IL5RA-AF17121 peptide complex
02 Feb 2024
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The eutectic of IL5RA-AF17121 peptide complex was too small to meet the required crystal size for synchrotron radiation. Finally, the structure with a resolution of 3Å was successfully resolved using the MicroED.

  • Background

    IL-5 is a cytokine secreted by Th2 cells. It is involved in regulating eosinophil function and is associated with T2-low asthma and hypereosinophilic syndrome. IL-5 binding to the IL-5 receptor to activates IL-5 signalling, so interfering with IL-5 receptor activation has become one of the directions of research.

    AF17121, an inhibitory peptide of IL-5, was reported in 2019 to bind to the alpha subunit of IL-5 receptor (IL5RA), thereby inhibiting the binding of IL-5 to IL-5 receptor.

    ReadCrystal successfully resolved the structure of IL5RA-AF17121 peptide complex by using MicroED.

  • Results
    1. Protein expression & purification
    IL5RA protein expression is prone to inclusion bodies. After the inclusion bodies were obtained, they were purified and renatured to finally obtain IL5RA soluble protein with >90% purity. IL5RA and peptide AF17121 were incubated and the IL5RA-AF17121 peptide complex was purified by gel filtration chromatography.
    Fig. 1 Results of gel filtration chromatography and SDS-PAGE
  • 2. Protein crystallization and diffraction data collection
    Several crystallization methods were tried, but we still could not obtain crystals of suitable size for X-ray diffraction.
    We laid 10 μM-sized crystals onto a copper grid, cut a 200 nm-thick slice with Cryo-FIB, and collected diffraction data with MicroED.
    Fig. 2 Cryo-FIB and MicroED diffraction data
    A 10μM size crystal; B Crystal laid onto the copper grid and sliced to 200nm thickness with Cryo-FIB; C MicroED diffraction data
  • 3. Structure determination
    The structure of IL5RA-AF17121 peptide complex was determined based on the collected diffraction data combined with data processing software with a resolution of 3Å.
    Fig. 3 Three-dimensional structure of IL5RA-AF17121 peptide complex
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