing factor 38 / U6 snRNA-associated Sm-like protein LSm3 / Small nuclear ribonucleoprotein F / U6 snRNA-associated Sm-like protein LSm7 / U2 snRNP component HSH155 / Pre-mRNA-processing factor 31 / U6 snRNA-associated Sm-like protein LSm8 / Spliceosomal protein DIB1 / Pre-mRNA-splicing factor 8
Biological speciesSaccharomyces cerevisiae (baker's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.2 Å
AuthorsPlaschka, C. / Lin, P.-C. / Nagai, K.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MC_U105184330 United Kingdom
European Research Council693087 - SPLICE3D United Kingdom
CitationJournal: Nature / Year: 2017
Title: Structure of a pre-catalytic spliceosome.
Authors: Clemens Plaschka / Pei-Chun Lin / Kiyoshi Nagai /
Abstract: Intron removal requires assembly of the spliceosome on precursor mRNA (pre-mRNA) and extensive remodelling to form the spliceosome's catalytic centre. Here we report the cryo-electron microscopy ...Intron removal requires assembly of the spliceosome on precursor mRNA (pre-mRNA) and extensive remodelling to form the spliceosome's catalytic centre. Here we report the cryo-electron microscopy structure of the yeast Saccharomyces cerevisiae pre-catalytic B complex spliceosome at near-atomic resolution. The mobile U2 small nuclear ribonucleoprotein particle (snRNP) associates with U4/U6.U5 tri-snRNP through the U2/U6 helix II and an interface between U4/U6 di-snRNP and the U2 snRNP SF3b-containing domain, which also transiently contacts the helicase Brr2. The 3' region of the U2 snRNP is flexibly attached to the SF3b-containing domain and protrudes over the concave surface of tri-snRNP, where the U1 snRNP may reside before its release from the pre-mRNA 5' splice site. The U6 ACAGAGA sequence forms a hairpin that weakly tethers the 5' splice site. The B complex proteins Prp38, Snu23 and Spp381 bind the Prp8 N-terminal domain and stabilize U6 ACAGAGA stem-pre-mRNA and Brr2-U4 small nuclear RNA interactions. These results provide important insights into the events leading to active site formation.
Validation Report
SummaryFull reportAbout validation report
History
DepositionApr 24, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 31, 2017Provider: repository / Type: Initial release
Revision 1.1Jul 5, 2017Group: Database references / Derived calculations / Category: citation / pdbx_struct_conn_angle / struct_conn
Item: _citation.country / _citation.journal_id_ASTM ..._citation.country / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_volume / _citation.page_first / _citation.page_last / _struct_conn.details / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_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
Revision 1.2Jul 19, 2017Group: Database references / Category: pdbx_database_related
Revision 1.3Aug 30, 2017Group: Author supporting evidence / Data collection / Category: em_imaging_optics / pdbx_audit_support
Item: _em_imaging_optics.energyfilter_name / _pdbx_audit_support.funding_organization
Revision 1.4Mar 21, 2018Group: Advisory / Derived calculations ...Advisory / Derived calculations / Source and taxonomy / Structure summary
Category: entity / entity_src_nat ...entity / entity_src_nat / pdbx_validate_close_contact / struct_conn
Item: _entity.pdbx_description / _entity_src_nat.pdbx_organism_scientific / _entity_src_nat.strain
Revision 1.5Nov 28, 2018Group: Advisory / Data collection ...Advisory / Data collection / Database references / Derived calculations
Category: pdbx_database_related / pdbx_validate_close_contact / struct_conn
Item: _pdbx_database_related.content_type

