Project

Discipline

membrane protein; 2D crystal; image processing; electron crystallography; maximum likelihood; single particle

Lead
Membrane proteins are central to health and disease, and represent the major focus of intensive research efforts. The structure of two-dimensionally crystallized membrane proteins can be determined by electron microscopy and image processing. We develop a software package for this task, called 2dx, which we extend to improve resolution and speed of the structure determination.
Lay summary
Background: The structure determination of human membrane proteins from electron microscopy images usually requires the proteins to be available in well-ordered two-dimensional crystals (side-by-side densely packed in a membrane). These crystals are imaged in the electron microscope from different viewing angles, and the 3D structure of the proteins is reconstructed through a combination of images from different viewing angles. So far, this required images of perfectly ordered 2D crystals that were prepared on extremely flat supports. Aims: Here we will extend the 2dx software package by implementing a single particle-based approach, where we treat the images as collection of particles, instead of crystals. This allows to refine the individual location and orientation of each membrane protein particle, thereby giving access to a higher resolution of the final reconstruction. Significance: The software will be available as open-source software in our 2dx package, which now enjoys over 600 external users from life sciences and materials sciences electron microscopy fields (http://2dx.org). As such, the algorithms will immediately be available in a user-friendly and heavily used environment to other research labs.

Direct link to Lay Summary

Last update: 07.11.2012

Self-organised

Communication	Title	Media	Place	Year

Number	Title	Start	Funding scheme

Membrane proteins are central to health and disease. Electron crystallography can deter-mine the high-resolution structure of membrane proteins from membrane-reconstituted and two-dimensionally crystallized protein. The Transmission Electron Microscope (TEM) is used to study membrane proteins in the lipid membrane environment, which is a powerful tool to study the structure and dynamic of membrane proteins, and of membrane protein complexes that still resist high-resolution 3D crystallization or large-quantity expression efforts.Two-dimensional membrane crystals are imaged by cryo-electron microscopy (cryo-EM). The high-resolution 3D membrane protein structure can then be reconstructed by computer im-age processing. Until recently, the only available software for that task was the MRC software (Crowther et al., 1996). Starting in 2003, my group has created a software system, called 2dx, which embeds the MRC software into a user-friendly graphical user interface, and in addition offers a multitude of additional functions that enable full automation of the processing task, user guidance, interactive documentation and help, and allows automated iterative refinement of the processing results. 2dx received good acceptance in and excellent feedback from the electron crystallography community, and we now count more than 240 external users (as of 1/15/2009).We here propose to extend the 2dx software by new algorithms, to increase resolution and speed of the membrane protein structure determination by electron crystallography, and to en-able high-resolution structure determination also from badly ordered 2D crystals. We will imple-ment a single-particle approach for 3D reconstructions from tilted 2D crystal images, and extend this by a maximum likelihood algorithm. We will develop a hybrid input output algorithm imple-mentation to improve the Z resolution of electron crystallography, and to reduce the need to record images of highly tilted 2D crystals. We will also modernize 2dx, by enabling cluster CPU and cluster GPU computing, and interface it with other systems like Eman2 and IPLT.