Efficient expression, purification, and visualization by cryo-EM of unliganded near full-length HER3
Devan Diwanji1, Raphael Trenker2, Natalia Jura3
1Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA, United States; Medical Scientist Training Program, University of California San Francisco, San Francisco, CA, United States.
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Biochemical analyses of membrane receptor kinases have been limited by challenges in obtaining sufficient homogeneous receptor samples for downstream structural and biophysical characterization. Here, we report a suite of methods for the efficient expression, purification, and visualization by cryo-electron microscopy (cryo-EM) of near full-length Human Epidermal Growth Factor Receptor 3 (HER3), a receptor tyrosine pseudokinase, in the unliganded state. Through transient mammalian cell expression, a two-step purification with detergent exchange into lauryl maltose neopentyl glycol (LMNG), and freezing devoid of background detergent micelle, we obtained ~6Å reconstructions of the ~60kDa fully-glycosylated unliganded extracellular domain of HER3 from just 30mL of suspension culture. The reconstructions reveal previously unappreciated extracellular domain dynamics and glycosylation sites.