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Structure visualization

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Assembly

Deposited unit
2: U2 snRNA
3: U6 snRNA-associated Sm-like protein LSm3
4: U4 snRNA
5: U5 snRNA
6: U6 snRNA
7: U6 snRNA-associated Sm-like protein LSm7
8: U6 snRNA-associated Sm-like protein LSm8
A: Pre-mRNA-splicing factor 8
B: Pre-mRNA-splicing helicase BRR2
C: Pre-mRNA-splicing factor SNU114
D: Spliceosomal protein DIB1
E: 66 kDa U4/U6.U5 small nuclear ribonucleoprotein component
F: Pre-mRNA-processing factor 31
G: U4/U6 small nuclear ribonucleoprotein PRP3
H: U4/U6 small nuclear ribonucleoprotein PRP4
I: Yeast UBC4 gene for ubiquitin-conjugating enzyme
J: Pre-mRNA-splicing factor 6
K: 13 kDa ribonucleoprotein-associated protein
L: 23 kDa U4/U6.U5 small nuclear ribonucleoprotein component
M: Pre-mRNA-splicing factor 38
N: Pre-mRNA-splicing factor SPP381
O: U2 snRNP component HSH155
P: Pre-mRNA-splicing factor RSE1
Q: Cold sensitive U2 snRNA suppressor 1
R: Protein HSH49
S: Pre-mRNA-splicing factor RDS3
T: Pre-mRNA-splicing factor PRP9
U: Pre-mRNA-splicing factor PRP11
V: Pre-mRNA-splicing factor PRP21
W: U2 small nuclear ribonucleoprotein A'
X: Unknown
Y: U2 small nuclear ribonucleoprotein B''
Z: RDS3 complex subunit 10
a: U6 snRNA-associated Sm-like protein LSm2
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
i: Small nuclear ribonucleoprotein Sm D2
j: U6 snRNA-associated Sm-like protein LSm4
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: U6 snRNA-associated Sm-like protein LSm5
p: Small nuclear ribonucleoprotein E
q: Small nuclear ribonucleoprotein F
r: Small nuclear ribonucleoprotein G
s: Small nuclear ribonucleoprotein-associated protein B
t: Small nuclear ribonucleoprotein Sm D1
u: Small nuclear ribonucleoprotein Sm D2
v: Small nuclear ribonucleoprotein Sm D3
w: Small nuclear ribonucleoprotein E
x: Small nuclear ribonucleoprotein F
y: Small nuclear ribonucleoprotein G
z: U6 snRNA-associated Sm-like protein LSm6
hetero molecules


Theoretical massNumber of molelcules
Total (without water)2,551,70966
Polymers2,550,72858
Non-polymers9818
Water0
1


  • Idetical with deposited unit
  • defined by software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area242730 Å2
ΔGint-1387 kcal/mol
Surface area639050 Å2
MethodPISA

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Components

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RNA chain , 5 types, 5 molecules 2456I

#1: RNA chain U2 snRNA / U2 spliceosomal RNA


Mass: 376267.406 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Saccharomyces cerevisiae U2 snRNA / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: GenBank: 536627
#3: RNA chain U4 snRNA / U4 spliceosomal RNA


Mass: 51186.023 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Saccharomyces cerevisiae U4 snRNA / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: GenBank: 1039024031
#4: RNA chain U5 snRNA / U5 spliceosomal RNA


Mass: 68643.344 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Saccharomyces cerevisiae U5 snRNA / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: GenBank: 1039024040
#5: RNA chain U6 snRNA / U6 spliceosomal RNA


Mass: 35883.176 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Saccharomyces cerevisiae U6 snRNA / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: GenBank: 1039023280
#16: RNA chain Yeast UBC4 gene for ubiquitin-conjugating enzyme


Mass: 30200.730 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: Saccharomyces cerevisiae UBC4 pre-mRNA for ubiquitin-conjugating enzyme, in vitro transcribed.
Source: (synth.) Saccharomyces cerevisiae (baker's yeast) / References: GenBank: 4718

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U6 snRNA-associated Sm-like protein ... , 7 types, 7 molecules 378ajoz

#2: Protein U6 snRNA-associated Sm-like protein LSm3 / SmX4 protein


Mass: 10039.262 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: U6 snRNA-associated Sm-like protein LSm3 / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P57743
#6: Protein U6 snRNA-associated Sm-like protein LSm7


Mass: 13027.045 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: U6 snRNA-associated Sm-like protein LSm7 / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P53905
#7: Protein U6 snRNA-associated Sm-like protein LSm8


Mass: 12403.378 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: U6 snRNA-associated Sm-like protein LSm8 / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P47093
#34: Protein U6 snRNA-associated Sm-like protein LSm2 / Small nuclear ribonucleoprotein D homolog SNP3


Mass: 11177.888 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: U6 snRNA-associated Sm-like protein LSm2 / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P38203
#42: Protein U6 snRNA-associated Sm-like protein LSm4


Mass: 21298.070 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: U6 snRNA-associated Sm-like protein LSm4 / Source: (natural) Saccharomyces cerevisiae / References: UniProt: P40070
#43: Protein U6 snRNA-associated Sm-like protein LSm5


Mass: 10432.954 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: U6 snRNA-associated Sm-like protein LSm5 / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P40089
#44: Protein U6 snRNA-associated Sm-like protein LSm6


