The analysis of protein structures by cryo electron microscopy

We applied cryo electron microscopy to addressing several topics of protein structures including protein conformational change, heterogeneity, physiological oligomeric state and aggregation stages of fibrils. The first topic is the chloride channel (ClC) family including chloride-proton transporters and chloride channels. How protons are transported in the chloride-proton exchanger and the scale of protein movement of ClC members in ion permeation are still unclear. We utilized 2D crystallization and observed 2D crystals of the E. coli chloride-proton transporter (ClC-ec1) form as multilayers at pH=6 and >0.6nm wide tubular crystals at pH=5, implying the conformational changes of ClC-ec1 in response to protons. It also shows the possibility of using electron crystallography to probe the conformational change at low pH. The other three topics are studying the protein oligomeric state and heterogeneity by using cryo electron microscopy and the single particle analysis. (1) Serum amyloid P-component (SAP) as a member of pentraxin family plays an important role in innate inflammatory system in invertebrates. Both heptameric and octameric forms of Limulus SAP demonstrate that the heptameric form is dominant. (2) Ethanol generated from the fermentation of xylose, the second most abundant carbohydrate on earth, benefits utilizing agricultural waste to produce fuel. Among the steps of fermentation pathway in prokaryotic cells, phosphorylation catalyzed by xylulose kinase is rate-determine step. The X-ray crystal structure doesn't show its oligomeric state clearly. We found the dimeric form in vitrified ice and obtained 36 A-resolution structure. (3) Human synuclein with the size of 140 amino acids unfolds and aggregates as fibrils resulting in Parkinson's disease. We observed the straight and twisted forms of the wild-type and the truncated (30-110) one and showed the protofilament aggregates at different levels.