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- PDB-5gan: The overall structure of the yeast spliceosomal U4/U6.U5 tri-snRN... -

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Basic information

Entry
Database: PDB / ID: 5gan
TitleThe overall structure of the yeast spliceosomal U4/U6.U5 tri-snRNP at 3.7 Angstrom
Components
  • (Pre-mRNA-splicing factor ...) x 3
  • (Small nuclear ribonucleoprotein ...SnRNP) x 6
  • (U4/U6 small nuclear ribonucleoprotein ...) x 2
  • (U6 snRNA-associated Sm-like protein ...) x 7
  • 13 kDa ribonucleoprotein-associated protein
  • Pre-mRNA-processing factor 31
  • Pre-mRNA-splicing helicase BRR2
  • Small nuclear ribonucleoprotein-associated protein B
  • Snu66
  • Spliceosomal protein DIB1Spliceosome
  • U4 snRNAU4 spliceosomal RNA
  • U5 snRNAU5 spliceosomal RNA
  • U6 snRNAU6 spliceosomal RNA
  • Unknown protein
KeywordsTRANSCRIPTION / pre-mRNA splicing / snRNP / GTPase / U5 snRNA / Prp8 / spliceosome / U4/U6 snRNP / Brr2 / Snu114
Function / homology
Function and homology information


