To each their own: Overcoming challenges in structural characterisation of closely related protein-protein complexes using single particle cryo-EM

Hagen Sülzen^1,2, Sami Kereiche¹, Jitka Votrubova¹, Petr Pompach³, Sebastian Zoll¹

¹Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo namesti 2, 16610 Prague 6, Czech Republic

²Charles University, Faculty of Science, Albertov 6, 12800 Prague 2, Czech Republic

³Charles University, Faculty of Science, Mass Spectroscopy, BIOCEV, Vestec, Czech Republic

sebastian.zoll@uochb.cas.cz

With an increasing demand for structural characterisation of large protein-protein complexes, single-particle cryo-EM has been gaining popularity over the recent years. Due to widespread access to high powered microscopes and the continuous development of user-friendly data processing software, the main bottlenecks limiting the success of structure determination by cryo-EM, are now sample preparation and vitrification. Here we describe the technical challenges we faced during the structural characterisation of two closely-related protein-protein complexes, both entailing the same trypanosome surface protein but different naturally occurring variants of human complement factor 3.