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- PDB-6g90: Prespliceosome structure provides insight into spliceosome assemb... -

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Basic information

Entry
Database: PDB / ID: 6g90
TitlePrespliceosome structure provides insight into spliceosome assembly and regulation (map A2)
Components
  • (Pre-mRNA-splicing factor ...) x 5
  • (Small nuclear ribonucleoprotein ...SnRNP) x 6
  • (U1 small nuclear ribonucleoprotein ...) x 5
  • (U2 small nuclear ribonucleoprotein ...) x 2
  • 56 kDa U1 small nuclear ribonucleoprotein component
  • Cold sensitive U2 snRNA suppressor 1
  • Pre-mRNA-processing factor 39
  • Protein HSH49
  • Protein LUC7,Protein LUC7,Protein LUC7
  • Protein NAM8
  • RDS3 complex subunit 10
  • Small nuclear ribonucleoprotein-associated protein B
  • U1 snRNA,U1 snRNA,U1 snRNA,U1 snRNA,U1 snRNA
  • U2 snRNAU2 spliceosomal RNA
  • U2 snRNP component HSH155
  • Unknown
  • Yeast UBC4 pre-mRNA (mutant)
KeywordsSPLICING / Spliceosome / A complex / RNA processing / RNA-protein complex
Function / homology
Function and homology information


