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- PDB-5mq0: Structure of a spliceosome remodeled for exon ligation -

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Entry
Database: PDB / ID: 5mq0
TitleStructure of a spliceosome remodeled for exon ligation
Components
  • (Pre-mRNA-processing factor ...) x 2
  • (Pre-mRNA-splicing factor ...) x 17
  • (Saccharomyces cerevisiae strain ...) x 2
  • (Small nuclear ribonucleoprotein ...) x 6
  • (U2 small nuclear ribonucleoprotein ...) x 2
  • 3'-EXON OF UBC4 PRE-MRNA, BOUND BY PRP22 HELICASE
  • 5'-EXON OF UBC4 PRE-MRNA
  • Pre-mRNA-processing protein 45
  • S.cerevisiae chromosome II reading frame ORF YBR230c
  • Small nuclear ribonucleoprotein-associated protein B
  • UNKNOWN PROTEIN
  • Yeast UBC4 gene for ubiquitin-conjugating enzyme
KeywordsSPLICING / pre-mRNA splicing / trans-esterification / lariat intermediate / complex C-star
Function / homology
Function and homology information


U2-type post-spliceosomal complex / mRNA branch site recognition / U2-type post-mRNA release spliceosomal complex / spliceosomal complex disassembly / 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 / mRNA branch site recognition / U2-type post-mRNA release spliceosomal complex / spliceosomal complex disassembly / 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 / U4/U6 snRNP / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) / 7-methylguanosine cap hypermethylation / Prp19 complex / pICln-Sm protein complex / snRNP binding / U2-type catalytic step 1 spliceosome / small nuclear ribonucleoprotein complex / pre-mRNA binding / SMN-Sm protein complex / spliceosomal tri-snRNP complex / U2-type spliceosomal complex / mRNA cis splicing, via spliceosome / poly(U) RNA binding / commitment complex / U2-type prespliceosome assembly / U2-type catalytic step 2 spliceosome / U4 snRNP / U2 snRNP / U1 snRNP / U2-type prespliceosome / precatalytic spliceosome / Formation of TC-NER Pre-Incision Complex / generation of catalytic spliceosome for second transesterification step / spliceosomal complex assembly / DNA replication origin binding / mRNA 5'-splice site recognition / Gap-filling DNA repair synthesis and ligation in TC-NER / protein K63-linked ubiquitination / mRNA 3'-splice site recognition / Dual incision in TC-NER / spliceosomal tri-snRNP complex assembly / DNA replication initiation / U5 snRNA binding / positive regulation of cell cycle / U5 snRNP / U2 snRNA binding / U6 snRNA binding / spliceosomal snRNP assembly / pre-mRNA intronic binding / U1 snRNA binding / U4/U6 x U5 tri-snRNP complex / catalytic step 2 spliceosome / nuclear periphery / positive regulation of RNA splicing / spliceosomal complex / RING-type E3 ubiquitin transferase / mRNA splicing, via spliceosome / ubiquitin-protein transferase activity / metallopeptidase activity / ubiquitin protein ligase activity / 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 / identical protein binding / nucleus / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Prp18 / Pre-mRNA-splicing factor 18 / Prp18 domain / Pre-mRNA-splicing factor SLU7 domain / Pre-mRNA-splicing factor SLU7 / Pre-mRNA splicing Prp18-interacting factor / DHX8/ Prp22, DEXH-box helicase domain / : / : / cwf21 ...Prp18 / Pre-mRNA-splicing factor 18 / Prp18 domain / Pre-mRNA-splicing factor SLU7 domain / Pre-mRNA-splicing factor SLU7 / Pre-mRNA splicing Prp18-interacting factor / DHX8/ Prp22, DEXH-box helicase domain / : / : / 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 / HAT (Half-A-TPR) repeat / Pre-mRNA-splicing factor 19 / Pre-mRNA-processing factor 19 / Prp19/Pso4-like / BUD31/G10-related, conserved site / G10 protein signature 1. / G10 protein signature 2. / : / STL11, N-terminal / SKI-interacting protein SKIP, SNW domain / SKI-interacting protein, SKIP / SKIP/SNW domain / Pre-mRNA-splicing factor Cwf15/Cwc15 / Suppressor of forked / Cwf15/Cwc15 cell cycle control protein / Suppressor of forked protein (Suf) / 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. / Middle domain of eukaryotic initiation factor 4G (eIF4G) / MIF4G-like, type 3 / U-box domain profile. / Modified RING finger domain / U-box domain / U2A'/phosphoprotein 32 family A, C-terminal / occurring C-terminal to leucine-rich repeats / : / Helicase associated domain (HA2), ratchet-like / Leucine-rich repeat / DEAD-box helicase, OB fold / Oligonucleotide/oligosaccharide-binding (OB)-fold / Helicase associated domain (HA2), winged-helix / Helicase-associated domain / Pre-mRNA-splicing factor Syf1-like / Helicase associated domain (HA2) Add an annotation / 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 / : / Small nuclear ribonucleoprotein Sm D3 / Small nuclear ribonucleoprotein Sm D2 / Small