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- EMDB-8014: Body region of the U4/U6.U5 tri-snRNP -

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Basic information

Entry
Database: EMDB / ID: EMD-8014
TitleBody region of the U4/U6.U5 tri-snRNP
Map dataCentral region of the U4/U6.U5 tri-snRNP comprising Prp8, Dib1, Prp31, Prp6, Prp4, Prp3, Snu13, U4 snRNA and U6 snRNA
Sample
  • Complex: Body region of the U4/U6.U5 tri-snRNP complex
    • RNA: x 3 types
    • Protein or peptide: x 9 types
KeywordsTranscription / snRNP / spliceosome / RNA-protein complex / U4/U6.U5 snRNP
Function / homology
Function and homology information


spliceosomal conformational changes to generate catalytic conformation / snoRNA splicing / snoRNA guided rRNA 2'-O-methylation / box C/D sno(s)RNA 3'-end processing / box C/D methylation guide snoRNP complex / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) / U4/U6 snRNP / U4 snRNA binding / spliceosomal tri-snRNP complex / U4 snRNP ...spliceosomal conformational changes to generate catalytic conformation / snoRNA splicing / snoRNA guided rRNA 2'-O-methylation / box C/D sno(s)RNA 3'-end processing / box C/D methylation guide snoRNP complex / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) / U4/U6 snRNP / U4 snRNA binding / spliceosomal tri-snRNP complex / U4 snRNP / U3 snoRNA binding / precatalytic spliceosome / generation of catalytic spliceosome for second transesterification step / Major pathway of rRNA processing in the nucleolus and cytosol / spliceosomal complex assembly / mRNA 5'-splice site recognition / mRNA 3'-splice site recognition / spliceosomal tri-snRNP complex assembly / Prp19 complex / U5 snRNA binding / U5 snRNP / U2 snRNA binding / U6 snRNA binding / pre-mRNA intronic binding / spliceosomal snRNP assembly / U1 snRNA binding / U4/U6 x U5 tri-snRNP complex / catalytic step 2 spliceosome / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / maturation of SSU-rRNA / spliceosomal complex / small-subunit processome / mRNA splicing, via spliceosome / metallopeptidase activity / nucleic acid binding / RNA helicase activity / RNA helicase / mRNA binding / nucleolus / ATP hydrolysis activity / mitochondrion / RNA binding / nucleoplasm / ATP binding / identical protein binding / nucleus / cytoplasm
Similarity search - Function
Pre-mRNA processing factor 4 (PRP4)-like / Splicing Factor Motif, present in Prp18 and Pr04 / Pre-mRNA-splicing factor 3 / U4/U6 small nuclear ribonucleoprotein Prp3 / pre-mRNA processing factor 3 domain / Prp31 C-terminal / U4/U6 small nuclear ribonucleoprotein Prp31 / Prp31 C terminal domain / Small nuclear ribonucleoprotein Prp3, C-terminal domain / Small nuclear ribonucleoprotein Prp3, C-terminal domain ...Pre-mRNA processing factor 4 (PRP4)-like / Splicing Factor Motif, present in Prp18 and Pr04 / Pre-mRNA-splicing factor 3 / U4/U6 small nuclear ribonucleoprotein Prp3 / pre-mRNA processing factor 3 domain / Prp31 C-terminal / U4/U6 small nuclear ribonucleoprotein Prp31 / Prp31 C terminal domain / Small nuclear ribonucleoprotein Prp3, C-terminal domain / Small nuclear ribonucleoprotein Prp3, C-terminal domain / Dim1 family / Mitosis protein DIM1 / Mitosis protein DIM1 / PRP1 splicing factor, N-terminal / PRP1 splicing factor, N-terminal / NOSIC / NOSIC (NUC001) domain / Nop domain / Nop domain superfamily / Nop, C-terminal domain / snoRNA binding domain, fibrillarin / Nop domain profile. / Brr2, N-terminal helicase PWI domain / : / N-terminal helicase PWI domain / Pre-mRNA-splicing helicase BRR2 plug domain / H/ACA ribonucleoprotein complex, subunit Nhp2-like / Winged Helix-turn-helix domain / Sec63 Brl domain / : / Pre-mRNA-splicing factor Syf1-like / Sec63 domain / Sec63 Brl domain / HAT (Half-A-TPR) repeat / HAT (Half-A-TPR) repeats / 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 / JAB/MPN domain / JAB1/MPN/MOV34 metalloenzyme domain / MPN domain / MPN domain profile. / Tetratricopeptide repeats / Ribosomal protein L7Ae conserved site / Ribosomal protein L7Ae signature. / C2 domain superfamily / DEAD/DEAH box helicase domain / DEAD/DEAH box helicase / Tetratricopeptide repeat / Ribosomal protein L7Ae/L8/Nhp2 family / : / Ribosomal protein L7Ae/L30e/S12e/Gadd45 / Ribosomal protein L7Ae/L30e/S12e/Gadd45 family / 50S ribosomal protein L30e-like / Helicase conserved C-terminal domain / Tetratricopeptide-like helical domain superfamily / helicase superfamily c-terminal domain / Immunoglobulin E-set / WD domain, G-beta repeat / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / Thioredoxin-like superfamily / Ribonuclease H-like superfamily / G-protein beta WD-40 repeat / Winged helix DNA-binding domain superfamily / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / Winged helix-like DNA-binding domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Pre-mRNA-splicing factor 6 / U4/U6 small nuclear ribonucleoprotein PRP4 / Pre-mRNA-splicing helicase BRR2 / Pre-mRNA-splicing factor 8 / 13 kDa ribonucleoprotein-associated protein / Pre-mRNA-processing factor 31 / U4/U6 small nuclear ribonucleoprotein PRP3 / Spliceosomal protein DIB1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsNguyen THD / Galej WP
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom) United Kingdom
CitationJournal: 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.
History
DepositionDec 15, 2015-
Header (metadata) releaseJan 27, 2016-
Map releaseJan 27, 2016-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5gap
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8014.png