positive regulation of RNA binding / splicing factor binding / mRNA splice site recognition / U4/U6 snRNP / 7-methylguanosine cap hypermethylation / pICln-Sm protein complex / spliceosomal tri-snRNP complex / small nuclear ribonucleoprotein complex / SMN-Sm protein complex / mRNA cis splicing, via spliceosome ...positive regulation of RNA binding / splicing factor binding / mRNA splice site recognition / U4/U6 snRNP / 7-methylguanosine cap hypermethylation / pICln-Sm protein complex / spliceosomal tri-snRNP complex / small nuclear ribonucleoprotein complex / SMN-Sm protein complex / mRNA cis splicing, via spliceosome / positive regulation of mRNA splicing, via spliceosome / U2-type spliceosomal complex / U2-type prespliceosome assembly / commitment complex / U4 snRNP / U2 snRNP / poly(U) RNA binding / U1 snRNP / U2-type prespliceosome / pre-mRNA 5'-splice site binding / precatalytic spliceosome / spliceosomal complex assembly / mRNA 5'-splice site recognition / U5 snRNP / spliceosomal snRNP assembly / U2 snRNA binding / U1 snRNA binding / U4/U6 x U5 tri-snRNP complex / catalytic step 2 spliceosome / spliceosomal complex / mRNA splicing, via spliceosome / response to xenobiotic stimulus / mRNA binding / RNA binding / zinc ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Single alpha-helices involved in coiled-coils or other helix-helix interfaces - #1320 / Surp module / Luc7-related / LUC7 N_terminus / Snu56-like U1 small nuclear ribonucleoprotein component / Snu56-like U1 small nuclear ribonucleoprotein component / Pre-mRNA-splicing factor Prp9, N-terminal / Pre-mRNA-splicing factor PRP9 N-terminus / U1 small nuclear ribonucleoprotein C / U1 small nuclear ribonucleoprotein of 70kDa N-terminal ...Single alpha-helices involved in coiled-coils or other helix-helix interfaces - #1320 / Surp module / Luc7-related / LUC7 N_terminus / Snu56-like U1 small nuclear ribonucleoprotein component / Snu56-like U1 small nuclear ribonucleoprotein component / Pre-mRNA-splicing factor Prp9, N-terminal / Pre-mRNA-splicing factor PRP9 N-terminus / U1 small nuclear ribonucleoprotein C / U1 small nuclear ribonucleoprotein of 70kDa N-terminal / U1 small nuclear ribonucleoprotein of 70kDa MW N terminal / U1-C, C2H2-type zinc finger / U1 zinc finger / PWI domain / PWI, domain in splicing factors / Splicing factor 3A subunit 1 / Splicing factor 3A subunit 1, conserved domain / Pre-mRNA splicing factor PRP21 like protein / SF3A2 domain / Pre-mRNA-splicing factor SF3a complex subunit 2 (Prp11) / Splicing factor SF3a60 /Prp9 subunit, C-terminal / SF3A3 domain / SF3B4, RNA recognition motif 1 / SF3a60/Prp9 C-terminal / Pre-mRNA-splicing factor SF3A3, of SF3a complex, Prp9 / SWAP/Surp / SWAP/Surp superfamily / Surp module / SURP motif repeat profile. / Suppressor-of-White-APricot splicing regulator / Domain of unknown function DUF382 / Domain of unknown function (DUF382) / RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) / Zinc-finger of C2H2 type / PHF5-like / PHF5-like protein / PSP, proline-rich / PSP / proline-rich domain in spliceosome associated proteins / Splicing factor 3B subunit 5/RDS3 complex subunit 10 / Splicing factor 3B subunit 10 (SF3b10) / Matrin/U1-C, C2H2-type zinc finger / Splicing factor 3B subunit 1-like / Zinc finger matrin-type profile. / U2A'/phosphoprotein 32 family A, C-terminal / occurring C-terminal to leucine-rich repeats / Leucine-rich repeat / Zinc-finger double-stranded RNA-binding / Zinc finger, double-stranded RNA binding / 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 / Matrin/U1-C-like, C2H2-type zinc finger / U1-like zinc finger / Sm-like protein Lsm6/SmF / LSM domain / LSM domain, eukaryotic/archaea-type / snRNP Sm proteins / HAT (Half-A-TPR) repeat / HAT (Half-A-TPR) repeats / Cleavage/polyadenylation specificity factor, A subunit, N-terminal / Mono-functional DNA-alkylating methyl methanesulfonate N-term / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / CPSF A subunit region / : / Sm domain profile. / LSM domain superfamily / Single alpha-helices involved in coiled-coils or other helix-helix interfaces / zinc finger / RRM (RNA recognition motif) domain / Zinc finger C2H2 superfamily / Leucine-rich repeat profile. / Zinc finger C2H2-type / Leucine-rich repeat / RNA recognition motif / RNA recognition motif / Eukaryotic RNA Recognition Motif (RRM) profile. / Helix non-globular / RNA recognition motif domain / Leucine-rich repeat domain superfamily / RNA-binding domain superfamily / Special / SH3 type barrels. / Armadillo-like helical / Tetratricopeptide-like helical domain superfamily / Arc Repressor Mutant, subunit A / Armadillo-type fold / Nucleotide-binding alpha-beta plait domain superfamily / Roll / Alpha-Beta Plaits / WD40/YVTN repeat-like-containing domain superfamily / 2-Layer Sandwich / Orthogonal Bundle / Mainly Beta / Mainly Alpha
Similarity search - Domain/homology
: / : / RNA / RNA (> 10) / RNA (> 100) / RDS3 complex subunit 10 / Pre-mRNA-splicing factor PRP9 / Pre-mRNA-splicing factor PRP21 / U1 small nuclear ribonucleoprotein A / Pre-mRNA-processing factor 39 ...: / : / RNA / RNA (> 10) / RNA (> 100) / RDS3 complex subunit 10 / Pre-mRNA-splicing factor PRP9 / Pre-mRNA-splicing factor PRP21 / U1 small nuclear ribonucleoprotein A / Pre-mRNA-processing factor 39 / Small nuclear ribonucleoprotein-associated protein B / Small nuclear ribonucleoprotein G / U2 small nuclear ribonucleoprotein B'' / Small nuclear ribonucleoprotein Sm D3 / U2 snRNP component HSH155 / U1 small nuclear ribonucleoprotein component SNU71 / Small nuclear ribonucleoprotein F / Protein NAM8 / U1 small nuclear ribonucleoprotein 70 kDa homolog / Small nuclear ribonucleoprotein Sm D1 / Cold sensitive U2 snRNA suppressor 1 / U1 small nuclear ribonucleoprotein component PRP42 / 56 kDa U1 small nuclear ribonucleoprotein component / Pre-mRNA-splicing factor RSE1 / U1 small nuclear ribonucleoprotein C / Small nuclear ribonucleoprotein Sm D2 / Pre-mRNA-splicing factor RDS3 / Pre-mRNA-splicing factor PRP11 / Protein LUC7 / U2 small nuclear ribonucleoprotein A' / Small nuclear ribonucleoprotein E / Protein HSH49
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsPlaschka, C. / Lin, P.-C. / Charenton, C. / Nagai, K.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MC_U105184330 United Kingdom
European Research Council693087 - SPLICE3D United Kingdom
CitationJournal: Nature / Year: 2018
Title: Prespliceosome structure provides insights into spliceosome assembly and regulation.
Authors: Clemens Plaschka / Pei-Chun Lin / Clément Charenton / Kiyoshi Nagai /
Abstract: The spliceosome catalyses the excision of introns from pre-mRNA in two steps, branching and exon ligation, and is assembled from five small nuclear ribonucleoprotein particles (snRNPs; U1, U2, U4, ...The spliceosome catalyses the excision of introns from pre-mRNA in two steps, branching and exon ligation, and is assembled from five small nuclear ribonucleoprotein particles (snRNPs; U1, U2, U4, U5, U6) and numerous non-snRNP factors. For branching, the intron 5' splice site and the branch point sequence are selected and brought by the U1 and U2 snRNPs into the prespliceosome, which is a focal point for regulation by alternative splicing factors. The U4/U6.U5 tri-snRNP subsequently joins the prespliceosome to form the complete pre-catalytic spliceosome. Recent studies have revealed the structural basis of the branching and exon-ligation reactions, however, the structural basis of the early events in spliceosome assembly remains poorly understood. Here we report the cryo-electron microscopy structure of the yeast Saccharomyces cerevisiae prespliceosome at near-atomic resolution. The structure reveals an induced stabilization of the 5' splice site in the U1 snRNP, and provides structural insights into the functions of the human alternative splicing factors LUC7-like (yeast Luc7) and TIA-1 (yeast Nam8), both of which have been linked to human disease. In the prespliceosome, the U1 snRNP associates with the U2 snRNP through a stable contact with the U2 3' domain and a transient yeast-specific contact with the U2 SF3b-containing 5' region, leaving its tri-snRNP-binding interface fully exposed. The results suggest mechanisms for 5' splice site transfer to the U6 ACAGAGA region within the assembled spliceosome and for its subsequent conversion to the activation-competent B-complex spliceosome. Taken together, the data provide a working model to investigate the early steps of spliceosome assembly.
History
DepositionApr 10, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 22, 2018Provider: repository / Type: Initial release