maturation of 5S rRNA / spliceosomal conformational changes to generate catalytic conformation / U4/U6 snRNP / snoRNA splicing / Lsm1-7-Pat1 complex / U6 snRNP / rRNA 2'-O-methylation / positive regulation of RNA binding / generation of catalytic spliceosome for first transesterification step / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) ...maturation of 5S rRNA / spliceosomal conformational changes to generate catalytic conformation / U4/U6 snRNP / snoRNA splicing / Lsm1-7-Pat1 complex / U6 snRNP / rRNA 2'-O-methylation / positive regulation of RNA binding / generation of catalytic spliceosome for first transesterification step / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) / splicing factor binding / box C/D RNP complex / P-body assembly / pICln-Sm protein complex / U4 snRNP / nuclear-transcribed mRNA catabolic process / sno(s)RNA-containing ribonucleoprotein complex / small nuclear ribonucleoprotein complex / spliceosomal tri-snRNP complex / SMN-Sm protein complex / mRNA cis splicing, via spliceosome / U4 snRNA binding / U2-type catalytic step 2 spliceosome / commitment complex / U2 snRNP / U3 snoRNA binding / U12-type spliceosomal complex / generation of catalytic spliceosome for second transesterification step / U2-type prespliceosome / spliceosomal snRNP assembly / U1 snRNP / mRNA 3'-splice site recognition / mRNA 5'-splice site recognition / precatalytic spliceosome / RNA metabolic process / tRNA processing / spliceosomal tri-snRNP complex assembly / rRNA methylation / U5 snRNP / U5 snRNA binding / poly(U) RNA binding / U2 snRNA binding / U6 snRNA binding / pre-mRNA intronic binding / U4/U6 x U5 tri-snRNP complex / maturation of SSU-rRNA / U1 snRNA binding / small-subunit processome / isopeptidase activity / catalytic step 2 spliceosome / maturation of LSU-rRNA / spliceosomal complex / P-body / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / metallopeptidase activity / rRNA processing / mRNA splicing, via spliceosome / ribonucleoprotein complex / RNA helicase / RNA helicase activity / GTPase activity / mRNA binding / GTP binding / nucleolus / mitochondrion / RNA binding / nucleoplasm / ATP binding / identical protein binding / nucleus / cytosol / cytoplasm
WD40-repeat-containing domain / Pre-mRNA-processing-splicing factor 8, U5-snRNA-binding / WD40/YVTN repeat-like-containing domain superfamily / Sm-like protein Lsm6/SmF / U6 snRNA-associated Sm-like protein LSm2 / Sm-like protein Lsm7 / Ribosomal protein L7Ae/L8/Nhp2 family / Prp31 C-terminal / Pre-mRNA-processing-splicing factor 8, U6-snRNA-binding / RNA recognition motif, spliceosomal PrP8 ...WD40-repeat-containing domain / Pre-mRNA-processing-splicing factor 8, U5-snRNA-binding / WD40/YVTN repeat-like-containing domain superfamily / Sm-like protein Lsm6/SmF / U6 snRNA-associated Sm-like protein LSm2 / Sm-like protein Lsm7 / Ribosomal protein L7Ae/L8/Nhp2 family / Prp31 C-terminal / Pre-mRNA-processing-splicing factor 8, U6-snRNA-binding / RNA recognition motif, spliceosomal PrP8 / Immunoglobulin E-set / Tetratricopeptide repeat / WD40 repeat, conserved site / G-protein beta WD-40 repeat / Ribosomal protein S5 domain 2-type fold / PRP8 domain IV core / Small nuclear ribonucleoprotein E / U4/U6 small nuclear ribonucleoprotein Prp3 / Pre-mRNA processing factor 4 (PRP4)-like / Ribosomal protein S5 domain 2-type fold, subgroup / Pre-mRNA-processing factor 6/Prp1/STA1 / LSM domain superfamily / Translation elongation factor EFTu-like, domain 2 / Sec63 domain / SNU66/SART1 family / Translation elongation factor EFG/EF2, domain IV / Translation protein, beta-barrel domain superfamily / PRP1 splicing factor, N-terminal / Domain of unknown function DUF1115 / DEAD/DEAH box helicase domain / Helicase superfamily 1/2, ATP-binding domain / Tetratricopeptide-like helical domain superfamily / Ribonuclease H-like superfamily / PRO8NT domain / PROCN domain / NOSIC / PROCT domain / Tetratricopeptide repeat-containing domain / Pre-mRNA-splicing factor 3 / U4/U6 small nuclear ribonucleoprotein Prp31 / Like-Sm (LSM) domain containing protein, LSm4/SmD1/SmD3 / PRP8 domain IV core / pre-mRNA processing factor 3 (PRP3) / Nop, C-terminal domain / Pre-mRNA-processing-splicing factor 8, U5-snRNA-binding domain superfamily / Prp8 RNase domain IV, palm region / Prp8 RNase domain IV, fingers region / Snu114, GTP-binding domain / PRP1 splicing factor, N-terminal / WD domain, G-beta repeat / Protein of unknown function (DUF1115) / U6 snRNA-associated Sm-like protein Lsm3 / Prp31 C terminal domain / Mitosis protein DIM1 / 116 kDa U5 small nuclear ribonucleoprotein component N-terminus / Elongation factor Tu domain 2 / Elongation factor G C-terminus / N-terminal helicase PWI domain / Sec63 Brl domain / Brr2, N-terminal helicase PWI domain / MPN domain / Small nuclear ribonucleoprotein Sm D2 / Small nuclear ribonucleoprotein F / P-loop containing nucleoside triphosphate hydrolase / Pre-mRNA-processing-splicing factor 8 / 50S ribosomal protein L30e-like / 116kDa U5 small nuclear ribonucleoprotein component, N-terminal / Sm-like protein LSm5 / Small nuclear ribonucleoprotein G / Small nuclear ribonucleoprotein Sm D3 / Sm-like protein Lsm4 / Winged helix DNA-binding domain superfamily / Sm-like protein Lsm8 / Sm-like protein Lsm3 / EF-G domain III/V-like / 116kDa U5 small nuclear ribonucleoprotein component, C-terminal / C2 domain superfamily / Nop domain superfamily / Thioredoxin-like superfamily / WD40-repeat-containing domain superfamily / Dim1 family / Ribosomal protein L7Ae/L30e/S12e/Gadd45 / Ribosomal protein L7Ae conserved site / Translational (tr)-type GTP-binding domain / HAT (Half-A-TPR) repeat / Ribosomal protein L7Ae/L30e/S12e/Gadd45 family / JAB1/MPN/MOV34 metalloenzyme domain / Elongation factor EFG, domain V-like / Helicase conserved C-terminal domain / LSM domain, eukaryotic/archaea-type / WD40 repeat / H/ACA ribonucleoprotein complex, subunit Nhp2, eukaryote / Nop domain / Helicase, C-terminal / Sec63 N-terminal domain-like domain / Sec63 N-terminal domain-like fold / SH3 type barrels. - #100 / C2 domain / Ribosomal protein L30/S12 / 60s Ribosomal Protein L30; Chain: A;
U6 snRNA-associated Sm-like protein LSm6 / U6 snRNA-associated Sm-like protein LSm8 / Pre-mRNA-processing factor 31 / gb:807071957: / U6 snRNA-associated Sm-like protein LSm7 / Small nuclear ribonucleoprotein F / U6 snRNA-associated Sm-like protein LSm3 / Small nuclear ribonucleoprotein Sm D1 / U4/U6 small nuclear ribonucleoprotein PRP3 / Small nuclear ribonucleoprotein Sm D2 ...U6 snRNA-associated Sm-like protein LSm6 / U6 snRNA-associated Sm-like protein LSm8 / Pre-mRNA-processing factor 31 / gb:807071957: / U6 snRNA-associated Sm-like protein LSm7 / Small nuclear ribonucleoprotein F / U6 snRNA-associated Sm-like protein LSm3 / Small nuclear ribonucleoprotein Sm D1 / U4/U6 small nuclear ribonucleoprotein PRP3 / Small nuclear ribonucleoprotein Sm D2 / U6 snRNA-associated Sm-like protein LSm6 / Spliceosomal protein DIB1 / Small nuclear ribonucleoprotein E / 66 kDa U4/U6.U5 small nuclear ribonucleoprotein component / gb:807071964: / U4/U6 small nuclear ribonucleoprotein PRP4 / gb:807071959: / Small nuclear ribonucleoprotein Sm D3 / Small nuclear ribonucleoprotein G / U6 snRNA-associated Sm-like protein LSm5 / U6 snRNA-associated Sm-like protein LSm4 / Small nuclear ribonucleoprotein-associated protein B / 13 kDa ribonucleoprotein-associated protein / U6 snRNA-associated Sm-like protein LSm2 / Pre-mRNA-splicing factor SNU114 / Pre-mRNA-splicing factor 8 / Pre-mRNA-splicing helicase BRR2 / Pre-mRNA-splicing factor 6
Biological speciesSaccharomyces cerevisiae (baker's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / Resolution: 3.7 Å
AuthorsNguyen, T.H.D. / Galej, W.P. / Bai, X.C. / Oubridge, C. / Scheres, S.H.W. / Newman, A.J. / Nagai, K.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom) United Kingdom
Citation
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.
#1: 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.
Validation Report
SummaryFull reportAbout validation report
History
DepositionDec 15, 2015Deposition site: RCSB / Processing site: PDBE
Revision 1.0Jan 27, 2016Provider: repository / Type: Initial release
Revision 1.1Aug 30, 2017Group: Advisory / Author supporting evidence ...Advisory / Author supporting evidence / Data collection / Derived calculations
Category: em_imaging_optics / em_software ...em_imaging_optics / em_software / pdbx_audit_support / pdbx_validate_close_contact / struct_conn
Item: _em_imaging_optics.energyfilter_name / _em_software.name ..._em_imaging_optics.energyfilter_name / _em_software.name / _em_software.version / _pdbx_audit_support.funding_organization
Revision 1.2Mar 21, 2018Group: Other / Structure summary / Category: cell / entity / Item: _cell.Z_PDB / _entity.pdbx_description
Revision 1.3Feb 20, 2019Group: Advisory / Data collection / Derived calculations
Category: em_admin / pdbx_data_processing_status ...em_admin / pdbx_data_processing_status / pdbx_database_proc / pdbx_seq_map_depositor_info / pdbx_validate_close_contact / struct_conn / struct_conn_type
Item: _em_admin.last_update / _pdbx_seq_map_depositor_info.one_letter_code / _pdbx_seq_map_depositor_info.one_letter_code_mod
Revision 1.4Dec 11, 2019Group: Other / Category: atom_sites
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]

