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- PDB-5lj3: Structure of the core of the yeast spliceosome immediately after ... -

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Basic information

Entry
Database: PDB / ID: 5lj3
TitleStructure of the core of the yeast spliceosome immediately after branching
Components
  • (Pre-mRNA-splicing factor ...) x 6
  • (Small nuclear ribonucleoprotein ...SnRNP) x 6
  • (U2 small nuclear ribonucleoprotein ...) x 2
  • CEF1
  • CLF1
  • CWC15
  • CWC2
  • CWC22
  • Exon 1 (5' exon) of UBC4 pre-mRNA
  • ISY1
  • Intron of UBC4 pre-mRNA
  • PRP46
  • Pre-mRNA-processing protein 45
  • Protein CWC16
  • SYF1
  • Small nuclear ribonucleoprotein-associated protein B
  • U2 snRNA (small nuclear RNA)
  • U5 snRNA (small nuclear RNA)
  • U6 snRNA (small nuclear RNA)
  • unknown
KeywordsSPLICING / spliceosome / snRNP / pre-mRNA splicing / trans-esterification / lariat intermediate / complex C
Function / homology
Function and homology information


post-spliceosomal complex / U2-type post-mRNA release spliceosomal complex / cellular bud site selection / post-mRNA release spliceosomal complex / U4/U6 snRNP / cis assembly of pre-catalytic spliceosome / generation of catalytic spliceosome for first transesterification step / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) / splicing factor binding / 7-methylguanosine cap hypermethylation ...post-spliceosomal complex / U2-type post-mRNA release spliceosomal complex / cellular bud site selection / post-mRNA release spliceosomal complex / U4/U6 snRNP / cis assembly of pre-catalytic spliceosome / generation of catalytic spliceosome for first transesterification step / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) / splicing factor binding / 7-methylguanosine cap hypermethylation / pre-mRNA binding / U2-type catalytic step 1 spliceosome / pICln-Sm protein complex / Prp19 complex / spliceosomal tri-snRNP complex / U4 snRNP / 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 / 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 / positive regulation of RNA splicing / 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 / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
CBF1-interacting co-repressor CIR, N-terminal domain / Pre-mRNA splicing factor / N-terminal domain of CBF1 interacting co-repressor CIR / Pre-mRNA splicing factor / N-terminal domain of CBF1 interacting co-repressor CIR / Saf4/Yju2 protein / Splicing factor Yju2 / Saf4/Yju2 protein / Pre-mRNA-splicing factor Isy1 / Pre-mRNA-splicing factor Isy1 superfamily ...CBF1-interacting co-repressor CIR, N-terminal domain / Pre-mRNA splicing factor / N-terminal domain of CBF1 interacting co-repressor CIR / Pre-mRNA splicing factor / N-terminal domain of CBF1 interacting co-repressor CIR / Saf4/Yju2 protein / Splicing factor Yju2 / Saf4/Yju2 protein / Pre-mRNA-splicing factor Isy1 / Pre-mRNA-splicing factor Isy1 superfamily / Isy1-like splicing family / Slt11, RNA recognition motif / cwf21 / Torus domain / Pre-mRNA-splicing factor Cwc2, RNA recognition motif / Torus domain / Helix hairpin bin domain superfamily / mRNA splicing factor Cwf21 domain / cwf21 domain / : / RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) / 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 / U2A'/phosphoprotein 32 family A, C-terminal / occurring C-terminal to leucine-rich repeats / Leucine-rich repeat / Pre-mRNA-splicing factor Syf1-like / 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 / RRM (RNA recognition motif) domain / Elongation factor Tu domain 2 / 7 Propeller
Similarity search - Domain/homology
GUANOSINE-5'-TRIPHOSPHATE / : / : / : / : / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / : ...GUANOSINE-5'-TRIPHOSPHATE / : / : / : / : / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / : / : / : / : / : / : / : / : / Pre-mRNA-splicing factor ISY1 / Pre-mRNA-splicing factor BUD31 / Pre-mRNA-processing protein 45 / Splicing factor YJU2 / Pre-mRNA-splicing factor 8 / Pre-mRNA-splicing factor SNU114 / Pre-mRNA-splicing factor SLT11 / Small nuclear ribonucleoprotein-associated protein B / Small nuclear ribonucleoprotein G / U2 small nuclear ribonucleoprotein B'' / Small nuclear ribonucleoprotein Sm D3 / Pre-mRNA-splicing factor CWC25 / Small nuclear ribonucleoprotein F / Small nuclear ribonucleoprotein Sm D1 / Pre-mRNA-splicing factor CWC21 / Pre-mRNA-splicing factor CEF1 / Pre-mRNA-splicing factor CWC15 / Pre-mRNA-splicing factor SYF1 / 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: 3.8 Å