nuclear ribonucleoprotein E / Small nuclear ribonucleoprotein G / Small nuclear ribonucleoprotein F / Myb-type HTH DNA-binding domain profile. / Like-Sm (LSM) domain containing protein, LSm4/SmD1/SmD3 / Sm-like protein Lsm7/SmG / Zinc finger, CCCH-type / Zinc finger C3H1-type profile. / Sm-like protein Lsm6/SmF / LSM domain / LSM domain, eukaryotic/archaea-type / snRNP Sm proteins / HAT (Half-A-TPR) repeat / HAT (Half-A-TPR) repeats / Myb domain / Myb-like DNA-binding domain / : / Sm domain profile. / DNA/RNA helicase, ATP-dependent, DEAH-box type, conserved site / DEAH-box subfamily ATP-dependent helicases signature. / LSM domain superfamily / Translation elongation factor EFG/EF2, domain IV / Elongation factor G, domain IV / Elongation factor G, domain IV / Elongation factor G C-terminus / Elongation factor EFG, domain V-like / Elongation factor G C-terminus
Similarity search - Domain/homology
GUANOSINE-5'-TRIPHOSPHATE / INOSITOL HEXAKISPHOSPHATE / : / : / : / : / : / RNA / RNA (> 10) / RNA (> 100) ...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 / 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 (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.17 Å
AuthorsFica, S.M. / Oubridge, C. / Galej, W.P. / Wilkinson, M.E. / Newman, A.J. / Bai, X.-C. / Nagai, K.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MC_U105184330 United Kingdom
European Research Council693087
EMBO and Marie Sklodowska-Curie FellowshipRef. S.M.Fica
Citation
Journal: Nature / Year: 2017
Title: Structure of a spliceosome remodelled for exon ligation.
Authors: Sebastian M Fica / Chris Oubridge / Wojciech P Galej / Max E Wilkinson / Xiao-Chen Bai / Andrew J Newman / Kiyoshi Nagai /
Abstract: The spliceosome excises introns from pre-mRNAs in two sequential transesterifications-branching and exon ligation-catalysed at a single catalytic metal site in U6 small nuclear RNA (snRNA). Recently ...The spliceosome excises introns from pre-mRNAs in two sequential transesterifications-branching and exon ligation-catalysed at a single catalytic metal site in U6 small nuclear RNA (snRNA). Recently reported structures of the spliceosomal C complex with the cleaved 5' exon and lariat-3'-exon bound to the catalytic centre revealed that branching-specific factors such as Cwc25 lock the branch helix into position for nucleophilic attack of the branch adenosine at the 5' splice site. Furthermore, the ATPase Prp16 is positioned to bind and translocate the intron downstream of the branch point to destabilize branching-specific factors and release the branch helix from the active site. Here we present, at 3.8 Å resolution, the cryo-electron microscopy structure of a Saccharomyces cerevisiae spliceosome stalled after Prp16-mediated remodelling but before exon ligation. While the U6 snRNA catalytic core remains firmly held in the active site cavity of Prp8 by proteins common to both steps, the branch helix has rotated by 75° compared to the C complex and is stabilized in a new position by Prp17, Cef1 and the reoriented Prp8 RNase H-like domain. This rotation of the branch helix removes the branch adenosine from the catalytic core, creates a space for 3' exon docking, and restructures the pairing of the 5' splice site with the U6 snRNA ACAGAGA region. Slu7 and Prp18, which promote exon ligation, bind together to the Prp8 RNase H-like domain. The ATPase Prp22, bound to Prp8 in place of Prp16, could interact with the 3' exon, suggesting a possible basis for mRNA release after exon ligation. Together with the structure of the C complex, our structure of the C* complex reveals the two major conformations of the spliceosome during the catalytic stages of splicing.
#1: Journal: Science / Year: 2015
Title: Structural basis of pre-mRNA splicing.
Authors: Jing Hang / Ruixue Wan / Chuangye Yan / Yigong Shi /
Abstract: Splicing of precursor messenger RNA is performed by the spliceosome. In the cryogenic electron microscopy structure of the yeast spliceosome, U5 small nuclear ribonucleoprotein acts as a central ...Splicing of precursor messenger RNA is performed by the spliceosome. In the cryogenic electron microscopy structure of the yeast spliceosome, U5 small nuclear ribonucleoprotein acts as a central scaffold onto which U6 and U2 small nuclear RNAs (snRNAs) are intertwined to form a catalytic center next to Loop I of U5 snRNA. Magnesium ions are coordinated by conserved nucleotides in U6 snRNA. The intron lariat is held in place through base-pairing interactions with both U2 and U6 snRNAs, leaving the variable-length middle portion on the solvent-accessible surface of the catalytic center. The protein components of the spliceosome anchor both 5' and 3' ends of the U2 and U6 snRNAs away from the active site, direct the RNA sequences, and allow sufficient flexibility between the ends and the catalytic center. Thus, the spliceosome is in essence a protein-directed ribozyme, with the protein components essential for the delivery of critical RNA molecules into close proximity of one another at the right time for the splicing reaction.
