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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 / homology
Function and homology information


U11/U12 snRNP / B-WICH complex / splicing factor binding / U12-type spliceosomal complex / RNA splicing, via transesterification reactions / U2-type spliceosomal complex / U2-type precatalytic spliceosome / U2-type prespliceosome assembly / U2 snRNP / SAGA complex ...U11/U12 snRNP / B-WICH complex / splicing factor binding / U12-type spliceosomal complex / RNA splicing, via transesterification reactions / U2-type spliceosomal complex / U2-type precatalytic spliceosome / U2-type prespliceosome assembly / U2 snRNP / SAGA complex / positive regulation of transcription by RNA polymerase III / U2-type prespliceosome / precatalytic spliceosome / positive regulation of transcription by RNA polymerase I / mRNA Splicing - Minor Pathway / spliceosomal complex assembly / regulation of RNA splicing / U2 snRNA binding / regulation of DNA repair / catalytic step 2 spliceosome / mRNA Splicing - Major Pathway / RNA splicing / stem cell differentiation / spliceosomal complex / B-WICH complex positively regulates rRNA expression / negative regulation of protein catabolic process / mRNA splicing, via spliceosome / nuclear matrix / nuclear speck / chromatin remodeling / mRNA binding / protein-containing complex binding / nucleolus / positive regulation of DNA-templated transcription / positive regulation of transcription by RNA polymerase II / DNA binding / RNA binding / zinc ion binding / nucleoplasm / nucleus
Similarity search - Function
Splicing factor 3B, subunit 5 / Splicing factor 3B subunit 1 / Splicing factor 3B subunit 1 / PHF5-like / PHF5-like protein / Splicing factor 3B subunit 5/RDS3 complex subunit 10 / Splicing factor 3B subunit 10 (SF3b10) / Splicing factor 3B subunit 1-like / PPP2R1A-like HEAT repeat / Cleavage/polyadenylation specificity factor, A subunit, N-terminal ...Splicing factor 3B, subunit 5 / Splicing factor 3B subunit 1 / Splicing factor 3B subunit 1 / PHF5-like / PHF5-like protein / Splicing factor 3B subunit 5/RDS3 complex subunit 10 / Splicing factor 3B subunit 10 (SF3b10) / Splicing factor 3B subunit 1-like / PPP2R1A-like HEAT repeat / 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 / Armadillo-like helical / Armadillo-type fold / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
Chem-9B0 / : / Splicing factor 3B subunit 1 / Splicing factor 3B subunit 3 / PHD finger-like domain-containing protein 5A / Splicing factor 3B subunit 5
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.95 Å
AuthorsFinci, L.I. / Larsen, N.A.
Funding support China, 1items
OrganizationGrant numberCountry
31650110470 China
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 ...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.
History
DepositionMay 23, 2018Deposition site: PDBJ / Processing site: PDBJ
SupersessionJun 20, 2018ID: 5ZD5
Revision 1.0Jun 20, 2018Provider: repository / Type: Initial release
Revision 1.1Nov 6, 2019Group: Data collection / Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB
Revision 1.2Mar 27, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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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
Polymers302,0414
Non-polymers9545
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area13420 Å2
ΔGint-53 kcal/mol
Surface area96870 Å2

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Components

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

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


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


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


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

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Protein , 1 types, 1 molecules D

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


Mass: 9394.955 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
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 / Source method: obtained synthetically / Formula: C40H66N2O9
#6: Chemical ChemComp-ZN / ZINC ION


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


Mass: 39.098 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: K

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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: 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 mg/ml / 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
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 55 e/Å2 / 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 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 241288 / Symmetry type: POINT
RefinementHighest resolution: 3.95 Å
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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