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

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Basic information

Entry
Database: PDB / ID: 5mps
TitleStructure of a spliceosome remodeled for exon ligation
Components
  • (Pre-mRNA-processing ...) x 2
  • (Pre-mRNA-splicing factor ...) x 15
  • (Small nuclear ribonucleoprotein ...SnRNP) x 6
  • Saccharomyces cerevisiae strain T.52_2H chromosome XII sequence
  • Small nuclear ribonucleoprotein-associated protein B
  • U2 snRNAU2 spliceosomal RNA
  • U5 snRNAU5 spliceosomal RNA
  • UBC4 gene exon
  • Unknown
  • 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 / U2-type post-mRNA release spliceosomal complex / mRNA branch site recognition / cellular bud site selection / pre-mRNA 3'-splice site binding / post-mRNA release spliceosomal complex / cis assembly of pre-catalytic spliceosome / generation of catalytic spliceosome for first transesterification step / nuclear mRNA surveillance / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) ...U2-type post-spliceosomal complex / U2-type post-mRNA release spliceosomal complex / mRNA branch site recognition / cellular bud site selection / pre-mRNA 3'-splice site binding / post-mRNA release spliceosomal complex / cis assembly of pre-catalytic spliceosome / generation of catalytic spliceosome for first transesterification step / nuclear mRNA surveillance / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) / splicing factor binding / U4/U6 snRNP / 7-methylguanosine cap hypermethylation / pre-mRNA binding / U2-type catalytic step 1 spliceosome / pICln-Sm protein complex / Prp19 complex / spliceosomal tri-snRNP complex / small nuclear ribonucleoprotein complex / SMN-Sm protein complex / mRNA cis splicing, via spliceosome / U2-type spliceosomal complex / U2-type prespliceosome assembly / commitment complex / U2-type catalytic step 2 spliceosome / U4 snRNP / U2 snRNP / poly(U) RNA binding / U1 snRNP / U2-type prespliceosome / precatalytic spliceosome / spliceosomal complex assembly / Dual incision in TC-NER / DNA replication origin binding / generation of catalytic spliceosome for second transesterification step / Gap-filling DNA repair synthesis and ligation in TC-NER / mRNA 3'-splice site recognition / mRNA 5'-splice site recognition / DNA replication initiation / spliceosomal tri-snRNP complex assembly / U5 snRNA binding / U5 snRNP / spliceosomal snRNP assembly / U2 snRNA binding / U6 snRNA binding / pre-mRNA intronic binding / positive regulation of cell cycle / U1 snRNA binding / U4/U6 x U5 tri-snRNP complex / catalytic step 2 spliceosome / nuclear periphery / positive regulation of RNA splicing / spliceosomal complex / mRNA splicing, via spliceosome / metallopeptidase activity / cell cycle / mRNA binding / GTPase activity / chromatin binding / chromatin / GTP binding / DNA binding / RNA binding / zinc ion binding / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
RNA Binding Protein, Prp18; Chain A / Functional domain of the splicing factor Prp18 / Prp18 / Pre-mRNA-splicing factor 18 / Prp18 domain / Pre-mRNA-splicing factor SLU7 domain / Pre-mRNA-splicing factor SLU7 / Pre-mRNA splicing Prp18-interacting factor / Slt11, RNA recognition motif / cwf21 ...RNA Binding Protein, Prp18; Chain A / Functional domain of the splicing factor Prp18 / 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 / Slt11, RNA recognition motif / cwf21 / Torus domain / Pre-mRNA-splicing factor Cwc2, RNA recognition motif / Torus domain / mRNA splicing factor SYF2 / SYF2 splicing factor / mRNA splicing factor Cwf21 domain / cwf21 domain / Pre-mRNA-processing factor 17 / : / STL11, N-terminal / WD repeat Prp46/PLRG1-like / BUD31/G10-related, conserved site / : / : / G10 protein signature 1. / G10 protein signature 2. / SKI-interacting protein SKIP, SNW domain / SKI-interacting protein, SKIP / SKIP/SNW domain / Pre-mRNA-splicing factor Cwf15/Cwc15 / HAT (Half-A-TPR) repeat / Cwf15/Cwc15 cell cycle control protein / 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. / Pre-mRNA-splicing factor Syf1-like / Middle domain of eukaryotic initiation factor 4G (eIF4G) / MIF4G-like, type 3 / Snu114, GTP-binding domain / 116kDa U5 small nuclear ribonucleoprotein component, N-terminal / 116kDa U5 small nuclear ribonucleoprotein component, C-terminal / 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 / Sm-like protein Lsm7/SmG / Like-Sm (LSM) domain containing protein, LSm4/SmD1/SmD3 / SH3 type barrels. - #100 / 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. / Myb-like DNA-binding domain / Translation elongation factor EFG/EF2, domain IV / Elongation factor G, domain IV / Elongation factor G, domain IV / LSM domain superfamily / Elongation factor G C-terminus / Elongation factor EFG, domain V-like / Elongation factor G C-terminus / EF-G domain III/V-like / SANT SWI3, ADA2, N-CoR and TFIIIB'' DNA-binding domains / SANT/Myb domain / PROCT domain / Prp8 RNase domain IV, fingers region / PROCT (NUC072) domain / PRO8NT domain / PROCN domain / Pre-mRNA-processing-splicing factor 8, U6-snRNA-binding / Pre-mRNA-processing-splicing factor 8, U5-snRNA-binding / RNA recognition motif, spliceosomal PrP8 / PRP8 domain IV core / Pre-mRNA-processing-splicing factor 8, U5-snRNA-binding domain superfamily / Prp8 RNase domain IV, palm region / PRO8NT (NUC069), PrP8 N-terminal domain / PROCN (NUC071) domain / U6-snRNA interacting domain of PrP8 / U5-snRNA binding site 2 of PrP8 / RNA recognition motif of the spliceosomal PrP8 / PRP8 domain IV core / Pre-mRNA-processing-splicing factor 8 / YVTN repeat-like/Quinoprotein amine dehydrogenase / Elongation factor Tu domain 2 / 7 Propeller
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 BUD31 / Pre-mRNA-processing protein 45 / 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 / 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 / Small nuclear ribonucleoprotein Sm D2 / 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: 3.85 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 CouncilAdG-693087-SPLICE3D
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 18, 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 17, 2018Group: Data collection / Refinement description / Category: refine
Revision 1.5Oct 30, 2019Group: Advisory / Data collection / Derived calculations
Category: pdbx_validate_close_contact / struct_conn / struct_conn_type
Revision 1.6Dec 11, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 2.0Oct 7, 2020Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Non-polymer description / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / entity / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / struct_conn / struct_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.type_symbol / _chem_comp.id / _chem_comp.name / _chem_comp.pdbx_synonyms / _entity.pdbx_description / _pdbx_entity_nonpoly.comp_id / _pdbx_entity_nonpoly.name / _pdbx_nonpoly_scheme.mon_id / _pdbx_nonpoly_scheme.pdb_mon_id / _struct_conn.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: UBC4 gene exon
2: U2 snRNA
6: Saccharomyces cerevisiae strain T.52_2H chromosome XII sequence
5: U5 snRNA
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
a: Pre-mRNA-splicing factor 18
c: Pre-mRNA-splicing factor SLU7
o: Pre-mRNA-processing factor 17
X: Unknown
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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,696,09543
Polymers1,694,36830
Non-polymers1,72713
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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RNA chain , 5 types, 5 molecules IE265

