A Journey Structural Determination of Human MLC1

초록

MLC1 is a membrane protein with eight transmembrane domains and is mainly expressed in astrocytes (Hwang J et al., Open Biology, 2021). Genetic mutants on Mlc1 are associated with an early-onset rare genetic neurological disease called Megalencephalic Leukoencephalopathy with Subcortical Cysts (MLC), which leads to progressive neurodegeneration. MLC typically presents with infantile macrocephaly, followed by progressive gait disturbances and cognitive impairment. However, the structure and function of MLC1 have not been fully understood. Thus, we aimed to determine a 3D-structure of human MLC1 protein using cryo-EM technique. We successfully expressed MLC1 proteins, in HEK293S GnTI - cells and purified them for cryo-EM microscopic imaging. We have obtained ~3.3 Å dataset, though the orientation preference in the EM-grid hinder fine structural determination. Currently, we have been screening various conditions to identify an optimal condition for overcoming orientation preference. The addition of 0.0005~0.001% CTAB to the MLC1 in LMNG micelle showed improved cryograph. In addition, reconstitution of MLC1 protein into PE/PC/PG lipid nanodisc with 1E3 belt protein increase orientation diversity.