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- PDB-6y50: 5'domain of human 17S U2 snRNP -

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Entry
Database: PDB / ID: 6y50
Title5'domain of human 17S U2 snRNP
Components
  • (Splicing factor 3B subunit ...) x 4
  • HIV Tat-specific factor 1
  • PHD finger-like domain-containing protein 5A
  • Probable ATP-dependent RNA helicase DDX46
  • Splicing factor 3A subunit 3
  • U2 snRNAU2 spliceosomal RNA
KeywordsSPLICING / 17S U2 snRNP
Function / homology
Function and homology information


U2-type prespliceosome assembly / U11/U12 snRNP / RNA splicing, via transesterification reactions / U2-type spliceosomal complex / regulation of DNA-templated transcription, elongation / splicing factor binding / U2-type precatalytic spliceosome / U2 snRNP / U12-type spliceosomal complex / positive regulation of gene expression, epigenetic ...U2-type prespliceosome assembly / U11/U12 snRNP / RNA splicing, via transesterification reactions / U2-type spliceosomal complex / regulation of DNA-templated transcription, elongation / splicing factor binding / U2-type precatalytic spliceosome / U2 snRNP / U12-type spliceosomal complex / positive regulation of gene expression, epigenetic / spliceosomal complex assembly / precatalytic spliceosome / U2-type prespliceosome / mRNA 3'-splice site recognition / viral genome replication / stem cell differentiation / catalytic step 2 spliceosome / Cajal body / spliceosomal complex / RNA splicing / fibrillar center / mRNA splicing, via spliceosome / mRNA processing / negative regulation of protein catabolic process / nuclear matrix / RNA helicase / nucleic acid binding / RNA helicase activity / nuclear speck / mRNA binding / regulation of transcription by RNA polymerase II / nucleolus / protein-containing complex binding / DNA-binding transcription factor activity / viral process / positive regulation of transcription, DNA-templated / RNA binding / DNA binding / zinc ion binding / membrane / nucleoplasm / ATP binding / nucleus
DEAD/DEAH box helicase domain / Splicing factor SF3a60 binding domain / RNA recognition motif domain / Nucleotide-binding alpha-beta plait domain superfamily / Helicase superfamily 1/2, ATP-binding domain / Helicase, C-terminal / RNA helicase, DEAD-box type, Q motif / Armadillo-like helical / Splicing factor 3B subunit 1 / WD40/YVTN repeat-like-containing domain superfamily ...DEAD/DEAH box helicase domain / Splicing factor SF3a60 binding domain / RNA recognition motif domain / Nucleotide-binding alpha-beta plait domain superfamily / Helicase superfamily 1/2, ATP-binding domain / Helicase, C-terminal / RNA helicase, DEAD-box type, Q motif / Armadillo-like helical / Splicing factor 3B subunit 1 / WD40/YVTN repeat-like-containing domain superfamily / SAP domain / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / Matrin/U1-C, C2H2-type zinc finger / PHF5-like / Splicing factor 3B, subunit 5 / Armadillo-type fold / ATP-dependent RNA helicase DEAD-box, conserved site / Domain of unknown function DUF382 / PSP, proline-rich / Domain of unknown function DUF3449 / P-loop containing nucleoside triphosphate hydrolase / SF3A3 domain / Splicing factor 3A subunit 3 / TatSF1-like, RNA recognition motif 1 / TatSF1-like / RNA-binding domain superfamily / WD40-repeat-containing domain superfamily / Splicing factor 3B subunit 1-like / Splicing factor 3B subunit 5/RDS3 complex subunit 10
HIV Tat-specific factor 1 / Splicing factor 3B subunit 1 / Splicing factor 3A subunit 3 / Splicing factor 3B subunit 2 / Splicing factor 3B subunit 3 / Probable ATP-dependent RNA helicase DDX46 / PHD finger-like domain-containing protein 5A / Splicing factor 3B subunit 5 / gb:36516:
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsZhang, Z. / Will, C.L. / Bertram, K. / Luehrmann, R. / Stark, H.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation (DFG) Germany
CitationJournal: Nature / Year: 2020
Title: Molecular architecture of the human 17S U2 snRNP.
Authors: Zhenwei Zhang / Cindy L Will / Karl Bertram / Olexandr Dybkov / Klaus Hartmuth / Dmitry E Agafonov / Romina Hofele / Henning Urlaub / Berthold Kastner / Reinhard Lührmann / Holger Stark /
Abstract: The U2 small nuclear ribonucleoprotein (snRNP) has an essential role in the selection of the precursor mRNA branch-site adenosine, the nucleophile for the first step of splicing. Stable addition of ...The U2 small nuclear ribonucleoprotein (snRNP) has an essential role in the selection of the precursor mRNA branch-site adenosine, the nucleophile for the first step of splicing. Stable addition of U2 during early spliceosome formation requires the DEAD-box ATPase PRP5. Yeast U2 small nuclear RNA (snRNA) nucleotides that form base pairs with the branch site are initially sequestered in a branchpoint-interacting stem-loop (BSL), but whether the human U2 snRNA folds in a similar manner is unknown. The U2 SF3B1 protein, a common mutational target in haematopoietic cancers, contains a HEAT domain (SF3B1) with an open conformation in isolated SF3b, but a closed conformation in spliceosomes, which is required for stable interaction between U2 and the branch site. Here we report a 3D cryo-electron microscopy structure of the human 17S U2 snRNP at a core resolution of 4.1 Å and combine it with protein crosslinking data to determine the molecular architecture of this snRNP. Our structure reveals that SF3B1 interacts with PRP5 and TAT-SF1, and maintains its open conformation in U2 snRNP, and that U2 snRNA forms a BSL that is sandwiched between PRP5, TAT-SF1 and SF3B1. Thus, substantial remodelling of the BSL and displacement of BSL-interacting proteins must occur to allow formation of the U2-branch-site helix. Our studies provide a structural explanation of why TAT-SF1 must be displaced before the stable addition of U2 to the spliceosome, and identify RNP rearrangements facilitated by PRP5 that are required for stable interaction between U2 and the branch site.
Validation Report
SummaryFull reportAbout validation report
History
DepositionFeb 24, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 1, 2020Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
9: Splicing factor 3A subunit 3
y: PHD finger-like domain-containing protein 5A
x: Splicing factor 3B subunit 5
8: Splicing factor 3B subunit 2
v: Splicing factor 3B subunit 3
u: Splicing factor 3B subunit 1
2: U2 snRNA
q: HIV Tat-specific factor 1
p: Probable ATP-dependent RNA helicase DDX46
hetero molecules


