Title:	Endophilin-B1 on nanodiscs
DNr:	Berzelius-2023-196
Project Type:	LiU Berzelius
Principal Investigator:	Anna Sundborger <anna-sundborger-lunna@icm.uu.se>
Affiliation:	Uppsala universitet
Duration:	2023-08-16 – 2023-12-01
Classification:	10601
Keywords:

Abstract

Endophilin-B1 is a BAR domain containing protein that plays a key role in multiple fundamental processes that are important for cell homeostasis, including Golgi scission, endosomal trafficking, mitochondrial dynamics, autophagy, and intrinsic apoptosis. EnB1 dysregulation has been implicated in various diseases, including multiple types of cancer. Endophilin-B1 has high affinity for membranes containing cardiolipin, a key signalling molecule that is also involved in the regulation of cell death. How endophilin-B1 can serve all these diverse roles in the cell remains unclear and the close involvement of endophilin-B1 to key steps that determine the fate of cells makes it an interesting subject. Endophilin-B1 was recombinantly expressed in E. coli cells and purified. The protein was incubated with MSP2N2 nanodiscs containing DOPS and cardiolipin and the complex was purified using size-exclusion chromatography. Two data-sets were collected, using a Titan Krios cryo-electron microscope at the Sci-Life facility in Solna. A small subset of the particles contained in the data set have already yielded two atomic models of EnB1 (in different conformations), however, the size and complexity of the data-set has made it difficult to fully analyze. The data-set contains not only continuous conformational hetereogeneity, but also compositional heterogeneity, which makes 3D classification difficult. Initial testing with the neural network method cryoDRGN on a small subset of the data had very interesting results. cryoDRGN was much more successful at sorting particles into discrete states than cryoSPARC. Access to a larger cluster would allow us to analyze the whole data set with cryoDRGN, and hopefully give us a clearer picture of the underlying dynamics, the protein-lipid interface and how EnB1 interacts with the model membrane.