#2: Journal: Nature / Year: 2015
Title: The architecture of the spliceosomal U4/U6.U5 tri-snRNP.
Authors: Thi Hoang Duong Nguyen / Wojciech P Galej / Xiao-chen Bai / Christos G Savva / Andrew J Newman / Sjors H W Scheres / Kiyoshi Nagai /
Abstract: U4/U6.U5 tri-snRNP is a 1.5-megadalton pre-assembled spliceosomal complex comprising U5 small nuclear RNA (snRNA), extensively base-paired U4/U6 snRNAs and more than 30 proteins, including the key ...U4/U6.U5 tri-snRNP is a 1.5-megadalton pre-assembled spliceosomal complex comprising U5 small nuclear RNA (snRNA), extensively base-paired U4/U6 snRNAs and more than 30 proteins, including the key components Prp8, Brr2 and Snu114. The tri-snRNP combines with a precursor messenger RNA substrate bound to U1 and U2 small nuclear ribonucleoprotein particles (snRNPs), and transforms into a catalytically active spliceosome after extensive compositional and conformational changes triggered by unwinding of the U4 and U6 (U4/U6) snRNAs. Here we use cryo-electron microscopy single-particle reconstruction of Saccharomyces cerevisiae tri-snRNP at 5.9 Å resolution to reveal the essentially complete organization of its RNA and protein components. The single-stranded region of U4 snRNA between its 3' stem-loop and the U4/U6 snRNA stem I is loaded into the Brr2 helicase active site ready for unwinding. Snu114 and the amino-terminal domain of Prp8 position U5 snRNA to insert its loop I, which aligns the exons for splicing, into the Prp8 active site cavity. The structure provides crucial insights into the activation process and the active site of the spliceosome.
#3: Journal: Nature / Year: 2016
Title: Cryo-EM structure of the yeast U4/U6.U5 tri-snRNP at 3.7 Å resolution.
Authors: Thi Hoang Duong Nguyen / Wojciech P Galej / Xiao-Chen Bai / Chris Oubridge / Andrew J Newman / Sjors H W Scheres / Kiyoshi Nagai /
Abstract: U4/U6.U5 tri-snRNP represents a substantial part of the spliceosome before activation. A cryo-electron microscopy structure of Saccharomyces cerevisiae U4/U6.U5 tri-snRNP at 3.7 Å resolution led ...U4/U6.U5 tri-snRNP represents a substantial part of the spliceosome before activation. A cryo-electron microscopy structure of Saccharomyces cerevisiae U4/U6.U5 tri-snRNP at 3.7 Å resolution led to an essentially complete atomic model comprising 30 proteins plus U4/U6 and U5 small nuclear RNAs (snRNAs). The structure reveals striking interweaving interactions of the protein and RNA components, including extended polypeptides penetrating into subunit interfaces. The invariant ACAGAGA sequence of U6 snRNA, which base-pairs with the 5'-splice site during catalytic activation, forms a hairpin stabilized by Dib1 and Prp8 while the adjacent nucleotides interact with the exon binding loop 1 of U5 snRNA. Snu114 harbours GTP, but its putative catalytic histidine is held away from the γ-phosphate by hydrogen bonding to a tyrosine in the amino-terminal domain of Prp8. Mutation of this histidine to alanine has no detectable effect on yeast growth. The structure provides important new insights into the spliceosome activation process leading to the formation of the catalytic centre.
#4: Journal: 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
DepositionDec 19, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 18, 2017Provider: repository / Type: Initial release
Revision 1.1Mar 1, 2017Group: Database references
Revision 1.2Jul 5, 2017Group: Data collection / Category: em_imaging / Item: _em_imaging.details
Revision 1.3Aug 30, 2017Group: Author supporting evidence / Data collection
Category: em_image_scans / em_imaging_optics ...em_image_scans / em_imaging_optics / em_software / pdbx_audit_support
Item: _em_imaging_optics.energyfilter_name / _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.4Oct 24, 2018Group: Advisory / Data collection / Derived calculations
Category: pdbx_unobs_or_zero_occ_atoms / pdbx_validate_close_contact / struct_conn
Revision 1.5Nov 21, 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 1.6Dec 11, 2019Group: Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB
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 / pdbx_struct_conn_angle / struct_conn / struct_site
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _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 / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_site.details / _struct_site.pdbx_auth_comp_id

