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

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Database: PDB / ID: 5mq0
TitleStructure of a spliceosome remodeled for exon ligation
DescriptorPre-mRNA-processing protein 45
Pre-mRNA-processing factor 17
U2 small nuclear ribonucleoprotein A'
U2 small nuclear ribonucleoprotein B''
Unknown protein/RNA Complex
(Pre-mRNA-splicing factor ...) x 16
(Small nuclear ribonucleoprotein ...) x 7
KeywordsSPLICING / pre-mRNA splicing / trans-esterification / lariat intermediate / complex C-star / Splicing
Specimen sourceSaccharomyces cerevisiae / yeast / Baker's yeast / サッカロミセス・セレビシエ /
MethodElectron microscopy (4.17 A resolution / Single particle)
AuthorsFica, S.M. / Oubridge, C. / Galej, W.P. / Wilkinson, M.E. / Newman, A.J. / Bai, X.-C. / Nagai, K.
CitationNature, 2017, 542, 377-380

primary. Nature, 2017, 542, 377-380 StrPapers
Structure of a spliceosome remodelled for exon ligation.
Sebastian M Fica / Chris Oubridge / Wojciech P Galej / Max E Wilkinson / Xiao-Chen Bai / Andrew J Newman / Kiyoshi Nagai

#1. Science, 2015, 349, 1191-1198 StrPapers
Structural basis of pre-mRNA splicing.
Hang, J. / Wan, R. / Yan, C. / Shi, Y.

#2. Nature, 2015, 523, 47-52 StrPapers
The architecture of the spliceosomal U4/U6.U5 tri-snRNP.
Nguyen, T.H. / Galej, W.P. / Bai, X.C. / Savva, C.G. / Newman, A.J. / Scheres, S.H. / Nagai, K.

#3. Nature, 2016, 530, 298-302 StrPapers
Cryo-EM structure of the yeast U4/U6.U5 tri-snRNP at 3.7 A resolution.
Nguyen, T.H. / Galej, W.P. / Bai, X.C. / Oubridge, C. / Newman, A.J. / Scheres, S.H. / Nagai, K.

#4. Nature, 2016, 537, 197-201 StrPapers
Cryo-EM structure of the spliceosome immediately after branching.
Galej, W.P. / Wilkinson, M.E. / Fica, S.M. / Oubridge, C. / Newman, A.J. / Nagai, K.

DateDeposition: Dec 19, 2016 / Release: Jan 18, 2017 / Last modification: Mar 1, 2017

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Assembly

Deposited unit
I: Yeast UBC4 gene for ubiquitin-conjugating enzyme
E: 5'-EXON OF UBC4 PRE-MRNA
2: S.cerevisiae chromosome II reading frame ORF YBR230c
6: Saccharomyces cerevisiae strain T.52_2H chromosome XII sequence
5: Saccharomyces cerevisiae strain WI_C_MBSP_4 chromosome VII sequence
A: Pre-mRNA-splicing factor 8
C: Pre-mRNA-splicing factor SNU114
H: Pre-mRNA-splicing factor CWC22
J: Pre-mRNA-splicing factor PRP46
K: Pre-mRNA-processing protein 45
L: Pre-mRNA-splicing factor BUD31
M: Pre-mRNA-splicing factor CWC2
N: Pre-mRNA-splicing factor SLT11
O: Pre-mRNA-splicing factor CEF1
P: Pre-mRNA-splicing factor CWC15
R: Pre-mRNA-splicing factor CWC21
S: Pre-mRNA-splicing factor CLF1
T: Pre-mRNA-splicing factor SYF1,PRE-MRNA-SPLICING FACTOR SYF1
a: Pre-mRNA-splicing factor 18
c: Pre-mRNA-splicing factor SLU7
o: Pre-mRNA-processing factor 17
X: UNKNOWN PROTEIN
y: Pre-mRNA-splicing factor SYF2
b: Small nuclear ribonucleoprotein-associated protein B
d: Small nuclear ribonucleoprotein Sm D3
e: Small nuclear ribonucleoprotein E
f: Small nuclear ribonucleoprotein F
g: Small nuclear ribonucleoprotein G
h: Small nuclear ribonucleoprotein Sm D1
j: Small nuclear ribonucleoprotein Sm D2
V: Pre-mRNA-splicing factor ATP-dependent RNA helicase PRP22
k: Small nuclear ribonucleoprotein-associated protein B
l: Small nuclear ribonucleoprotein Sm D1
m: Small nuclear ribonucleoprotein Sm D2
n: Small nuclear ribonucleoprotein Sm D3
r: Small nuclear ribonucleoprotein G
W: U2 small nuclear ribonucleoprotein A'
Y: U2 small nuclear ribonucleoprotein B''
p: Small nuclear ribonucleoprotein E
q: Small nuclear ribonucleoprotein F
3: 3'-EXON OF UBC4 PRE-MRNA, BOUND BY PRP22 HELICASE
s: Pre-mRNA-splicing factor SNT309
t: Pre-mRNA-processing factor 19
u: Pre-mRNA-processing factor 19
v: Pre-mRNA-processing factor 19
w: Pre-mRNA-processing factor 19
hetero molecules