Downloads & links

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Map

FileDownload / File: emd_8014.map.gz / Format: CCP4 / Size: 209.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCentral region of the U4/U6.U5 tri-snRNP comprising Prp8, Dib1, Prp31, Prp6, Prp4, Prp3, Snu13, U4 snRNA and U6 snRNA
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.43 Å/pix.
x 380 pix.
= 543.4 Å		1.43 Å/pix.
x 380 pix.
= 543.4 Å		1.43 Å/pix.
x 380 pix.
= 543.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.43 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.06
Minimum - Maximum-0.2323884 - 0.32634056
Average (Standard dev.)-0.0001666498 (±0.0078094634)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions380380380
Spacing380380380
CellA=B=C: 543.39996 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.431.431.43
M x/y/z380380380
origin x/y/z0.0000.0000.000
length x/y/z543.400543.400543.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-190-190-190
NX/NY/NZ380380380
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS380380380
D min/max/mean-0.2320.326-0.000

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Supplemental data

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Sample components

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Entire : Body region of the U4/U6.U5 tri-snRNP complex

EntireName: Body region of the U4/U6.U5 tri-snRNP complex
Components
  • Complex: Body region of the U4/U6.U5 tri-snRNP complex
    • RNA: U4 snRNA, 5' region, nucleotides 1-67
    • RNA: U6 snRNA
    • RNA: U5 snRNA
    • Protein or peptide: unknown protein
    • Protein or peptide: Pre-mRNA-splicing factor 8
    • Protein or peptide: U4/U6 small nuclear ribonucleoprotein PRP4
    • Protein or peptide: Pre-mRNA-splicing factor 6
    • Protein or peptide: Spliceosomal protein DIB1
    • Protein or peptide: Pre-mRNA-processing factor 31
    • Protein or peptide: U4/U6 small nuclear ribonucleoprotein PRP3
    • Protein or peptide: 13 kDa ribonucleoprotein-associated protein
    • Protein or peptide: Pre-mRNA-splicing helicase BRR2

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Supramolecule #1: Body region of the U4/U6.U5 tri-snRNP complex

SupramoleculeName: Body region of the U4/U6.U5 tri-snRNP complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 500 KDa

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Macromolecule #1: U4 snRNA, 5' region, nucleotides 1-67