AuthorsGalej, W.P. / Wilkinson, M.F. / Fica, S.M. / Oubridge, C. / Newman, A.J. / Nagai, K.
CitationJournal: Nature / Year: 2016
Title: Cryo-EM structure of the spliceosome immediately after branching.
Authors: Wojciech P Galej / Max E Wilkinson / Sebastian M Fica / Chris Oubridge / Andrew J Newman / Kiyoshi Nagai /
Abstract: Precursor mRNA (pre-mRNA) splicing proceeds by two consecutive transesterification reactions via a lariat-intron intermediate. Here we present the 3.8 Å cryo-electron microscopy structure of the ...Precursor mRNA (pre-mRNA) splicing proceeds by two consecutive transesterification reactions via a lariat-intron intermediate. Here we present the 3.8 Å cryo-electron microscopy structure of the spliceosome immediately after lariat formation. The 5'-splice site is cleaved but remains close to the catalytic Mg site in the U2/U6 small nuclear RNA (snRNA) triplex, and the 5'-phosphate of the intron nucleotide G(+1) is linked to the branch adenosine 2'OH. The 5'-exon is held between the Prp8 amino-terminal and linker domains, and base-pairs with U5 snRNA loop 1. Non-Watson-Crick interactions between the branch helix and 5'-splice site dock the branch adenosine into the active site, while intron nucleotides +3 to +6 base-pair with the U6 snRNA ACAGAGA sequence. Isy1 and the step-one factors Yju2 and Cwc25 stabilize docking of the branch helix. The intron downstream of the branch site emerges between the Prp8 reverse transcriptase and linker domains and extends towards the Prp16 helicase, suggesting a plausible mechanism of remodelling before exon ligation.
History
DepositionJul 17, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 3, 2016Provider: repository / Type: Initial release
Revision 1.1Sep 21, 2016Group: Database references
Revision 1.2Aug 30, 2017Group: Data collection / Derived calculations / Category: em_imaging_optics / em_software / struct_conn
Item: _em_imaging_optics.energyfilter_name / _em_software.name
Revision 1.3Oct 3, 2018Group: Data collection / Refinement description
Category: refine / refine_hist ...refine / refine_hist / refine_ls_restr / refine_ls_shell
Item: _refine.pdbx_refine_id / _refine_hist.pdbx_refine_id ..._refine.pdbx_refine_id / _refine_hist.pdbx_refine_id / _refine_ls_restr.pdbx_refine_id / _refine_ls_shell.pdbx_refine_id
Revision 1.4Oct 24, 2018Group: Advisory / Data collection / Derived calculations
Category: pdbx_validate_close_contact / struct_conn / struct_conn_type
Revision 1.5Oct 23, 2019Group: Data collection / Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
U: U5 snRNA (small nuclear RNA)
E: Exon 1 (5' exon) of UBC4 pre-mRNA
I: Intron of UBC4 pre-mRNA
Z: U2 snRNA (small nuclear RNA)
V: U6 snRNA (small nuclear RNA)
A: Pre-mRNA-splicing factor 8
D: Protein CWC16
F: Pre-mRNA-splicing factor CWC25
C: Pre-mRNA-splicing factor SNU114
G: ISY1
H: CWC22
J: PRP46
K: Pre-mRNA-processing protein 45
L: Pre-mRNA-splicing factor BUD31
M: CWC2
N: Pre-mRNA-splicing factor SLT11
O: CEF1
P: CWC15
R: Pre-mRNA-splicing factor CWC21
S: CLF1
T: SYF1
b: Small nuclear ribonucleoprotein-associated protein B
d: Small nuclear ribonucleoprotein Sm D3
e: Small nuclear ribonucleoprotein E
f: Small nuclear ribonucleoprotein F
g: Small nuclear ribonucleoprotein G
h: Small nuclear ribonucleoprotein Sm D1
j: Small nuclear ribonucleoprotein Sm D2
W: U2 small nuclear ribonucleoprotein A'
Y: U2 small nuclear ribonucleoprotein B''
k: Small nuclear ribonucleoprotein-associated protein B
l: Small nuclear ribonucleoprotein Sm D1
m: Small nuclear ribonucleoprotein Sm D2
n: Small nuclear ribonucleoprotein Sm D3
p: Small nuclear ribonucleoprotein E
q: Small nuclear ribonucleoprotein F
r: Small nuclear ribonucleoprotein G
x: unknown
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,755,83048
Polymers1,754,80138
Non-polymers1,03010
Water0
1