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Structure visualization

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Assembly

Deposited unit
I: Yeast UBC4 gene for ubiquitin-conjugating enzyme
E: 5'-EXON OF UBC4 PRE-MRNA
2: S.cerevisiae chromosome II reading frame ORF YBR230c
6: Saccharomyces cerevisiae strain T.52_2H chromosome XII sequence
5: Saccharomyces cerevisiae strain WI_C_MBSP_4 chromosome VII sequence
A: Pre-mRNA-splicing factor 8
C: Pre-mRNA-splicing factor SNU114
H: Pre-mRNA-splicing factor 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: Pre-mRNA-splicing factor CWC15
R: Pre-mRNA-splicing factor CWC21
S: Pre-mRNA-splicing factor CLF1
T: Pre-mRNA-splicing factor SYF1,PRE-MRNA-SPLICING FACTOR SYF1
a: Pre-mRNA-splicing factor 18
c: Pre-mRNA-splicing factor SLU7
o: Pre-mRNA-processing factor 17
X: UNKNOWN PROTEIN
y: Pre-mRNA-splicing factor SYF2
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
V: Pre-mRNA-splicing factor ATP-dependent RNA helicase PRP22
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
r: Small nuclear ribonucleoprotein G
W: U2 small nuclear ribonucleoprotein A'
Y: U2 small nuclear ribonucleoprotein B''
p: Small nuclear ribonucleoprotein E
q: Small nuclear ribonucleoprotein F
3: 3'-EXON OF UBC4 PRE-MRNA, BOUND BY PRP22 HELICASE
s: Pre-mRNA-splicing factor SNT309
t: Pre-mRNA-processing factor 19
u: Pre-mRNA-processing factor 19
v: Pre-mRNA-processing factor 19
w: Pre-mRNA-processing factor 19
hetero molecules


Theoretical massNumber of molelcules
Total (without water)2,205,88659
Polymers2,204,15946
Non-polymers1,72713
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 , 4 types, 4 molecules IE23

#1: RNA chain Yeast UBC4 gene for ubiquitin-conjugating enzyme


Mass: 30200.730 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Production host: IN VITRO TRANSCRIPTION VECTOR PT7-FLUC(DELTAI) (others)
References: GenBank: 4718
#2: RNA chain 5'-EXON OF UBC4 PRE-MRNA


Mass: 6518.976 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Production host: IN VITRO TRANSCRIPTION VECTOR PT7-FLUC(DELTAI) (others)
#3: RNA chain S.cerevisiae chromosome II reading frame ORF YBR230c


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
#34: RNA chain 3'-EXON OF UBC4 PRE-MRNA, BOUND BY PRP22 HELICASE


Mass: 873.540 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Production host: IN VITRO TRANSCRIPTION VECTOR PT7-FLUC(DELTAI) (others)

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Saccharomyces cerevisiae strain ... , 2 types, 2 molecules 65

