On the following pages, we will explain how we try to answer different biological questions using our main experimental method called cryo-electron tomography (cryo-ET). This information is supposed to provide basic information for non-experts.
The word tomography has its origin in ancient greek, and consists of the word τόμος (tomos), “slice” and γράφω (grapho), “to write”. Tomography has found its application in a variety of fields, including medicine (radiology), material science and of course biology.
Cryo-ET is a specialized application of cryo-electron microscopy (cryo-EM). Cryo-EM is a method to study the structure of proteins and macromolecular complexes in their native hydrated state. This is achieved via a process called vitrification, i.e. the freezing without the formation of ice crystals.
Contrary to x-ray crystallography, single-particle cryo-EM allows studying specimens that do not form a regular crystalline lattice and it is therefore possible to visualize different conformational states of the proteins. Recent breakthroughs in hardware and software developments make it now possible to visualize proteins at atomic resolutions, which has truly opened up new exciting possibilities to do understand protein structure and function.
Still, standard (single-particle) cryo-EM suffers similar limitations compared to the other conventional structural biology techniques, e.g. it requires the purification of proteins and complexes, which is often detrimental to their structure or function.
The method of choice to study demanding specimens is therefore cryo-ET, in combination with a image processing method called subtomogram averaging. Cryo-ET can provide three-dimensional insights into the unperturbed organization of tissues, cells and viruses. The method provides 3D-volumes (so called tomograms) that give information of the structural arrangement of certain features in their native environment.
In case proteins are present in multiple copies within a tomogram, sophisticated image processing (subtomogram averaging) can be performed. In this process proteins are extracted into subtomograms that then are iteratively aligned to generate a higher resolved structure.
Click on the links to get more info on the individual steps of cryo-ET data acquisition, tomogram reconstruction and subtomogram averaging
|Cryo-ET data acquisition||Tomogram reconstruction||Subtomogram averaging|