#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
Details: Saccharomyces cerevisiae UBC4 pre-mRNA intron, in vitro transcribed.
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Production host: in vitro transcription vector pT7-Fluc(deltai) (others)
References: GenBank: 4718
#2: RNA chain UBC4 gene exon


Mass: 6518.976 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Saccharomyces cerevisiae UBC4 pre-mRNA exon, in vitro transcribed.
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: UBC4
Production host: in vitro transcription vector pT7-Fluc(deltai) (others)
#3: RNA chain U2 snRNA / U2 spliceosomal RNA


Mass: 376267.406 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Saccharomyces cerevisiae U2 snRNA
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Production host: Saccharomyces cerevisiae (brewer's yeast) / References: GenBank: 536627
#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 genetically manipulated source
Details: Saccharomyces cerevisiae U6 snRNA
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Production host: Saccharomyces cerevisiae (brewer's yeast) / References: GenBank: 1039022925
#5: RNA chain U5 snRNA / U5 spliceosomal RNA


Mass: 57444.875 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Saccharomyces cerevisiae U5 snRNA
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Production host: Saccharomyces cerevisiae (brewer's yeast) / References: GenBank: 1039023700

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Pre-mRNA-splicing factor ... , 15 types, 15 molecules ACHJLMNOPRSTacy

#6: Protein Pre-mRNA-splicing factor 8


Mass: 279867.469 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Saccharomyces cerevisiae Prp8 protein / 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 / Details: c Snu114 protein / 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 / Details: Saccharomyces cerevisiae Cwc22 protein / 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 / Details: Saccharomyces cerevisiae Prp46 protein / 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 / Details: Saccharomyces cerevisiae Bud31 protein / 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 / Details: Saccharomyces cerevisiae Cwc2 protein / 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 / Details: Saccharomyces cerevisiae Ecm2 protein / 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 / Details: Saccharomyces cerevisiae Cef1 protein / 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 / Details: Saccharomyces cerevisiae Cwc15 protein / 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 / Details: Saccharomyces cerevisiae Cwc21 protein / 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 / Details: Saccharomyces cerevisiae Clf1 protein / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q12309
#18: Protein 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 / Details: Saccharomyces cerevisiae Syf1 protein / 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 / Details: Saccharomyces cerevisiae Prp18 protein / 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 / Details: Saccharomyces cerevisiae Slu7 protein / 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 / Details: Saccharomyces cerevisiae Syf2 protein / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P53277