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

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Components

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RNA chain , 5 types, 5 molecules UEIZV

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


Mass: 57444.875 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: GenBank: 1039022920
#2: RNA chain Exon 1 (5' exon) of UBC4 pre-mRNA


Mass: 5170.152 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Details: This RNA was produced from an in vitro transcribed yeast UBC4 pre-mRNA with two stem-loops added at the 5' end for binding by the MS2 bacteriophage coat protein. This pre-mRNA is added to a ...Details: This RNA was produced from an in vitro transcribed yeast UBC4 pre-mRNA with two stem-loops added at the 5' end for binding by the MS2 bacteriophage coat protein. This pre-mRNA is added to a yeast splicing extract and the first step of splicing yields the 5' exon and the lariat intron-3' exon intermediate, which both remain associated with the spliceosome.
Source: (natural) Saccharomyces cerevisiae (brewer's yeast)
#3: RNA chain Intron of UBC4 pre-mRNA


Mass: 24109.115 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Details: This RNA was produced from an in vitro transcribed yeast UBC4 pre-mRNA with two stem-loops added at the 5' end for binding by the MS2 bacteriophage coat protein. This pre-mRNA is added to a ...Details: This RNA was produced from an in vitro transcribed yeast UBC4 pre-mRNA with two stem-loops added at the 5' end for binding by the MS2 bacteriophage coat protein. This pre-mRNA is added to a yeast splicing extract and the first step of splicing yields the 5' exon and the lariat intron-3' exon intermediate (this RNA), which both remain associated with the spliceosome.
Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: GenBank: 4718
#4: RNA chain U2 snRNA (small nuclear RNA)


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


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

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Pre-mRNA-splicing factor ... , 6 types, 6 molecules AFCLNR

#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 / References: UniProt: P33334
#8: Protein Pre-mRNA-splicing factor CWC25 / Complexed with CEF1 protein 25


Mass: 20412.477 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae / References: UniProt: P53854
#9: 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 / References: UniProt: P36048
#14: Protein Pre-mRNA-splicing factor BUD31 / Bud site selection protein 31 / Complexed with CEF1 protein 14


Mass: 18484.502 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P25337
#16: Protein Pre-mRNA-splicing factor SLT11 / Extracellular mutant protein 2 / Synthetic lethality with U2 protein 11 / Ecm2


Mass: 40988.590 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae / References: UniProt: P38241
#19: 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 / References: UniProt: Q03375

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Protein , 12 types, 13 molecules DGHJKMOPSTbkx

#7: Protein Protein CWC16 / Yju2


Mass: 32371.086 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae / References: UniProt: P28320
#10: Protein ISY1


Mass: 28120.896 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A165UGV0, UniProt: P21374*PLUS
#11: Protein CWC22


Mass: 69118.984 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A162ERE7
#12: Protein PRP46


Mass: 50801.316 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A162HKW4, UniProt: Q12417*PLUS
#13: 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
#15: Protein CWC2


Mass: 38458.508 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A162HWE4, UniProt: Q12046*PLUS
#17: Protein CEF1


Mass: 67819.789 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A165TLN2, UniProt: Q03654*PLUS
#18: Protein CWC15


Mass: 19970.195 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A162GIZ3, UniProt: Q03772*PLUS
#20: Protein CLF1


Mass: 82532.867 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A165TU20, UniProt: Q12309*PLUS
#21: Protein SYF1


Mass: 100356.141 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A165VVY6, UniProt: Q04048*PLUS
#22: 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 / References: UniProt: P40018
#31: Protein unknown


Mass: 11251.861 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast)

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

#23: 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 / References: UniProt: P43321
#24: 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 / References: UniProt: Q12330
#25: 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
#26: 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 / References: UniProt: P40204
#27: 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
Details: MKLVNFLKKLRNEQVTIELKNGTTVWGTLQSVSPQMNAILTDVKLTLPQPRLNKLNSNGIAMASLYLTGGQQPTASDNIA SLQYINIRGNTIRQIILPDSLNLDSLLVDQKQLNSLRRSGQIANDPSKKRRRDFGAPANKRPRRGL
Source: (natural) Saccharomyces cerevisiae / References: UniProt: Q02260
#28: 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 / References: UniProt: Q06217

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

#29: 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
#30: 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 / References: UniProt: P40567

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Non-polymers , 3 types, 10 molecules