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Structure visualization

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Assembly

Deposited unit
V: U4 snRNA
W: U6 snRNA
A: Pre-mRNA-splicing factor 8
H: U4/U6 small nuclear ribonucleoprotein PRP4
J: Pre-mRNA-splicing factor 6
D: Spliceosomal protein DIB1
F: Pre-mRNA-processing factor 31
G: U4/U6 small nuclear ribonucleoprotein PRP3
B: Pre-mRNA-splicing helicase BRR2
x: Unknown protein
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
E: Snu66
b: Small nuclear ribonucleoprotein-associated protein B
e: Small nuclear ribonucleoprotein E
f: Small nuclear ribonucleoprotein F
g: Small nuclear ribonucleoprotein G
d: Small nuclear ribonucleoprotein Sm D3
h: Small nuclear ribonucleoprotein Sm D1
j: Small nuclear ribonucleoprotein Sm D2
U: U5 snRNA
K: 13 kDa ribonucleoprotein-associated protein
2: U6 snRNA-associated Sm-like protein LSm2
3: U6 snRNA-associated Sm-like protein LSm3
4: U6 snRNA-associated Sm-like protein LSm4
5: U6 snRNA-associated Sm-like protein LSm5
6: U6 snRNA-associated Sm-like protein LSm6
7: U6 snRNA-associated Sm-like protein LSm7
8: U6 snRNA-associated Sm-like protein LSm8
C: Pre-mRNA-splicing factor SNU114
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,404,06636
Polymers1,403,54335
Non-polymers5231
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area152010 Å2
ΔGint-952 kcal/mol
Surface area420400 Å2
MethodPISA