Theoretical massNumber of molelcules
Total (without water)727,55812
Polymers727,3629
Non-polymers1963
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy, mass-spectrometry & EM. For details, see publication
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TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 4 types, 4 molecules 9yqp

#1: Protein Splicing factor 3A subunit 3 / SF3a60 / Spliceosome-associated protein 61 / SAP 61


Mass: 58934.844 Da / Num. of mol.: 1 / Details: SF3A3, SAP61 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q12874
#2: Protein PHD finger-like domain-containing protein 5A / PHD finger-like domain protein 5A / Splicing factor 3B-associated 14 kDa protein / SF3b14b


Mass: 12427.524 Da / Num. of mol.: 1 / Details: PHF5A / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q7RTV0
#8: Protein HIV Tat-specific factor 1 / Tat-SF1


Mass: 85965.961 Da / Num. of mol.: 1 / Details: HTATSF1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: O43719
#9: Protein Probable ATP-dependent RNA helicase DDX46 / DEAD box protein 46 / PRP5 homolog


Mass: 117576.484 Da / Num. of mol.: 1 / Details: DDX46, KIAA0801 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q7L014, RNA helicase

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Splicing factor 3B subunit ... , 4 types, 4 molecules x8vu

#3: Protein Splicing factor 3B subunit 5 / SF3b5 / Pre-mRNA-splicing factor SF3b 10 kDa subunit


Mass: 10149.369 Da / Num. of mol.: 1 / Details: SF3B5, SF3B10 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q9BWJ5
#4: Protein Splicing factor 3B subunit 2 / Pre-mRNA-splicing factor SF3b 145 kDa subunit / SF3b145 / Spliceosome-associated protein 145 / SAP 145


Mass: 100377.812 Da / Num. of mol.: 1 / Details: SF3B2, SAP145 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q13435
#5: Protein Splicing factor 3B subunit 3 / Pre-mRNA-splicing factor SF3b 130 kDa subunit / SF3b130 / STAF130 / Spliceosome-associated protein ...Pre-mRNA-splicing factor SF3b 130 kDa subunit / SF3b130 / STAF130 / Spliceosome-associated protein 130 / SAP 130


Mass: 135718.844 Da / Num. of mol.: 1 / Details: SF3B3, KIAA0017, SAP130 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q15393
#6: Protein Splicing factor 3B subunit 1 / Pre-mRNA-splicing factor SF3b 155 kDa subunit / SF3b155 / Spliceosome-associated protein 155 / SAP 155


Mass: 146024.938 Da / Num. of mol.: 1 / Details: SF3B1, SAP155 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: O75533

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RNA chain / Non-polymers , 2 types, 4 molecules 2

#10: Chemical ChemComp-ZN / ZINC ION / Zinc


Mass: 65.409 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Formula: Zn
#7: RNA chain U2 snRNA / U2 spliceosomal RNA


Mass: 60186.445 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: GenBank: 36516

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Details

Has ligand of interestN

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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: human 17S U2 snRNP / Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5, 6, 7, 8, 9 / Source: NATURAL
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 7.9
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R3.5/1
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 0.01 mm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1 sec. / Electron dose: 72 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

EM software
IDNameVersionCategory
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 120070 / Symmetry type: POINT
Atomic model buildingSpace: REAL

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