MacromoleculeName: U4 snRNA, 5' region, nucleotides 1-67 / type: rna / ID: 1 / Details: U4 snRNA 5' region, nucleotides 1-67. / Number of copies: 1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 21.528688 KDa
SequenceString:
AUCCUUAUGC ACGGGAAAUA CGCAUAUCAG UGAGGAUUCG UCCGAGAUUG UGUUUUUGCU GGUUGAA

GENBANK: GENBANK: CP011083.1

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Macromolecule #2: U6 snRNA

MacromoleculeName: U6 snRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 35.883176 KDa
SequenceString:
GUUCGCGAAG UAACCCUUCG UGGACAUUUG GUCAAUUUGA AACAAUACAG AGAUGAUCAG CAGUUCCCCU GCAUAAGGAU GAACCGUUU UACAAAGAGA UUUAUUUCGU UUU

GENBANK: GENBANK: CP011090.1

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Macromolecule #3: U5 snRNA

MacromoleculeName: U5 snRNA / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 68.643344 KDa
SequenceString: AAGCAGCUUU ACAGAUCAAU GGCGGAGGGA GGUCAACAUC AAGAACUGUG GGCCUUUUAU UGCCUAUAGA ACUUAUAACG AACAUGGUU CUUGCCUUUU ACCAGAACCA UCCGGGUGUU GUCUCCAUAG AAACAGGUAA AGCUGUCCGU UACUGUGGGC U UGCCAUAU ...String:
AAGCAGCUUU ACAGAUCAAU GGCGGAGGGA GGUCAACAUC AAGAACUGUG GGCCUUUUAU UGCCUAUAGA ACUUAUAACG AACAUGGUU CUUGCCUUUU ACCAGAACCA UCCGGGUGUU GUCUCCAUAG AAACAGGUAA AGCUGUCCGU UACUGUGGGC U UGCCAUAU UUUUUGGAAC UUUUCUGCCC UUUUUCUCAA UGAGUAAGGA GGGCGU

GENBANK: GENBANK: CP011085.1

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Macromolecule #4: unknown protein

MacromoleculeName: unknown protein / type: protein_or_peptide / ID: 4
Details: Clear helical regions were built as poly(Ala) but could not be assigned to a specific protein.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 6.996616 KDa
SequenceString: (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) ...String:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK) (UNK)(UNK)

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Macromolecule #5: Pre-mRNA-splicing factor 8