#32: Chemical ChemComp-MG / MAGNESIUM ION


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


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

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Spliceosome immediately after branching / Type: COMPLEX
Details: Splicing extract was prepared from Prp18-HA or Slu7-TAPS yeast strains. An in vitro transcribed yeast UBC4 pre-mRNA substrate (with 2 x MS2 bacteriophage coat protein-binding stem loops at ...Details: Splicing extract was prepared from Prp18-HA or Slu7-TAPS yeast strains. An in vitro transcribed yeast UBC4 pre-mRNA substrate (with 2 x MS2 bacteriophage coat protein-binding stem loops at the 5' end and with the 3'-splice site sequence UAGAG mutated to UACAC) was pre-bound to an MS2-maltose binding protein fusion protein. This substrate-protein complex was added to the splicing extract. The splicing reaction proceeded through the first step but the second step was blocked by the 3' splice site mutation. Substrate-bound spliceosomes from the splicing extract were purified on amylose resin and eluted with maltose. Subsequently the spliceosomes were captured on anti-HA-agarose (for Prp18-HA-tagged) or streptactin resin (for Slu7-TAPS tagged) and eluted with HA peptide or desthiobiotin, respectively. Purified spliceosomes were then dialysed against 20 mM HEPES KOH pH 7.8, 75 mM KCl, 0.25 mM EDTA
Entity ID: #1-#31 / Source: NATURAL
Molecular weightValue: 2 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHepes.KOH pH 7.81
275 mMpotassium chlorideKCl1
3250 micromolarEDTAEthylenediaminetetraacetic acid1
41
51
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: 3 microlitres sample were applied to the grid, left for 30 seconds and then blotted for 2.5-3.0 seconds before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Calibrated magnification: 35714 X / Nominal defocus max: 4000 nm / Nominal defocus min: 500 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 0.8 sec. / Electron dose: 2 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of real images: 2213
Details: Total dose: 40 electrons/Angstrom^2 over 16 seconds. 20 movie frames collected at 1.25 frames per second.
EM imaging opticsEnergyfilter name: GIF Quantum
Image scansMovie frames/image: 20 / Used frames/image: 1-20

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Processing

SoftwareName: REFMAC / Version: 5.8.0124 / Classification: refinement
EM software
IDNameVersionCategoryDetails
1RELION1.4particle selection
4CTFFIND4CTF correction
7Coot0.8.3model fittingPaul Emsley's experimental version.
9REFMAC5.8model refinement
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: 248000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 93106 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: RECIPROCAL
Details: Used secondary structure restraints generated in ProSMART and LibG.
RefinementResolution: 3.8→274.56 Å / Cor.coef. Fo:Fc: 0.954 / SU B: 27.594 / SU ML: 0.398 / ESU R: 0.522
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.31901 --
obs0.31901 592776 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 180.1 Å2
Baniso -1Baniso -2Baniso -3
1-1.84 Å21.69 Å2-1.19 Å2
2---1.95 Å2-1.26 Å2
3---0.11 Å2
Refinement stepCycle: 1 / Total: 63166
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0070.01865428
ELECTRON MICROSCOPYr_bond_other_d0.0020.0253593
ELECTRON MICROSCOPYr_angle_refined_deg1.2521.83290631
ELECTRON MICROSCOPYr_angle_other_deg1.023122463
ELECTRON MICROSCOPYr_dihedral_angle_1_deg9.4125.2417501
ELECTRON MICROSCOPYr_dihedral_angle_2_deg36.16323.6681963
ELECTRON MICROSCOPYr_dihedral_angle_3_deg18.101157986
ELECTRON MICROSCOPYr_dihedral_angle_4_deg13.28515310
ELECTRON MICROSCOPYr_chiral_restr0.0890.20310628
ELECTRON MICROSCOPYr_gen_planes_refined0.0050.0268092
ELECTRON MICROSCOPYr_gen_planes_other0.0020.0214494
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it4.93120.95729635
ELECTRON MICROSCOPYr_mcbond_other4.9320.95729634
ELECTRON MICROSCOPYr_mcangle_it8.83931.34736846
ELECTRON MICROSCOPYr_mcangle_other8.83931.34836847
ELECTRON MICROSCOPYr_scbond_it5.32920.04235793
ELECTRON MICROSCOPYr_scbond_other5.32920.04235794
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other9.20830.54753786
ELECTRON MICROSCOPYr_long_range_B_refined19.477137799
ELECTRON MICROSCOPYr_long_range_B_other19.477137800
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.8→3.899 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork0.563 44142 -
Rfree-0 -
obs--100 %

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