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- PDB-5zya: SF3b spliceosomal complex bound to E7107 -

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Basic information

Entry
Database: PDB / ID: 5zya
TitleSF3b spliceosomal complex bound to E7107
Components
  • (Splicing factor 3B subunit ...) x 3
  • PHD finger-like domain-containing protein 5A
KeywordsSPLICING / Splicing Modulator / SF3b sub-complex
Function / homologyMono-functional DNA-alkylating methyl methanesulfonate N-term / Splicing factor 3B, subunit 5 / PHF5-like / Splicing factor 3B subunit 5/RDS3 complex subunit 10 / Armadillo-like helical / Splicing factor 3B subunit 1 / WD40/YVTN repeat-like-containing domain superfamily / Armadillo-type fold / WD40-repeat-containing domain superfamily / Splicing factor 3B subunit 1-like ...Mono-functional DNA-alkylating methyl methanesulfonate N-term / Splicing factor 3B, subunit 5 / PHF5-like / Splicing factor 3B subunit 5/RDS3 complex subunit 10 / Armadillo-like helical / Splicing factor 3B subunit 1 / WD40/YVTN repeat-like-containing domain superfamily / Armadillo-type fold / WD40-repeat-containing domain superfamily / Splicing factor 3B subunit 1-like / CPSF A subunit region / PHF5-like protein / Splicing factor 3B subunit 10 (SF3b10) / Splicing factor 3B subunit 1 / B-WICH complex positively regulates rRNA expression / mRNA Splicing - Major Pathway / mRNA Splicing - Minor Pathway / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / U11/U12 snRNP / RNA splicing, via transesterification reactions / U2-type precatalytic spliceosome / small nuclear ribonucleoprotein complex / U12-type spliceosomal complex / U2 snRNP / positive regulation of gene expression, epigenetic / spliceosomal complex assembly / U2-type prespliceosome / catalytic step 2 spliceosome / spliceosomal complex / mRNA splicing, via spliceosome / negative regulation of protein catabolic process / nuclear matrix / nucleic acid binding / mRNA binding / protein-containing complex binding / nuclear speck / nucleolus / DNA-binding transcription factor activity / positive regulation of transcription, DNA-templated / RNA binding / DNA binding / nucleoplasm / nucleus / metal ion binding / Splicing factor 3B subunit 1 / Splicing factor 3B subunit 3 / PHD finger-like domain-containing protein 5A / Splicing factor 3B subunit 5
Function and homology information
Specimen sourceHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.95 Å resolution
AuthorsFinci, L.I. / Larsen, N.A.
CitationJournal: Genes Dev. / Year: 2018
Title: The cryo-EM structure of the SF3b spliceosome complex bound to a splicing modulator reveals a pre-mRNA substrate competitive mechanism of action.
Authors: Lorenzo I Finci / Xiaofeng Zhang / Xiuliang Huang / Qiang Zhou / Jennifer Tsai / Teng Teng / Anant Agrawal / Betty Chan / Sean Irwin / Craig Karr / Andrew Cook / Ping Zhu / Dominic Reynolds / Peter G Smith / Peter Fekkes / Silvia Buonamici / Nicholas A Larsen
Abstract: Somatic mutations in spliceosome proteins lead to dysregulated RNA splicing and are observed in a variety of cancers. These genetic aberrations may offer a potential intervention point for targeted ...Somatic mutations in spliceosome proteins lead to dysregulated RNA splicing and are observed in a variety of cancers. These genetic aberrations may offer a potential intervention point for targeted therapeutics. SF3B1, part of the U2 small nuclear RNP (snRNP), is targeted by splicing modulators, including E7107, the first to enter clinical trials, and, more recently, H3B-8800. Modulating splicing represents a first-in-class opportunity in drug discovery, and elucidating the structural basis for the mode of action opens up new possibilities for structure-based drug design. Here, we present the cryogenic electron microscopy (cryo-EM) structure of the SF3b subcomplex (SF3B1, SF3B3, PHF5A, and SF3B5) bound to E7107 at 3.95 Å. This structure shows that E7107 binds in the branch point adenosine-binding pocket, forming close contacts with key residues that confer resistance upon mutation: SF3B1 and PHF5A The structure suggests a model in which splicing modulators interfere with branch point adenosine recognition and supports a substrate competitive mechanism of action (MOA). Using several related chemical probes, we validate the pose of the compound and support their substrate competitive MOA by comparing their activity against both strong and weak pre-mRNA substrates. Finally, we present functional data and structure-activity relationship (SAR) on the PHF5A mutation that sensitizes cells to some chemical probes but not others. Developing small molecule splicing modulators represents a promising therapeutic approach for a variety of diseases, and this work provides a significant step in enabling structure-based drug design for these elaborate natural products. Importantly, this work also demonstrates that the utilization of cryo-EM in drug discovery is coming of age.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: May 23, 2018 / Release: Jun 20, 2018