MacromoleculeName: Pre-mRNA-splicing factor 8 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 279.867469 KDa
SequenceString: MSGLPPPPPG FEEDSDLALP PPPPPPPGYE IEELDNPMVP SSVNEDTFLP PPPPPPSNFE INAEEIVDFT LPPPPPPPGL DELETKAEK KVELHGKRKL DIGKDTFVTR KSRKRAKKMT KKAKRSNLYT PKAEMPPEHL RKIINTHSDM ASKMYNTDKK A FLGALKYL ...String:
MSGLPPPPPG FEEDSDLALP PPPPPPPGYE IEELDNPMVP SSVNEDTFLP PPPPPPSNFE INAEEIVDFT LPPPPPPPGL DELETKAEK KVELHGKRKL DIGKDTFVTR KSRKRAKKMT KKAKRSNLYT PKAEMPPEHL RKIINTHSDM ASKMYNTDKK A FLGALKYL PHAILKLLEN MPHPWEQAKE VKVLYHTSGA ITFVNETPRV IEPVYTAQWS ATWIAMRREK RDRTHFKRMR FP PFDDDEP PLSYEQHIEN IEPLDPINLP LDSQDDEYVK DWLYDSRPLE EDSKKVNGTS YKKWSFDLPE MSNLYRLSTP LRD EVTDKN YYYLFDKKSF FNGKALNNAI PGGPKFEPLY PREEEEDYNE FNSIDRVIFR VPIRSEYKVA FPHLYNSRPR SVRI PWYNN PVSCIIQNDE EYDTPALFFD PSLNPIPHFI DNNSSLNVSN TKENGDFTLP EDFAPLLAEE EELILPNTKD AMSLY HSPF PFNRTKGKMV RAQDVALAKK WFLQHPDEEY PVKVKVSYQK LLKNYVLNEL HPTLPTNHNK TKLLKSLKNT KYFQQT TID WVEAGLQLCR QGHNMLNLLI HRKGLTYLHL DYNFNLKPTK TLTTKERKKS RLGNSFHLMR ELLKMMKLIV DTHVQFR LG NVDAFQLADG IHYILNHIGQ LTGIYRYKYK VMHQIRACKD LKHIIYYKFN KNLGKGPGCG FWQPAWRVWL NFLRGTIP L LERYIGNLIT RQFEGRSNEI VKTTTKQRLD AYYDLELRNS VMDDILEMMP ESIRQKKART ILQHLSEAWR CWKANIPWD VPGMPAPIKK IIERYIKSKA DAWVSAAHYN RERIKRGAHV EKTMVKKNLG RLTRLWIKNE QERQRQIQKN GPEITPEEAT TIFSVMVEW LESRSFSPIP FPPLTYKNDT KILVLALEDL KDVYASKVRL NASEREELAL IEEAYDNPHD TLNRIKKYLL T QRVFKPVD ITMMENYQNI SPVYSVDPLE KITDAYLDQY LWYEADQRKL FPNWIKPSDS EIPPLLVYKW TQGINNLSEI WD VSRGQSA VLLETTLGEM AEKIDFTLLN RLLRLIVDPN IADYITAKNN VVINFKDMSH VNKYGLIRGL KFASFIFQYY GLV IDLLLL GQERATDLAG PANNPNEFMQ FKSKEVEKAH PIRLYTRYLD RIYMLFHFEE DEGEELTDEY LAENPDPNFE NSIG YNNRK CWPKDSRMRL IRQDVNLGRA VFWEIQSRVP TSLTSIKWEN AFVSVYSKNN PNLLFSMCGF EVRILPRQRM EEVVS NDEG VWDLVDERTK QRTAKAYLKV SEEEIKKFDS RIRGILMASG STTFTKVAAK WNTSLISLFT YFREAIVATE PLLDIL VKG ETRIQNRVKL GLNSKMPTRF PPAVFYTPKE LGGLGMISAS HILIPASDLS WSKQTDTGIT HFRAGMTHED EKLIPTI FR YITTWENEFL DSQRVWAEYA TKRQEAIQQN RRLAFEELEG SWDRGIPRIS TLFQRDRHTL AYDRGHRIRR EFKQYSLE R NSPFWWTNSH HDGKLWNLNA YRTDVIQALG GIETILEHTL FKGTGFNSWE GLFWEKASGF EDSMQFKKLT HAQRTGLSQ IPNRRFTLWW SPTINRANVY VGFLVQLDLT GIFLHGKIPT LKISLIQIFR AHLWQKIHES IVFDICQILD GELDVLQIES VTKETVHPR KSYKMNSSAA DITMESVHEW EVSKPSLLHE TNDSFKGLIT NKMWFDVQLR YGDYDSHDIS RYVRAKFLDY T TDNVSMYP SPTGVMIGID LAYNMYDAYG NWFNGLKPLI QNSMRTIMKA NPALYVLRER IRKGLQIYQS SVQEPFLNSS NY AELFNND IKLFVDDTNV YRVTVHKTFE GNVATKAING CIFTLNPKTG HLFLKIIHTS VWAGQKRLSQ LAKWKTAEEV SAL VRSLPK EEQPKQIIVT RKAMLDPLEV HMLDFPNIAI RPTELRLPFS AAMSIDKLSD VVMKATEPQM VLFNIYDDWL DRIS SYTAF SRLTLLLRAL KTNEESAKMI LLSDPTITIK SYHLWPSFTD EQWITIESQM RDLILTEYGR KYNVNISALT QTEIK DIIL GQNIKAPSVK RQKMAELEAA RSEKQNDEEA AGASTVMKTK TINAQGEEIV VVASADYESQ TFSSKNEWRK SAIANT LLY LRLKNIYVSA DDFVEEQNVY VLPKNLLKKF IEISDVKIQV AAFIYGMSAK DHPKVKEIKT VVLVPQLGHV GSVQISN IP DIGDLPDTEG LELLGWIHTQ TEELKFMAAS EVATHSKLFA DKKRDCIDIS IFSTPGSVSL SAYNLTDEGY QWGEENKD I MNVLSEGFEP TFSTHAQLLL SDRITGNFII PSGNVWNYTF MGTAFNQEGD YNFKYGIPLE FYNEMHRPVH FLQFSELAG DEELEAEQID VFS