#4: RNA chain Saccharomyces cerevisiae strain T.52_2H chromosome XII sequence


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
#5: RNA chain Saccharomyces cerevisiae strain WI_C_MBSP_4 chromosome VII sequence


Mass: 57444.875 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: GenBank: 1039023924

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Pre-mRNA-splicing factor ... , 17 types, 17 molecules ACHJLMNOPRSTacyVs

#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
#7: 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
#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) / References: UniProt: P53333
#9: 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
#11: 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
#12: 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
#13: 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
#14: 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
#15: Protein Pre-mRNA-splicing factor CWC15 / Complexed with CEF1 protein 15


Mass: 19975.195 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q03772
#16: 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
#17: 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
#18: Protein Pre-mRNA-splicing factor SYF1,PRE-MRNA-SPLICING FACTOR SYF1 / PRP19-associated complex protein 90 / Synthetic lethal with CDC40 protein 1


Mass: 101875.852 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q04048
#19: Protein Pre-mRNA-splicing factor 18


Mass: 28414.391 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P33411
#20: 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 (brewer's yeast) / References: UniProt: Q02775
#23: 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 (brewer's yeast) / References: UniProt: P53277
#31: 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 (brewer's yeast) / References: UniProt: P24384, RNA helicase
#35: 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 , 3 types, 4 molecules KXbk

#10: 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
#22: Protein UNKNOWN PROTEIN


Mass: 5805.147 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast)
#24: 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

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

#21: Protein Pre-mRNA-processing factor 17 / Cell division control protein 40


Mass: 52128.762 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40968
#36: 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

#25: 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
#26: 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
#27: 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
#28: 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
#29: 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
#30: 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

#32: 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
#33: 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 , 5 types, 13 molecules

#37: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg
#38: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: K
#39: Chemical ChemComp-IHP / INOSITOL HEXAKISPHOSPHATE / MYO-INOSITOL HEXAKISPHOSPHATE / INOSITOL 1,2,3,4,5,6-HEXAKISPHOSPHATE


Mass: 660.035 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H18O24P6
#40: 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
#41: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 6 / 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: Saccharomyces cerevisiae spliceosome. Complex C just after Prp16-mediated remodeling
Type: COMPLEX
Details: Splicing extract was prepared from 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 ...Details: Splicing extract was prepared from 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 a 2'-deoxy substitution at the 3'-splice site sequence UAG sequence (UA-2'dG) 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 deoxy substitution. Substrate-bound spliceosomes from the splicing extract were purified on amylose resin and eluted with maltose. Subsequently the spliceosomes were captured on streptactin resin and eluted with desthiobiotin. Purified spliceosomes were concentrated in 20 mM HEPES KOH pH 7.9, 100 mM KCl, 0.25 mM EDTA.
Entity ID: #1-#30 / Source: MULTIPLE SOURCES
Buffer solutionpH: 7.9 / Details: NP-40 is also called IGEPAL CA-630
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHepes.KOH pH 7.91
2100 mMpotassium chlorideKCl1
3250 micromolarEDTA1
41 % v/vglycerol1
50.0025 % v/vNonidet P-40 (NP-40)1
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 R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: 3.5 microlitres sample were applied to the grid, left for 25 seconds and then blotted for 3.0-3.5 seconds before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: GIF Quantum energy filter, 20 eV slit width
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 4500 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 SUMMIT (4k x 4k) / Num. of real images: 3596
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

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Processing

EM software
IDNameVersionCategoryDetails
1RELION1.4particle selection
4CTFFIND4CTF correction
7Coot0.8.3model fittingPaul Emsley's experimental version.
9REFMAC5.8model refinementUsed "SOURCE EM" command to get correct electron form factors for refinement of electron microscopy structure.
10RELION1.4initial Euler assignment
11RELION1.4final Euler assignment
12RELION1.4classification
13RELION1.43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 350000
Details: Selected initial particles automatically using C complex 2D class averages, low-pass filtered to 20 Angstrom for automatic particle picking.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.17 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 29527
Details: A 3D reconstruction was obtained after refinement of a subset of particles obtained by classification with a mask around Prp22.
Num. of class averages: 4 / Symmetry type: POINT
Atomic model buildingB value: 330 / Protocol: FLEXIBLE FIT / Space: RECIPROCAL / Target criteria: Fourier Shell Correlation
Details: Used secondary structure restraints generated in ProSMART and LibG.

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