High-Throughput methods for Electron Crystallography

The Stokes lab is part of TEMIMPS membrane center funded by the NIH Protein Structure Initiative (http://temimps.nysbc.org). Our goal is to develop technologies to facilitate screening of two-dimensional crystals and determination of 3D structures. A major motivation for using electron crytsallography is that these 2D crystals provide a lipid bilayer for the membrane proteins and thus has the potential of revealing the structure in a native conformation. Although electron crystallography has proven capable of achieving atomic resolution, the lack of high-throughput tools for screening crystallization trials presents a major disadvantage relative to X-ray crystallography and the process of structure determination is cumbersome and highly inefficient. A major stumbling block is the lack of high-throughput methods for evaluating large numbers of 2D crystallization trials. Indeed, X-ray crystallographers routinely screen thousands of conditions in order to obtain suitable crystals and our project starts with the development of comparable methods for electron crystallography. We have implemented methods for parallel microdialysis trials on a 96-well format followed by robotic imaging in the electron microscope. Ultimately, we hope not only to determine structures for a variety of membrane proteins, but also to establish fundamental parameters that govern the process of 2D crystallization.

dialysis block

Original design for 96-well dialysis block out of plexiglass. Each well holds 50 ul of protein sample and is dialyzed against a unique buffer

 

 

John Henry Sample Loading Robot

Schematic diagram of the sample loading robot, which consists of two elements. A SCARA robot picks EM grids from a 96-well tray and loads them into the sample holder. Next, a cartesian robot picks up the sample holder and inserts it through the airlock into the electron microscope. The process is controled with the Leginon software package, which also collects images from each sample.

EMIP Image Procssing Software

Screen shot of a GUI for helical image reconstruction called EMIP. Data for the helical crystal is entered into the GUI, which then guides the user through the step-by-step process of image reconstruction process.