Theoretical massNumber of molelcules
Total (without water)2,205,88659
Polyers2,204,15946
Non-polymers1,72713
Water0
#1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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RNA chain , 6 types, 6 molecules IE2653

#1: RNA chainYeast UBC4 gene for ubiquitin-conjugating enzyme


Mass: 30200.730 Da / Num. of mol.: 1 / Source: (gene. exp.) Saccharomyces cerevisiae / References: GenBank: 4718
#2: RNA chain5'-EXON OF UBC4 PRE-MRNA


Mass: 6518.976 Da / Num. of mol.: 1 / Source: (gene. exp.) Saccharomyces cerevisiae
#3: RNA chainS.cerevisiae chromosome II reading frame ORF YBR230c


Mass: 376267.406 Da / Num. of mol.: 1 / Source: (natural) Saccharomyces cerevisiae / References: GenBank: 536627
#4: RNA chainSaccharomyces cerevisiae strain T.52_2H chromosome XII sequence


Mass: 35883.176 Da / Num. of mol.: 1 / Source: (natural) Saccharomyces cerevisiae / References: GenBank: 1039022925
#5: RNA chainSaccharomyces cerevisiae strain WI_C_MBSP_4 chromosome VII sequence


Mass: 57444.875 Da / Num. of mol.: 1 / Source: (natural) Saccharomyces cerevisiae / References: GenBank: 1039023924
#34: RNA chain3'-EXON OF UBC4 PRE-MRNA, BOUND BY PRP22 HELICASE


Mass: 873.540 Da / Num. of mol.: 1 / Source: (gene. exp.) Saccharomyces cerevisiae

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

#6: Polypeptide(L)Pre-mRNA-splicing factor 8


Mass: 279867.469 Da / Num. of mol.: 1 / Source: (natural) Saccharomyces cerevisiae / References: UniProt: P33334

Cellular component

Molecular function

Biological process

  • generation of catalytic spliceosome for second transesterification step (GO: 0000350)
  • mRNA 3'-splice site recognition (GO: 0000389)
  • spliceosomal tri-snRNP complex assembly (GO: 0000244)
#7: Polypeptide(L)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: (natural) Saccharomyces cerevisiae / References: UniProt: P36048

Cellular component

Molecular function

Biological process

  • generation of catalytic spliceosome for first transesterification step (GO: 0000349)
  • mRNA splicing, via spliceosome (GO: 0000398)
  • spliceosomal tri-snRNP complex assembly (GO: 0000244)
  • spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) (GO: 0000388)
#8: Polypeptide(L)Pre-mRNA-splicing factor CWC22 / Complexed with CEF1 protein 22


Mass: 67386.062 Da / Num. of mol.: 1 / Source: (natural) Saccharomyces cerevisiae / References: UniProt: P53333

Cellular component

Molecular function

Biological process

#9: Polypeptide(L)Pre-mRNA-splicing factor PRP46 / 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: (natural) Saccharomyces cerevisiae / References: UniProt: Q12417

Cellular component

Biological process

#11: Polypeptide(L)Pre-mRNA-splicing factor BUD31 / Bud site selection protein 31 / Complexed with CEF1 protein 14


Mass: 18484.502 Da / Num. of mol.: 1 / Source: (natural) Saccharomyces cerevisiae / References: UniProt: P25337

Cellular component

Biological process

#12: Polypeptide(L)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: (natural) Saccharomyces cerevisiae / References: UniProt: Q12046

Cellular component

Molecular function

Biological process

#13: Polypeptide(L)Pre-mRNA-splicing factor SLT11 / Extracellular mutant protein 2 / Synthetic lethality with U2 protein 11


Mass: 40988.590 Da / Num. of mol.: 1 / Source: (natural) Saccharomyces cerevisiae / References: UniProt: P38241

Cellular component

Molecular function

Biological process

#14: Polypeptide(L)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: (natural) Saccharomyces cerevisiae / References: UniProt: Q03654

Cellular component

Molecular function

  • RNA polymerase II transcription factor activity, sequence-specific DNA binding (GO: 0000981)
  • sequence-specific DNA binding (GO: 0043565)
  • transcription factor activity, RNA polymerase II transcription factor recruiting (GO: 0001135)
  • transcription regulatory region DNA binding (GO: 0044212)