UniProtKB: Pre-mRNA-splicing factor 8

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Macromolecule #6: U4/U6 small nuclear ribonucleoprotein PRP4

MacromoleculeName: U4/U6 small nuclear ribonucleoprotein PRP4 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 52.506984 KDa
SequenceString: MSKYIALENL PVDLQHKGAT QNESTADILK QLPHERLQAV LEKIPEEDLE VRRLLSILKK PEVVENEDVQ QRRIRLAEIL MVDEIDLEN INNMENINGE EVDEEDDEDF FTPATSELIF ARRFLINYSL ERSRKRLQKE MERHQKFNTR QELLSRRTEL Q RMANLELA ...String:
MSKYIALENL PVDLQHKGAT QNESTADILK QLPHERLQAV LEKIPEEDLE VRRLLSILKK PEVVENEDVQ QRRIRLAEIL MVDEIDLEN INNMENINGE EVDEEDDEDF FTPATSELIF ARRFLINYSL ERSRKRLQKE MERHQKFNTR QELLSRRTEL Q RMANLELA GSQLVSTKPI SAVSLSTDDM VVATGSWAGD LQVLNSQTLQ PLTQKLDSHV GKIGAIDWHP DSNNQMISCA ED GLIKNFQ YSNEEGGLRL LGDLVGHERR ISDVKYHPSG KFIGSASHDM TWRLWDASTH QELLLQEGHD KGVFSLSFQC DGS LVCSGG MDSLSMLWDI RSGSKVMTLA GHSKPIYTVA WSPNGYQVAT GGGDGIINVW DIRKRDEGQL NQILAHRNIV TQVR FSKED GGKKLVSCGY DNLINVYSSD TWLKMGSLAG HTDKIISLDI SNNSHFLVSG GWDRSIKLWN

UniProtKB: U4/U6 small nuclear ribonucleoprotein PRP4

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Macromolecule #7: Pre-mRNA-splicing factor 6

MacromoleculeName: Pre-mRNA-splicing factor 6 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 104.370133 KDa
SequenceString: MERPSFLDQE PPAGYVPGIG RGATGFSTKE KQVVSNDDKG RRIPKRYREN LNNHLQSQPK DDEDDEAANV FKTLELKLAQ KKKKRANEK DDDNSVDSSN VKRQFADLKE SLAAVTESEW MDIPDATDFT RRNKRNRIQE QLNRKTYAAP DSLIPGNVDL N KLTEEREK ...String:
MERPSFLDQE PPAGYVPGIG RGATGFSTKE KQVVSNDDKG RRIPKRYREN LNNHLQSQPK DDEDDEAANV FKTLELKLAQ KKKKRANEK DDDNSVDSSN VKRQFADLKE SLAAVTESEW MDIPDATDFT RRNKRNRIQE QLNRKTYAAP DSLIPGNVDL N KLTEEREK LLQSQIDENL AQLTKNASNP IQVNKPNAAT DALSYLKDLE NDRVNSLSDA TLEDLQKMRT ILKSYRKADP TN PQGWIAS ARLEEKARKF SVAKKIIENG CQECPRSSDI WLENIRLHES DVHYCKTLVA TAINFNPTSP LLWFKAIDLE STT VNKYRV VRKALQEIPR DEGLWKLAVS FEADKAQVIK MLEKATQFIP QSMDLLTAYT NLQSYHNAKM TLNSFRKILP QEPE IWIIS TLLEERNNPD IPVDKLVSLL KEGLLELSKN GYKATLSAWL KRAEALNDAP NSNLTCQAIV YAILEWLRES GEYES ELNN VDQILEKMPH SKVQIAVLKK LIQWDPCDTV LWSRLKMATE SYHKIEELLA FFQELLFQTK NSDDIRANMR EKSPGL LMM YVSEYWKAQK GDTRQTLVLI DQIIDFAPHN LDLRFFKIKL LGRSLQLDEL RDFFQQTFSS LEDFKISGTE RLYYKYV NF LRYQDLNEEA IKFLNERCLK SFPICHKFFL QLGQIYHSMG NIEMSRETYL SGTRLVPNCP LLWVSLSKID EIDLKNPV R ARSILDRGLL KNPDDVLFYI AKIQMEIRLG NLDQAELLVT QALQKFPSNA LLWVEQIKLF KHGNKSSLKK TIFQDALRR TQNDHRVLLE IGVSFYAEAQ YETSLKWLER ALKKCSRYGD TWVWLFRTYA RLGKDTVDLY NMFDQCEPTY GPEWIAASKN VKMQYCTPR EILLRLMNDK