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Structure visualization

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Assembly

Deposited unit
B: Splicing factor 3B subunit 5
C: Splicing factor 3B subunit 1
D: PHD finger-like domain-containing protein 5A
A: Splicing factor 3B subunit 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)302,9959
Polyers302,0414
Non-polymers9545
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)13420
ΔGint (kcal/M)-53
Surface area (Å2)96870

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Components

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Splicing factor 3B subunit ... , 3 types, 3 molecules BCA

#1: Protein/peptide Splicing factor 3B subunit 5 / SF3b5


Mass: 10149.369 Da / Num. of mol.: 1 / Source: (gene. exp.) Homo sapiens (human) / Gene: SF3B5 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9BWJ5
#2: Protein/peptide Splicing factor 3B subunit 1 / Pre-mRNA-splicing factor SF3b 155 kDa subunit / SF3b155


Mass: 146024.938 Da / Num. of mol.: 1 / Source: (gene. exp.) Homo sapiens (human) / Gene: SF3B1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O75533
#4: Protein/peptide Splicing factor 3B subunit 3 / Pre-mRNA-splicing factor SF3b 130 kDa subunit / SF3b130


Mass: 136471.562 Da / Num. of mol.: 1 / Source: (gene. exp.) Homo sapiens (human) / Gene: SF3B3 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q15393

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Protein/peptide , 1 types, 1 molecules D

#3: Protein/peptide PHD finger-like domain-containing protein 5A / PHD finger-like domain protein 5A


Mass: 9394.955 Da / Num. of mol.: 1 / Source: (gene. exp.) Homo sapiens (human) / Gene: PHF5A / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q7RTV0

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Non-polymers , 3 types, 5 molecules

#5: Chemical ChemComp-9B0 / [(2~{S},3~{S},4~{E},6~{S},7~{R},10~{R})-3,7-dimethyl-2-[(2~{E},4~{E},6~{R})-6-methyl-6-oxidanyl-7-[(2~{R},3~{R})-3-[(2~{R},3~{S})-3-oxidanylpentan-2-yl]oxiran-2-yl]hepta-2,4-dien-2-yl]-7,10-bis(oxidanyl)-12-oxidanylidene-1-oxacyclododec-4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate


Mass: 718.960 Da / Num. of mol.: 1 / Formula: C40H66N2O9
#6: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 3 / Formula: Zn / Zinc
#7: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 1 / Formula: K / Potassium

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: SF3b Sub-complex / Type: COMPLEX / Entity ID: 1,2,4 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 8
SpecimenConc.: 1 / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 55 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.95 / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 241288 / Symmetry type: POINT
Least-squares processHighest resolution: 3.95
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00817290
ELECTRON MICROSCOPYf_angle_d1.23823429
ELECTRON MICROSCOPYf_dihedral_angle_d10.74510496
ELECTRON MICROSCOPYf_chiral_restr0.0652662
ELECTRON MICROSCOPYf_plane_restr0.0093021

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