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Assembly

Deposited unit
1: U1 snRNA,U1 snRNA,U1 snRNA,U1 snRNA,U1 snRNA
2: U2 snRNA
A: U1 small nuclear ribonucleoprotein A,U1 small nuclear ribonucleoprotein A,U1 small nuclear ribonucleoprotein A,U1 small nuclear ribonucleoprotein A,U1 small nuclear ribonucleoprotein A
B: U1 small nuclear ribonucleoprotein 70 kDa homolog,U1 small nuclear ribonucleoprotein 70 kDa homolog,U1 small nuclear ribonucleoprotein 70 kDa homolog
C: U1 small nuclear ribonucleoprotein C
D: Pre-mRNA-processing factor 39
E: U1 small nuclear ribonucleoprotein component PRP42
F: Protein NAM8
G: 56 kDa U1 small nuclear ribonucleoprotein component
H: Protein LUC7,Protein LUC7,Protein LUC7
I: Yeast UBC4 pre-mRNA (mutant)
J: U1 small nuclear ribonucleoprotein component SNU71
O: U2 snRNP component HSH155
P: Pre-mRNA-splicing factor RSE1
Q: Cold sensitive U2 snRNA suppressor 1
R: Protein HSH49
S: Pre-mRNA-splicing factor RDS3
T: Pre-mRNA-splicing factor PRP9
U: Pre-mRNA-splicing factor PRP11,Pre-mRNA-splicing factor PRP11,Pre-mRNA-splicing factor PRP11
V: Pre-mRNA-splicing factor PRP21
W: U2 small nuclear ribonucleoprotein A'
X: Unknown
Y: U2 small nuclear ribonucleoprotein B''
Z: RDS3 complex subunit 10
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
i: Small nuclear ribonucleoprotein Sm D2
s: Small nuclear ribonucleoprotein-associated protein B
t: Small nuclear ribonucleoprotein Sm D1
u: Small nuclear ribonucleoprotein Sm D2
v: Small nuclear ribonucleoprotein Sm D3
w: Small nuclear ribonucleoprotein E
x: Small nuclear ribonucleoprotein F
y: Small nuclear ribonucleoprotein G
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,346,39147
Polymers1,345,80338
Non-polymers5899
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 , 3 types, 3 molecules 12I

#1: RNA chain U1 snRNA,U1 snRNA,U1 snRNA,U1 snRNA,U1 snRNA


Mass: 126116.875 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: GenBank: 1039023797
#2: RNA chain U2 snRNA / U2 spliceosomal RNA


Mass: 45762.836 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: GenBank: 536627
#11: RNA chain Yeast UBC4 pre-mRNA (mutant)


Mass: 12045.173 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast)

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U1 small nuclear ribonucleoprotein ... , 5 types, 5 molecules ABCEJ