Biological process

  • cell differentiation (GO: 0030154)
  • generation of catalytic spliceosome for second transesterification step (GO: 0000350)
  • mRNA splicing, via spliceosome (GO: 0000398)
  • regulation of transcription from RNA polymerase II promoter (GO: 0006357)
#15: Polypeptide(L)Pre-mRNA-splicing factor CWC15 / Complexed with CEF1 protein 15


Mass: 19975.195 Da / Num. of mol.: 1 / Source: (natural) Saccharomyces cerevisiae / References: UniProt: Q03772

Cellular component

Molecular function

Biological process

#16: Polypeptide(L)Pre-mRNA-splicing factor CWC21 / Complexed with CEF1 protein 21


Mass: 15793.596 Da / Num. of mol.: 1 / Source: (natural) Saccharomyces cerevisiae / References: UniProt: Q03375

Cellular component

Biological process

#17: Polypeptide(L)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: (natural) Saccharomyces cerevisiae / References: UniProt: Q12309

Cellular component

Molecular function

Biological process

#18: Polypeptide(L)Pre-mRNA-splicing factor SYF1,PRE-MRNA-SPLICING FACTOR SYF1 / PRP19-associated complex protein 90 / Synthetic lethal with CDC40 protein 1


Mass: 101875.852 Da / Num. of mol.: 1 / Source: (natural) Saccharomyces cerevisiae / References: UniProt: Q04048

Cellular component

Biological process

  • generation of catalytic spliceosome for first transesterification step (GO: 0000349)
#19: Polypeptide(L)Pre-mRNA-splicing factor 18


Mass: 28414.391 Da / Num. of mol.: 1 / Source: (natural) Saccharomyces cerevisiae / References: UniProt: P33411

Cellular component

Biological process

  • generation of catalytic spliceosome for second transesterification step (GO: 0000350)
  • mRNA splicing, via spliceosome (GO: 0000398)
  • nuclear retention of unspliced pre-mRNA at the site of transcription (GO: 0071048)
#20: Polypeptide(L)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: (natural) Saccharomyces cerevisiae / References: UniProt: Q02775

Cellular component

Molecular function

Biological process

  • generation of catalytic spliceosome for second transesterification step (GO: 0000350)
#23: Polypeptide(L)Pre-mRNA-splicing factor SYF2 / PRP19 complex protein 31 / Synthetic lethal with CDC40 protein 2


Mass: 24850.719 Da / Num. of mol.: 1 / Source: (natural) Saccharomyces cerevisiae / References: UniProt: P53277

Cellular component

Biological process

#31: Polypeptide(L)Pre-mRNA-splicing factor ATP-dependent RNA helicase PRP22


Mass: 130187.359 Da / Num. of mol.: 1 / Source: (natural) Saccharomyces cerevisiae / References: UniProt: P24384, EC: 3.6.4.13

Cellular component

Molecular function

Biological process

  • generation of catalytic spliceosome for second transesterification step (GO: 0000350)
  • spliceosomal complex disassembly (GO: 0000390)
#35: Polypeptide(L)Pre-mRNA-splicing factor SNT309 / PRP19-associated complex protein 25 / Synergistic to PRP19 mutation protein 309


Mass: 20741.455 Da / Num. of mol.: 1 / Source: (natural) Saccharomyces cerevisiae / References: UniProt: Q06091

Cellular component

Biological process

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Polypeptide(L) , 6 types, 9 molecules KoXWYtuvw

#10: Polypeptide(L)Pre-mRNA-processing protein 45


Mass: 42548.727 Da / Num. of mol.: 1 / Source: (natural) Saccharomyces cerevisiae / References: UniProt: P28004

Cellular component

Molecular function

Biological process

  • generation of catalytic spliceosome for second transesterification step (GO: 0000350)
  • regulation of transcription from RNA polymerase II promoter (GO: 0006357)
#21: Polypeptide(L)Pre-mRNA-processing factor 17 / Cell division control protein 40


Mass: 52128.762 Da / Num. of mol.: 1 / Source: (natural) Saccharomyces cerevisiae / References: UniProt: P40968

Cellular component

Biological process

  • generation of catalytic spliceosome for second transesterification step (GO: 0000350)
  • mRNA 3'-splice site recognition (GO: 0000389)
  • mRNA branch site recognition (GO: 0000348)
#22: Polypeptide(L)UNKNOWN PROTEIN


Mass: 5805.147 Da / Num. of mol.: 1 / Source: (natural) Saccharomyces cerevisiae
#32: Polypeptide(L)U2 small nuclear ribonucleoprotein A' / U2 snRNP A' / Looks exceptionally like U2A protein 1