UniProtKB: Pre-mRNA-splicing factor 6

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Macromolecule #8: Spliceosomal protein DIB1

MacromoleculeName: Spliceosomal protein DIB1 / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 16.798387 KDa
SequenceString:
MASVLLPQLR TGWHVDQAIV TETKRLVVIR FGRKNDRQCM IMDELLSSIA ERVRNFAVIY LCDIDEVSDF DEMYELTDPM TVMFFYHNK HMMCDFGTGN NNKLNFIVDD KQEMIDILET IFRGARKNKG LVVSPYDYNH KRVS

UniProtKB: Spliceosomal protein DIB1

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Macromolecule #9: Pre-mRNA-processing factor 31

MacromoleculeName: Pre-mRNA-processing factor 31 / type: protein_or_peptide / ID: 9 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 56.382516 KDa
SequenceString: MSSEEDYFDE LEYDLADEVN EEKEDIQTKK LTTVNCQTEK FNPFEILPES IELFRTLALI SPDRLSLSET AQILPKIVDL KRILQQQEI DFIKLLPFFN EIIPLIKSNI KLMHNFLISL YSRRFPELSS LIPSPLQYSK VISILENENY SKNESDELFF H LENKAKLT ...String:
MSSEEDYFDE LEYDLADEVN EEKEDIQTKK LTTVNCQTEK FNPFEILPES IELFRTLALI SPDRLSLSET AQILPKIVDL KRILQQQEI DFIKLLPFFN EIIPLIKSNI KLMHNFLISL YSRRFPELSS LIPSPLQYSK VISILENENY SKNESDELFF H LENKAKLT REQILVLTMS MKTSFKNKEP LDIKTRTQIL EANSILENLW KLQEDIGQYI ASKISIIAPN VCFLVGPEIA AQ LIAHAGG VLEFSRIPSC NIASIGKNKH LSHELHTLES GVRQEGYLFA SDMIQKFPVS VHKQMLRMLC AKVSLAARVD AGQ KNGDRN TVLAHKWKAE LSKKARKLSE APSISETKAL PIPEDQPKKK RAGRKFRKYK EKFRLSHVRQ LQNRMEFGKQ EQTV LDSYG EEVGLGMSNT SLQQAVGATS GSRRSAGNQA KLTKVMKHRI SEANQQADEF LISLGHNTEQ PNLSPEMVQM HKKQH TNPE EETNWFSGHG

UniProtKB: Pre-mRNA-processing factor 31

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Macromolecule #10: U4/U6 small nuclear ribonucleoprotein PRP3