Mass: 9406.579 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: U6 snRNA-associated Sm-like protein LSm6 / Source: (natural) Saccharomyces cerevisiae / References: UniProt: A6ZYX7

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Pre-mRNA-splicing factor ... , 10 types, 10 molecules ACJMNPSTUV

#8: Protein Pre-mRNA-splicing factor 8


Mass: 279867.469 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Pre-mRNA-splicing factor 8 / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P33334
#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 / Details: Pre-mRNA-splicing factor SNU114 / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P36048
#17: Protein Pre-mRNA-splicing factor 6


Mass: 104370.133 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Pre-mRNA-splicing factor 6 / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P19735
#20: Protein Pre-mRNA-splicing factor 38 / Pre-mRNA-processing factor 38


Mass: 27995.619 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Pre-mRNA-splicing factor 38 / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: Q00723
#21: Protein Pre-mRNA-splicing factor SPP381 / Suppressor of PRP38-1 mutation


Mass: 33830.926 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Pre-mRNA-splicing factor SPP381 / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P38282
#23: Protein Pre-mRNA-splicing factor RSE1 / RNA splicing and ER to Golgi transport factor 1 / Spliceosome-associated protein 130


Mass: 153956.781 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Pre-mRNA-splicing factor RSE1 / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: Q04693
#26: Protein Pre-mRNA-splicing factor RDS3 / Regulator of drug sensitivity 3


Mass: 12283.573 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Pre-mRNA-splicing factor RDS3 / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: Q06835
#27: Protein Pre-mRNA-splicing factor PRP9


Mass: 63126.445 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Pre-mRNA-splicing factor PRP9 / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P19736
#28: Protein Pre-mRNA-splicing factor PRP11


Mass: 29962.809 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Pre-mRNA-splicing factor PRP11 / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: Q07350
#29: Protein Pre-mRNA-splicing factor PRP21


Mass: 33111.512 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Pre-mRNA-splicing factor PRP21 / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P32524

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Protein , 12 types, 14 molecules BDEFKLOQRXZbks

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


Mass: 246470.266 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Pre-mRNA-splicing helicase BRR2 / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P32639, RNA helicase
#11: Protein Spliceosomal protein DIB1 / Spliceosome


Mass: 16798.387 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Spliceosomal protein DIB1 / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: Q06819
#12: Protein 66 kDa U4/U6.U5 small nuclear ribonucleoprotein component


Mass: 66544.047 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Details: 66 kDa U4/U6.U5 small nuclear ribonucleoprotein component
Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: Q12420
#13: Protein Pre-mRNA-processing factor 31


Mass: 56382.516 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Pre-mRNA-processing factor 31 / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P49704
#18: Protein 13 kDa ribonucleoprotein-associated protein / Small nuclear ribonucleoprotein-associated protein 1


Mass: 13582.855 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: 13 kDa ribonucleoprotein-associated protein / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P39990
#19: Protein 23 kDa U4/U6.U5 small nuclear ribonucleoprotein component


Mass: 22717.799 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Details: 23 kDa U4/U6.U5 small nuclear ribonucleoprotein component
Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: Q12368
#22: Protein U2 snRNP component HSH155


Mass: 110166.672 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: U2 snRNP component HSH155 / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P49955
#24: Protein Cold sensitive U2 snRNA suppressor 1


Mass: 50210.699 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Cold sensitive U2 snRNA suppressor 1 / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: Q02554
#25: Protein Protein HSH49


Mass: 24534.152 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Protein HSH49 / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: Q99181
#31: Protein Unknown


Mass: 7907.637 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Unknown protein helices within B complex / Source: (natural) Saccharomyces cerevisiae (baker's yeast)
#33: Protein RDS3 complex subunit 10 / Splicing factor 3b subunit


Mass: 10045.401 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: RDS3 complex subunit 10 / Source: (natural) Saccharomyces cerevisiae / References: UniProt: P0C074
#35: Protein Small nuclear ribonucleoprotein-associated protein B / snRNP-B / Sm protein B / SmB


Mass: 22426.990 Da / Num. of mol.: 3 / Source method: isolated from a natural source
Details: Small nuclear ribonucleoprotein-associated protein B
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
References: UniProt: P40018

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U4/U6 small nuclear ribonucleoprotein ... , 2 types, 2 molecules GH

#14: Protein U4/U6 small nuclear ribonucleoprotein PRP3 / Pre-mRNA-splicing factor 3