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Pre-mRNA-processing ... , 2 types, 2 molecules Ko

#10: Protein Pre-mRNA-processing protein 45


Mass: 42548.727 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Saccharomyces cerevisiae Prp45 protein / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P28004
#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 / Details: Saccharomyces cerevisiae Prp17 protein / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40968

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Protein , 2 types, 2 molecules Xb

#22: Protein Unknown


Mass: 5805.147 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Unknown protein helices within C-star complex / 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.: 1 / Source method: isolated from a natural source / Details: Saccharomyces cerevisiae SmB protein / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40018

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

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


Mass: 11240.139 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Saccharomyces cerevisiae SmD3 protein / 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.: 1 / Source method: isolated from a natural source / Details: Saccharomyces cerevisiae SmE protein / 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.: 1 / Source method: isolated from a natural source / Details: Saccharomyces cerevisiae SmF protein / 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.: 1 / Source method: isolated from a natural source / Details: Saccharomyces cerevisiae SmG protein / 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.: 1 / Source method: isolated from a natural source / Details: Saccharomyces cerevisiae SmD1 protein / 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.: 1 / Source method: isolated from a natural source / Details: Saccharomyces cerevisiae SmD2 protein / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q06217

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Non-polymers , 5 types, 13 molecules

#31: Chemical ChemComp-MG / MAGNESIUM ION


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


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


Mass: 660.035 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H18O24P6
#34: 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
#35: 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 micromolarEDTAEthylenediaminetetraacetic acid1
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 FIELDBright-field microscopy / 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

SoftwareName: REFMAC / Version: 5.8.0124 / Classification: refinement
EM software
IDNameVersionCategoryDetails
1RELION1.4particle selection
4CTFFIND4CTF correction
7Coot0.8.3model fittingPaul Emsley's experimental version.
9RELION1.4initial Euler assignment
10RELION1.4final Euler assignment
11RELION1.4classification
12RELION1.43D reconstruction
13REFMAC5.8model refinementUsed "SOURCE EM" command to get correct electron form factors for refinement of electron microscopy structure.
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.
3D reconstructionResolution: 3.85 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 65824 / 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.
RefinementResolution: 3.85→257.4 Å / Cor.coef. Fo:Fc: 0.952 / SU B: 37.73 / SU ML: 0.526 / ESU R: 0.868
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.32177 --
obs0.32177 357071 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 170.644 Å2
Baniso -1Baniso -2Baniso -3
1-0 Å20.34 Å2-0.53 Å2
2---0.26 Å2-0.07 Å2
3---0.26 Å2
Refinement stepCycle: 1 / Total: 59157
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0080.01861218
ELECTRON MICROSCOPYr_bond_other_d0.0020.0253354
ELECTRON MICROSCOPYr_angle_refined_deg1.2871.85984337
ELECTRON MICROSCOPYr_angle_other_deg1.0123122254
ELECTRON MICROSCOPYr_dihedral_angle_1_deg9.7535.1836844
ELECTRON MICROSCOPYr_dihedral_angle_2_deg32.3223.4852126
ELECTRON MICROSCOPYr_dihedral_angle_3_deg14.397158722
ELECTRON MICROSCOPYr_dihedral_angle_4_deg14.12915340
ELECTRON MICROSCOPYr_chiral_restr0.0830.2029725
ELECTRON MICROSCOPYr_gen_planes_refined0.0050.0263652
ELECTRON MICROSCOPYr_gen_planes_other0.0020.0213902
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_it5.46717.89927134
ELECTRON MICROSCOPYr_mcbond_other5.46717.89827133
ELECTRON MICROSCOPYr_mcangle_it9.5826.79733767
ELECTRON MICROSCOPYr_mcangle_other9.5826.79833768
ELECTRON MICROSCOPYr_scbond_it5.40118.17334084
ELECTRON MICROSCOPYr_scbond_other5.40118.17334085
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other9.74927.20850571
ELECTRON MICROSCOPYr_long_range_B_refined19.018122798
ELECTRON MICROSCOPYr_long_range_B_other19.018122798
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.85→3.95 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork0.599 26341 -
Rfree-0 -
obs--100 %

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