Mass: 27232.252 Da / Num. of mol.: 1 / Source: (natural) Saccharomyces cerevisiae / References: UniProt: Q08963

Cellular component

Molecular function

Biological process

#33: Polypeptide(L)U2 small nuclear ribonucleoprotein B'' / U2 snRNP B''


Mass: 12850.944 Da / Num. of mol.: 1 / Source: (natural) Saccharomyces cerevisiae / References: UniProt: P40567

Cellular component

Molecular function

Biological process

#36: Polypeptide(L)
Pre-mRNA-processing factor 19


Mass: 56629.777 Da / Num. of mol.: 4 / Source: (natural) Saccharomyces cerevisiae / References: UniProt: P32523, EC: 6.3.2.-

Cellular component

Molecular function

Biological process

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Small nuclear ribonucleoprotein ... , 7 types, 14 molecules bkdnepfqgr...

#24: Polypeptide(L)Small nuclear ribonucleoprotein-associated protein B / snRNP-B / Sm protein B / SmB


Mass: 22426.990 Da / Num. of mol.: 2 / Source: (natural) Saccharomyces cerevisiae / References: UniProt: P40018

Cellular component

Molecular function

Biological process

#25: Polypeptide(L)Small nuclear ribonucleoprotein Sm D3 / Sm-D3 / snRNP core protein D3


Mass: 11240.139 Da / Num. of mol.: 2 / Source: (natural) Saccharomyces cerevisiae / References: UniProt: P43321

Cellular component

Molecular function

Biological process

#26: Polypeptide(L)Small nuclear ribonucleoprotein E / snRNP-E / Sm protein E / SmE


Mass: 10385.098 Da / Num. of mol.: 2 / Source: (natural) Saccharomyces cerevisiae / References: UniProt: Q12330

Cellular component

Molecular function

Biological process

#27: Polypeptide(L)Small nuclear ribonucleoprotein F / snRNP-F / Sm protein F / SmF


Mass: 9669.945 Da / Num. of mol.: 2 / Source: (natural) Saccharomyces cerevisiae / References: UniProt: P54999

Cellular component

Molecular function

Biological process

#28: Polypeptide(L)Small nuclear ribonucleoprotein G / snRNP-G / Sm protein G / SmG


Mass: 8490.809 Da / Num. of mol.: 2 / Source: (natural) Saccharomyces cerevisiae / References: UniProt: P40204

Cellular component

Molecular function

Biological process

#29: Polypeptide(L)Small nuclear ribonucleoprotein Sm D1 / Sm-D1 / snRNP core protein D1


Mass: 16296.798 Da / Num. of mol.: 2 / Source: (natural) Saccharomyces cerevisiae / References: UniProt: Q02260

Cellular component

Molecular function

Biological process

#30: Polypeptide(L)Small nuclear ribonucleoprotein Sm D2 / Sm-D2 / snRNP core protein D2


Mass: 12876.066 Da / Num. of mol.: 2 / Source: (natural) Saccharomyces cerevisiae / References: UniProt: Q06217

Cellular component

Molecular function

Biological process

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Non-polymers , 5 types, 13 molecules

#37: ChemicalChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Formula: Mg
#38: ChemicalChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 2 / Formula: K
#39: ChemicalChemComp-I6P / INOSITOL 1,2,3,4,5,6-HEXAKISPHOSPHATE / MYO-INOSITOL HEXAKISPHOSPHATE


Mass: 660.035 Da / Num. of mol.: 1 / Formula: C6H18O24P6
#40: ChemicalChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE / GTP *YM


Mass: 523.180 Da / Num. of mol.: 1 / Formula: C10H16N5O14P3
#41: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 6 / Formula: Zn

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentReconstruction method: SINGLE PARTICLE

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Sample preparation

ComponentName: Saccharomyces cerevisiae spliceosome. Complex C just after Prp16-mediated remodeling
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.
Type: COMPLEX
Buffer solutionDetails: NP-40 is also called IGEPAL CA-630
Specimen supportGrid material: COPPER / Grid mesh size: 400 /inch. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100
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

MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 4500 nm / Nominal defocus min: 500 nm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER

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Processing

3D reconstructionResolution: 4.17 A / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 29527
Details: A 3D reconstruction was obtained after refinement of a subset of particles obtained by classification with a mask around Prp22.
Number of class averages: 4
Atomic model buildingDetails: Used secondary structure restraints generated in ProSMART and LibG.
Overall b value: 330 / Ref protocol: FLEXIBLE FIT / Ref space: RECIPROCAL / Target criteria: Fourier Shell Correlation

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