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Components

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RNA chain , 3 types, 3 molecules VWU

#1: RNA chain U4 snRNA / U4 spliceosomal RNA / U4 snRNA


Mass: 51186.023 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / Strain: BCY123 / References: GenBank: 807071957
#2: RNA chain U6 snRNA / U6 spliceosomal RNA / U6 snRNA


Mass: 35883.176 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / Strain: BCY123 / References: GenBank: 807071964
#19: RNA chain U5 snRNA / U5 spliceosomal RNA


Mass: 68643.344 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / Strain: BCY123 / References: GenBank: 807071959

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Pre-mRNA-splicing factor ... , 3 types, 3 molecules AJC

#3: Protein Pre-mRNA-splicing factor 8 / Prp8


Mass: 279867.469 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / Strain: BCY123 / References: UniProt: P33334
#5: Protein Pre-mRNA-splicing factor 6 / Prp6


Mass: 104370.133 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / Strain: BCY123 / References: UniProt: P19735
#28: Protein Pre-mRNA-splicing factor SNU114 / 114 kDa U5 small nuclear ribonucleoprotein component / Growth inhibitory protein 10 / Snu114


Mass: 114132.086 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / Strain: BCY123 / References: UniProt: P36048

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

#4: Protein U4/U6 small nuclear ribonucleoprotein PRP4 / Pre-mRNA-processing protein 4 / Prp4


Mass: 52506.984 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / Strain: BCY123 / References: UniProt: P20053
#8: Protein U4/U6 small nuclear ribonucleoprotein PRP3 / Pre-mRNA-splicing factor 3 / Prp3


Mass: 55974.320 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / Strain: BCY123 / References: UniProt: Q03338

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Protein , 7 types, 8 molecules DFBxkbEK

#6: Protein Spliceosomal protein DIB1 / Spliceosome


Mass: 16798.387 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / Strain: BCY123 / References: UniProt: Q06819
#7: Protein Pre-mRNA-processing factor 31 / Prp31


Mass: 56382.516 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / Strain: BCY123 / References: UniProt: P49704
#9: Protein Pre-mRNA-splicing helicase BRR2 / Protein Snu246 / Brr2


Mass: 246470.266 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / Strain: BCY123 / References: UniProt: P32639, RNA helicase
#10: Protein Unknown protein


Mass: 8528.504 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / Strain: BCY123
#11: 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 (baker's yeast) / Strain: BCY123 / References: UniProt: P40018
#18: Protein Snu66


Mass: 28660.018 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Details: Snu66 is mostly fitted as polyAla into helices and extended polypeptides within the EM map. The authors do not believe that the sequence number is correct.
Source: (natural) Saccharomyces cerevisiae (baker's yeast) / Strain: BCY123 / References: UniProt: Q12420*PLUS
#20: Protein 13 kDa ribonucleoprotein-associated protein / Small nuclear ribonucleoprotein-associated protein 1 / Snu13


Mass: 13582.855 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / Strain: BCY123 / References: UniProt: P39990

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

#12: Protein Small nuclear ribonucleoprotein Sm D1 / Sm-D1 / snRNP core protein D1 / U4 SmD1


Mass: 16296.798 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / Strain: BCY123 / References: UniProt: Q02260
#13: Protein Small nuclear ribonucleoprotein Sm D2 / Sm-D2 / snRNP core protein D2 / SmD2


Mass: 12876.066 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / Strain: BCY123 / References: UniProt: Q06217
#14: Protein Small nuclear ribonucleoprotein Sm D3 / Sm-D3 / snRNP core protein D3 / SmD3


Mass: 11240.139 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / Strain: BCY123 / References: UniProt: P43321
#15: 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 (baker's yeast) / Strain: BCY123 / References: UniProt: Q12330
#16: 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 (baker's yeast) / Strain: BCY123 / References: UniProt: P54999
#17: 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 (baker's yeast) / Strain: BCY123 / References: UniProt: P40204

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U6 snRNA-associated Sm-like protein ... , 7 types, 7 molecules 2345678

