Development of new cryo-EM methods
A theme of our laboratory has been the development of new EM methods for visualizing DNA, DNA-protein complexes and cells for parallel EM and biochemical studies. We were recently awarded an NIH grant of over $300,000 to obtain several state of the art cryoEM instruments including a new cryostage for the EMs and an FEI Vitrobot freezing robot. Using a combination of the Vitrobot and a dedicated ultra high vacuum system we built in-house, we have been able to achieve a longstanding goal. This has been to grow human cells directly on an EM grid and then quickly freeze the cells followed by careful freeze-drying and finally high resolution metal shadowcasting. We term this method cryo-shadowing. It was recently used in our study of KSHV protein filaments (Ozgur et al 2011) and a study recently submitted with the group of Dr. Jim Bear, on the organization of growing actin filaments in mutant and normal mouse cells. We will be applying this method in the coming year to studies involved in asthma to show how human lung cells excrete mucin proteins and to look at high resolution about how cells make first contact with each other.