Postdoctoral Position in 3-D Electron Microscopy

Graduate Student Research Opportunities

(1) Structure of contracting insect flight muscle In this project, we utilize electron tomography to produce 3-D images of muscle in various states in various states of contraction. The approach allows us to obtain 3-D information of individual crossbridges. The project is a large collaboration between the laboratories of Drs. Yale Goldman at U. Pennsylvania School of Medicine and Dr. Michael Reedy at Duke University Medical Center. Specimens of contracting muscle are rapidly frozen at different stages of the contractile cycle, prepared for electron microscopy and viewed by electron tomography. Monitoring of the fiber mechanics facilitates correlations between structure and tension development. Projects would involve mostly 3-D image reconstruction, molecular model building and refinement. This is a good project to learn all aspects of molecular imaging by electron tomography.

(2) Structure of smooth muscle myosin and heavy meromyosin In this project we are studying the structure of various constructs of smooth muscle myosin II to determine the effects of S2 length on regulation and actin binding by two heads. This is a collaboration with Prof. Kathy Trybus at the University of Vermont. We are also studying the structure of the inhibited state which can be produced by dephosphorylation of the regulatory light chain and conversely the active state, which is produced by regulatory light chain phosphorylation. We produce specimens for 3-D imaging by growing 2-D arrays on lipid monolayers and obtain 3-D image data from frozen hydrated specimens. Quasiatomic models are produced using high resolution X-ray structures of the different domains which are then fit into the lower resolution 3-D envelope and then refined using various objective refinement methods.

(3)Structure of the unconventional myosin-V In this project we are studying the structure of the inhibited conformation of myosin-V, one of the unconventional myosins, which can be found in many kinds of eukaryotic cells. This is a collaboration with Prof. Kathy Trybus at the University of Vermont. This project is in many ways similar to the smooth muscle myosin studies in that it involves 2-D arrays of the inhibited conformation in a state that is not bound to actin and the structure of the actin bound state. Both approaches involve cryoelectron tomography, volume classification and averaging and atomic model building.

(4) Cryoelectron tomography We are using cryoelectron tomography to visualize proteins, protein complexes and cellular fragments that are involved in cell migration and cell adhesion. Current emphasis is on integrins incorporated into small unilamellar vesicles and protrusive membranes from migrating cells. Both of these involve development of novel alignment and 3-D image classification techniques to identify and obtain information on protein conformation in difficult to work with environments. We are also using cryoelectron tomography to study enveloped viruses. This effort is largely, but not completely funded by the Cell Migration Consortion.

(5) Structure of cell adhesion complexes This project is our contribution to the Cell Migration Consortium. In this project we are using lipid monolayers to form 2-D arrays of adhesion proteins and to assemble cell adhesion complexes for 3-D electron microscopy. The project would involve protein purification and expression and a lot of biochemistry to persuade the proteins to make paracrystalline or crystalline arrays. The proteins that will be investigated include talin, alpha-actinin, vinculin, integrins and many other proteins that have been identified (or will be identified) to be part of cell adhesion complexes. We are also studying the thin margins of cells such as lamellapodia and filopodia by cryoelectron tomography. The type of image processing utilized will depend on the kind of specimen produced. Everything from single particle methods to electron tomography of frozen hydrated specimens will be utilized to obtain the 3-D images. Quasiatomic models will be fit to determine the 3-D arrangement of molecules within reconstructions. There are excellent opportunities for collaboration within the framework of the consortium.