#3: Protein U1 small nuclear ribonucleoprotein A,U1 small nuclear ribonucleoprotein A,U1 small nuclear ribonucleoprotein A,U1 small nuclear ribonucleoprotein A,U1 small nuclear ribonucleoprotein A / U1A / Mutant U1 die protein 1


Mass: 33754.168 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P32605
#4: Protein U1 small nuclear ribonucleoprotein 70 kDa homolog,U1 small nuclear ribonucleoprotein 70 kDa homolog,U1 small nuclear ribonucleoprotein 70 kDa homolog / U1-70K / U1 small nuclear ribonucleoprotein SNP1 / U1 snRNP protein SNP1 / U1-70K / U1 small ...U1-70K / U1 small nuclear ribonucleoprotein SNP1 / U1 snRNP protein SNP1 / U1-70K / U1 small nuclear ribonucleoprotein SNP1 / U1 snRNP protein SNP1 / / U1-70K / U1 small nuclear ribonucleoprotein SNP1 / U1 snRNP protein SNP1


Mass: 33445.242 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q00916
#5: Protein U1 small nuclear ribonucleoprotein C / U1C


Mass: 27106.016 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q05900
#7: Protein U1 small nuclear ribonucleoprotein component PRP42 / U1 snRNP protein PRP42 / 65 kDa snRNP protein / Pre-mRNA-processing factor 42


Mass: 65222.020 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q03776
#12: Protein U1 small nuclear ribonucleoprotein component SNU71


Mass: 71484.555 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P53207

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Protein , 10 types, 11 molecules DFGHOQRXZbs

#6: Protein Pre-mRNA-processing factor 39


Mass: 74834.742 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P39682
#8: Protein Protein NAM8


Mass: 57010.840 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q00539
#9: Protein 56 kDa U1 small nuclear ribonucleoprotein component


Mass: 56575.277 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q03782
#10: Protein Protein LUC7,Protein LUC7,Protein LUC7


Mass: 28504.295 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q07508
#13: Protein U2 snRNP component HSH155


Mass: 110166.672 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P49955
#15: Protein Cold sensitive U2 snRNA suppressor 1


Mass: 50210.699 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q02554
#16: Protein Protein HSH49


Mass: 24534.152 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q99181
#22: Protein Unknown


Mass: 4358.364 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast)
#24: Protein RDS3 complex subunit 10 / Splicing factor 3b subunit


Mass: 10045.401 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P0C074
#25: 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 (brewer's yeast) / References: UniProt: P40018

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Pre-mRNA-splicing factor ... , 5 types, 5 molecules PSTUV

#14: Protein Pre-mRNA-splicing factor RSE1 / RNA splicing and ER to Golgi transport factor 1 / Spliceosome-associated protein 130


Mass: 153956.781 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q04693
#17: Protein Pre-mRNA-splicing factor RDS3 / Regulator of drug sensitivity 3


Mass: 12283.573 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q06835
#18: Protein Pre-mRNA-splicing factor PRP9


Mass: 63126.445 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P19736
#19: Protein Pre-mRNA-splicing factor PRP11,Pre-mRNA-splicing factor PRP11,Pre-mRNA-splicing factor PRP11


Mass: 29292.230 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q07350
#20: Protein Pre-mRNA-splicing factor PRP21


Mass: 33111.512 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P32524

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

#21: 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
#23: 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 (brewer's yeast) / References: UniProt: P40567

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

#26: 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 (brewer's yeast) / References: UniProt: P43321
#27: 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 (brewer's yeast) / References: UniProt: Q12330
#28: 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
#29: 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 (brewer's yeast) / References: UniProt: P40204
#30: 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 / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q02260
#31: 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 (brewer's yeast) / References: UniProt: Q06217

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Non-polymers , 1 types, 9 molecules

#32: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: Zn

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Prespliceosome A complex / Type: COMPLEX / Entity ID: #1-#31 / Source: NATURAL
Molecular weightValue: 1.8 MDa / Experimental value: YES
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.9
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES pH 7.9HEPES1
250 mMKCl1
31 %Glycerol1
42 mMMgCl21
50.001 %NP-401
61 mMDTT1
SpecimenConc.: 0.4 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 %
Details: Grids were blotted for 2-3.5 s and vitrified by plunging into liquid ethane with a FEI Vitrobot Mark III operated at 4 degree Celsius and 100% humidity.

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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: 105000 X / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 43 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 9407
Image scansMovie frames/image: 20

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4GctfCTF correction
7UCSF Chimeramodel fitting
10RELION2.1final Euler assignment
12RELION2.13D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 153556 / Symmetry type: POINT
Atomic model buildingSpace: REAL

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