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.
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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.
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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
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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
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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
02 Feb 
