maximum likelihood; software; image processing; 2dx; electron crystallography; structure; hybrid input output algorithm; cryo-electron microscopy; membrane protein
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Membrane proteins are central to health and disease. Membrane proteins thereby function within the lipid membranes of cells, where they are responsible for a plethora of functions. Among others, they facilitate or regulate transport of molecules across the membrane, or sense ligands, membrane potential, or buffer gra-dients to react. Cryo-transmission electron microscopy (cryo-EM) classically allows studying the high-resolution structure of membrane proteins, if these are lipid membrane-reconstituted and two-dimensionally crystallized. This method is called electron crystallography of membrane proteins. My group has developed an image processing package for the analysis of 2D crystals, called the 2dx software package. Cryo-EM of isolated single particles has seen a major breakthrough recently, through the introduction of di-rect electron detector (DED) cameras and the development of improved single particle image processing algorithms. Single-particle cryo-EM allows to rapidly determine the atomic resolution structure of detergent-solubilized membrane proteins without the need for crystallization. This, however, does not allow the direct study of the structure of membrane proteins in a full lipid membrane, as electron crystallography provides. In this project, we will extend the 2dx software suite to accelerate processing, to automate processing, to increase resolution of the 3D reconstructions, and to make this method largely independent of highly ordered 2D crystals, so that also membrane-reconstituted membrane proteins that are not - or only partly - in a crystalline arrangement can be studied at high resolution. This will be achieved by developing certain image processing algorithms and tools to apply iterative refine-ment procedures to 3D structure reconstructions, and by adopting some of the recent algorithmic develop-ments in the single particle field such as gold-standard FSC for reliable resolution estimation or drift-correction for dose-fractionation into the 2dx electron crystallography software suite. Finally, we will imple-ment an interface of the 2dx package to leading single particle software suites such as RELION and/or FREALIGN, and adapt the latter to the specific geometries and settings of cryo-EM data of membrane-embedded membrane proteins. For development purposes, a large high-resolution dataset of images of a potassium channel membrane protein system is available. These images are recorded of small 2D crystals that diffract only to about 12 Å resolution. Preliminary application of novel algorithms has already shown significant resolution improve-ments, so that the helical pitch of alpha helices and some side-chain densities are recognizable in the 3D reconstruction. This software will continue being available open-source under the GPL at 2dx.org, and we will organize regu-lar workshops for knowledge transfer and planning of further development targets.