Mass: 55974.320 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: U4/U6 small nuclear ribonucleoprotein PRP3 / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: Q03338
#15: Protein U4/U6 small nuclear ribonucleoprotein PRP4 / Pre-mRNA-processing protein 4


Mass: 52506.984 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: U4/U6 small nuclear ribonucleoprotein PRP4 / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P20053

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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 / Details: U2 small nuclear ribonucleoprotein A' / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: Q08963
#32: Protein U2 small nuclear ribonucleoprotein B'' / U2 snRNP B''


Mass: 12850.944 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: U2 small nuclear ribonucleoprotein B'' / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P40567

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Small nuclear ribonucleoprotein ... , 6 types, 18 molecules dnvepwfqxgryhltimu

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


Mass: 11240.139 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Details: Small nuclear ribonucleoprotein Sm D3
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
References: UniProt: P43321
#37: Protein Small nuclear ribonucleoprotein E / snRNP-E / Sm protein E / SmE


Mass: 10385.098 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Details: Small nuclear ribonucleoprotein E
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
References: UniProt: Q12330
#38: Protein Small nuclear ribonucleoprotein F / snRNP-F / Sm protein F / SmF


Mass: 9669.945 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Details: Small nuclear ribonucleoprotein F
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
References: UniProt: P54999
#39: Protein Small nuclear ribonucleoprotein G / snRNP-G / Sm protein G / SmG


Mass: 8490.809 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Details: Small nuclear ribonucleoprotein G
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
References: UniProt: P40204
#40: Protein Small nuclear ribonucleoprotein Sm D1 / Sm-D1 / snRNP core protein D1


Mass: 16296.798 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Details: Small nuclear ribonucleoprotein Sm D1
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
References: UniProt: Q02260
#41: Protein Small nuclear ribonucleoprotein Sm D2 / Sm-D2 / snRNP core protein D2


Mass: 12876.066 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Details: Small nuclear ribonucleoprotein Sm D2
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
References: UniProt: Q06217

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Non-polymers , 2 types, 8 molecules

#45: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE / Guanosine triphosphate


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


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

ComponentName: Pre-catalytic B complex Spliceosome / Type: COMPLEX / Entity ID: #1-#44 / Source: NATURAL
Molecular weightValue: 2.5 MDa / Experimental value: YES
Source (natural)Organism: Saccharomyces cerevisiae (baker's yeast) / Strain: BCY123
Buffer solutionpH: 7.9 / Details: Buffer pH: HEPES, 7.9; EDTA, 8.0
Buffer component
IDConc.FormulaBuffer-ID
120 mMHEPES1
250 mMKCl1
30.2 mMEDTAEthylenediaminetetraacetic acid1
41 mMDTT1
52.5 mMDesthiobiotin1
SpecimenConc.: 1.5 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 R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
Details: Grids were glow-discharged for 15 s before deposition of 3 microliter sample (~1.5 mg mL-1), and subsequently incubated for 2-3.5 s before blotting and vitrification by plunging into liquid ...Details: Grids were glow-discharged for 15 s before deposition of 3 microliter sample (~1.5 mg mL-1), and subsequently incubated for 2-3.5 s before blotting and vitrification by plunging into liquid ethane with a Vitrobot Mark III (FEI) operated at 4 degrees Celsius and 100% humidity.

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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 FIELDBright-field microscopy / Nominal magnification: 81000 X / Calibrated defocus min: 350 nm / Calibrated defocus max: 5300 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 16 sec. / Electron dose: 56 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 5115
EM imaging opticsEnergyfilter name: GIF Quantum
Image scansSampling size: 5 µm / Width: 3710 / Height: 3838 / Movie frames/image: 20 / Used frames/image: 1-20

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Processing

EM software
IDNameVersionCategoryDetails
1RELION2particle selection
2DigitalMicrographimage acquisition
4CTFFIND4.1CTF correctionCTF determination
5RELION2CTF correctionCTF correction
8Coot0.8.3model fitting
9UCSF Chimera1.10.2model fitting
10Situs2.8model fitting
12PHENIX1.10.1-2155-000model refinement
13RELION2initial Euler assignment
14RELION2final Euler assignment
15RELION2classification
16RELION23D reconstruction
Image processingDetails: Movies were binned once and aligned using MOTIONCORR.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 496581
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 7.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 9559
Details: A temperature factor of -170 A2 was applied (sharpened B2 map).
Symmetry type: POINT
Atomic model buildingB value: 170 / Space: REAL

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