Electron microscopy in three dimensions.

I would like to invite you to seminar by Dr. Sergey Ryazantsev, from California NanoSystems Institute, University of California Los Angeles, entitled: Electron microscopy in three dimensions. I hope to see many of you at the seminar.

 

Date: Tuesday, August 18

Time: 14:00 - 15:00

Venue: Meeting Room C016, Level C lab1

 

Speaker: Dr. Sergey Ryazantsev, from California NanoSystems Institute, University of California Los Angeles

Title: Electron microscopy in three dimensions.

 

Abstract:

My area of interest is imaging and 3D reconstruction of "difficult samples."  It includes macromolecules with MW less than 150 kDa such as immunoglobulins, small heat-shock proteins, etc. In order to visualize such relatively small objects in TEM, staining is necessary. In most cases, I use negative staining with uranyl-acetate. To minimize noise from stain, I use home-made super thin carbon support film 1.2-1.8 nm thick. Images are taken very close to focus to eliminate the necessity of CTF correction. Single-particle 3D reconstruction is performed using freely available EMAN software from Steven Ludke. Due to skepticism in the EM community regarding usefulness of negative staining, all my models were vigorously tested using independent physicochemical methods. For instance, the structure of human IgG subclasses was tested independently by microcalorimetry; and X-ray structure of subunits was used as internal standard. Obtained resolution of 1.2-2 nm is not enough to decipher secondary structure, but it is enough to see domain structure and to fit known structures into it. It is also the last resort when other methods fail. I will present a number of structures from very small proteins such as Hfq to relatively large filamentous proteins (decorated actin). In most cases, my approach was used because more "advanced" (and fancy) methods such as cryo-EM had failed. 3D reconstruction of stained samples is very easy and does not require any special equipment; it is also fast.
 I'll also show a few examples of tomography on fixed and stained plastic-embedded samples. The main message from my presentation is that methods of classical EM may be successfully used in combination with a modern 3D reconstruction approach.