#21: Protein U6 snRNA-associated Sm-like protein LSm2 / Small nuclear ribonucleoprotein D homolog SNP3


Mass: 11177.888 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / Strain: BCY123 / References: UniProt: P38203
#22: Protein U6 snRNA-associated Sm-like protein LSm3 / SmX4 protein


Mass: 10039.262 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / Strain: BCY123 / References: UniProt: P57743
#23: Protein U6 snRNA-associated Sm-like protein LSm4 / LSm4


Mass: 21298.070 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / Strain: BCY123 / References: UniProt: P40070
#24: Protein U6 snRNA-associated Sm-like protein LSm5 / LSm5


Mass: 10432.954 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / Strain: BCY123 / References: UniProt: P40089
#25: Protein U6 snRNA-associated Sm-like protein LSm6 / LSm6


Mass: 9406.579 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / Strain: BCY123 / References: UniProt: A6ZYX7, UniProt: Q06406*PLUS
#26: Protein U6 snRNA-associated Sm-like protein LSm7 / LSm7


Mass: 13027.045 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / Strain: BCY123 / References: UniProt: P53905
#27: Protein U6 snRNA-associated Sm-like protein LSm8 / LSm8


Mass: 12403.378 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / Strain: BCY123 / References: UniProt: P47093

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Non-polymers , 1 types, 1 molecules

#29: 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: The overall structure of the yeast spliceosomal U4/U6.U5 tri-snRNP
Type: COMPLEX / Details: 30 proteins and 3 snRNAs / Entity ID: #1-#29 / Source: NATURAL
Molecular weightValue: 1.5 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (baker's yeast) / Strain: BCY123
Buffer solutionpH: 7.9
Buffer componentConc.: 1 mM / Name: DTT
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: NO
Specimen supportDetails: Grids are made of holey carbon, carbon-coated and glow discharged in N-amylamine.
Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3

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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: 3500 nm / Nominal defocus min: 500 nm / Cs: 2 mm
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 16 sec. / Electron dose: 38 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 2477
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
IDNameVersionCategory
1RELION1.4particle selection
4CTFFIND4CTF correction
7Coot0.8.3model fitting
9REFMAC5.8model refinement
10RELION1.4initial Euler assignment
11RELION1.4final Euler assignment
12RELIONclassification
13RELION1.43D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 473827
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 140155 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingSpace: RECIPROCAL
RefinementResolution: 3.6→308.88 Å / Cor.coef. Fo:Fc: 0.974 / SU B: 26.326 / SU ML: 0.371 / ESU R: 0.409
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.29721 --
Obs0.29721 838746 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 397.886 Å2
Baniso -1Baniso -2Baniso -3
1--0.98 Å21.68 Å2-0.03 Å2
2--4.25 Å20.28 Å2
3----3.27 Å2
Refinement stepCycle: 1 / Total: 77375
Refine LS restraints
Refinement-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0080.01979802
ELECTRON MICROSCOPYr_bond_other_d0.0020.0272624
ELECTRON MICROSCOPYr_angle_refined_deg1.3391.886109442
ELECTRON MICROSCOPYr_angle_other_deg0.9823166950
ELECTRON MICROSCOPYr_dihedral_angle_1_deg13.2695.5059696
ELECTRON MICROSCOPYr_dihedral_angle_2_deg34.72924.2173014
ELECTRON MICROSCOPYr_dihedral_angle_3_deg16.8961512280
ELECTRON MICROSCOPYr_dihedral_angle_4_deg12.50615401
ELECTRON MICROSCOPYr_chiral_restr0.1720.20212649
ELECTRON MICROSCOPYr_gen_planes_refined0.0060.0284167
ELECTRON MICROSCOPYr_gen_planes_other0.0020.0217927
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_it17.54239.76135848
ELECTRON MICROSCOPYr_mcbond_other17.54239.76135847
ELECTRON MICROSCOPYr_mcangle_it26.84659.60144681
ELECTRON MICROSCOPYr_mcangle_other26.84659.60244682
ELECTRON MICROSCOPYr_scbond_it18.10640.93743954
ELECTRON MICROSCOPYr_scbond_other18.10340.94143942
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other29.67160.94864741
ELECTRON MICROSCOPYr_long_range_B_refined39.339141451
ELECTRON MICROSCOPYr_long_range_B_other39.339141445
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.6→3.693 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork3.416 62060 -
Rfree-0 -
Obs--100 %

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