MacromoleculeName: U4/U6 small nuclear ribonucleoprotein PRP3 / type: protein_or_peptide / ID: 10 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 55.97432 KDa
SequenceString: MPPRNTYEKG NPKRQNSPYY KPSFLRREET TNDEEKFQGH GLKTELHSAL KSSNLNLIRR TYQTGENPYL SDPHDRGSSS RFNRRYERG LKFYQKGEIS KRIAQERTLQ KQQEEEELKR KLKQEEDEKD KRKLIESGDL PNLELHEDKF LLDLSKFKIY Y DNNHGYEW ...String:
MPPRNTYEKG NPKRQNSPYY KPSFLRREET TNDEEKFQGH GLKTELHSAL KSSNLNLIRR TYQTGENPYL SDPHDRGSSS RFNRRYERG LKFYQKGEIS KRIAQERTLQ KQQEEEELKR KLKQEEDEKD KRKLIESGDL PNLELHEDKF LLDLSKFKIY Y DNNHGYEW WDTAYLDEKG ELMEKYDMNG TSPAEEKLAE DIDEVDDDDD DEHPSIRYVA HPLPEKINEA KVSIKAYLTQ HE RKRLRRN RRKMAREARE IKIKLGLLPK PEPKVKLSNM MSVFENDQNI TDPTAWEKVV KDQVDLRKRK HLEENERRHE DAI KRRKEA VNMNVEKPTV YHCKVFQFKN LQNPKIRFKL KMNSKELSLK GLCLRIRDDG PGIIIVVGNE KSCKFYENLV MKRI KWNED FELHTNTGDI KMDMHNNSIS KTWEGYLQDC KFKGWFMKVC NDQDSLLRTL GQFDSEHFYS PVQT

UniProtKB: U4/U6 small nuclear ribonucleoprotein PRP3

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Macromolecule #11: 13 kDa ribonucleoprotein-associated protein

MacromoleculeName: 13 kDa ribonucleoprotein-associated protein / type: protein_or_peptide / ID: 11 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 13.582855 KDa
SequenceString:
MSAPNPKAFP LADAALTQQI LDVVQQAANL RQLKKGANEA TKTLNRGISE FIIMAADCEP IEILLHLPLL CEDKNVPYVF VPSRVALGR ACGVSRPVIA ASITTNDASA IKTQIYAVKD KIETLLI

UniProtKB: 13 kDa ribonucleoprotein-associated protein

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Macromolecule #12: Pre-mRNA-splicing helicase BRR2

MacromoleculeName: Pre-mRNA-splicing helicase BRR2 / type: protein_or_peptide / ID: 12 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA helicase
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 246.470266 KDa
SequenceString: MTEHETKDKA KKIREIYRYD EMSNKVLKVD KRFMNTSQNP QRDAEISQPK SMSGRISAKD MGQGLCNNIN KGLKENDVAV EKTGKSASL KKIQQHNTIL NSSSDFRLHY YPKDPSNVET YEQILQWVTE VLGNDIPHDL IIGTADIFIR QLKENEENED G NIEERKEK ...String:
MTEHETKDKA KKIREIYRYD EMSNKVLKVD KRFMNTSQNP QRDAEISQPK SMSGRISAKD MGQGLCNNIN KGLKENDVAV EKTGKSASL KKIQQHNTIL NSSSDFRLHY YPKDPSNVET YEQILQWVTE VLGNDIPHDL IIGTADIFIR QLKENEENED G NIEERKEK IQHELGINID SLKFNELVKL MKNITDYETH PDNSNKQAVA ILADDEKSDE EEVTEMSNNA NVLGGEINDN ED DDEEYDY NDVEVNSKKK NKRALPNIEN DIIKLSDSKT SNIESVPIYS IDEFFLQRKL RSELGYKDTS VIQDLSEKIL NDI ETLEHN PVALEQKLVD LLKFENISLA EFILKNRSTI FWGIRLAKST ENEIPNLIEK MVAKGLNDLV EQYKFRETTH SKRE LDSGD DQPQSSEAKR TKFSNPAIPP VIDLEKIKFD ESSKLMTVTK VSLPEGSFKR VKPQYDEIHI PAPSKPVIDY ELKEI TSLP DWCQEAFPSS ETTSLNPIQS KVFHAAFEGD SNMLICAPTG SGKTNIALLT VLKALSHHYN PKTKKLNLSA FKIVYI APL KALVQEQVRE FQRRLAFLGI KVAELTGDSR LSRKQIDETQ VLVSTPEKWD ITTRNSNNLA IVELVRLLII DEIHLLH DD RGPVLESIVA RTFWASKYGQ EYPRIIGLSA TLPNYEDVGR FLRVPKEGLF YFDSSFRPCP LSQQFCGIKE RNSLKKLK A MNDACYEKVL ESINEGNQII VFVHSRKETS RTATWLKNKF AEENITHKLT KNDAGSKQIL KTEAANVLDP SLRKLIESG IGTHHAGLTR SDRSLSEDLF ADGLLQVLVC TATLAWGVNL PAHTVIIKGT DVYSPEKGSW EQLSPQDVLQ MLGRAGRPRY DTFGEGIII TDQSNVQYYL SVLNQQLPIE SQFVSKLVDN LNAEVVAGNI KCRNDAVNWL AYTYLYVRML ASPMLYKVPD I SSDGQLKK FRESLVHSAL CILKEQELVL YDAENDVIEA TDLGNIASSF YINHASMDVY NRELDEHTTQ IDLFRIFSMS EE FKYVSVR YEEKRELKQL LEKAPIPIRE DIDDPLAKVN VLLQSYFSQL KFEGFALNSD IVFIHQNAGR LLRAMFEICL KRG WGHPTR MLLNLCKSAT TKMWPTNCPL RQFKTCPVEV IKRLEASTVP WGDYLQLETP AEVGRAIRSE KYGKQVYDLL KRFP KMSVT CNAQPITRSV MRFNIEIIAD WIWDMNVHGS LEPFLLMLED TDGDSILYYD VLFITPDIVG HEFTLSFTYE LKQHN QNNL PPNFFLTLIS ENWWHSEFEI PVSFNGFKLP KKFPPPTPLL ENISISTSEL GNDDFSEVFE FKTFNKIQSQ VFESLY NSN DSVFVGSGKG TGKTAMAELA LLNHWRQNKG RAVYINPSGE KIDFLLSDWN KRFSHLAGGK IINKLGNDPS LNLKLLA KS HVLLATPVQF ELLSRRWRQR KNIQSLELMI YDDAHEISQG VYGAVYETLI SRMIFIATQL EKKIRFVCLS NCLANARD F GEWAGMTKSN IYNFSPSERI EPLEINIQSF KDVEHISFNF SMLQMAFEAS AAAAGNRNSS SVFLPSRKDC MEVASAFMK FSKAIEWDML NVEEEQIVPY IEKLTDGHLR APLKHGVGIL YKGMASNDER IVKRLYEYGA VSVLLISKDC SAFACKTDEV IILGTNLYD GAEHKYMPYT INELLEMVGL ASGNDSMAGK VLILTSHNMK AYYKKFLIEP LPTESYLQYI IHDTLNNEIA N SIIQSKQD CVDWFTYSYF YRRIHVNPSY YGVRDTSPHG ISVFLSNLVE TCLNDLVESS FIEIDDTEAE VTAEVNGGDD EA TEIISTL SNGLIASHYG VSFFTIQSFV SSLSNTSTLK NMLYVLSTAV EFESVPLRKG DRALLVKLSK RLPLRFPEHT SSG SVSFKV FLLLQAYFSR LELPVDFQND LKDILEKVVP LINVVVDILS ANGYLNATTA MDLAQMLIQG VWDVDNPLRQ IPHF NNKIL EKCKEINVET VYDIMALEDE ERDEILTLTD SQLAQVAAFV NNYPNVELTY SLNNSDSLIS GVKQKITIQL TRDVE PENL QVTSEKYPFD KLESWWLVLG EVSKKELYAI KKVTLNKETQ QYELEFDTPT SGKHNLTIWC VCDSYLDADK ELSFEI NVK

UniProtKB: Pre-mRNA-splicing helicase BRR2

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.2 mg/mL
BufferpH: 7.9 / Component - Concentration: 1.0 mM / Component - Name: DTT
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 6 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 70 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III
Details: Grids were blotted at 4 deg C for 2 seconds before plunging..
Details3.5 microlitre of sample was applied to grid.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-20 / Number real images: 2477 / Average exposure time: 16.0 sec. / Average electron dose: 38.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 35714 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 473827
Startup modelType of model: EMDB MAP
EMDB ID:

2966


Details: Low pass filtered startup model to 60 Angstroms.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 140155
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: OTHER
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: AB INITIO MODEL
Output model

PDB-5gap:
Body region of the U4/U6.U5 tri-snRNP

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