As structural biologists, our research is strongly driven by using cutting-edge technological advancements and methods.
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.
Cryo-ET is a specialized application of cryo-electron microscopy (cryo-EM). In brief, 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 (which can cause damage to biological specimens).
Single-particle cryo-EM also offers the advantage to study specimens in multiple conformational states. This allows to understand biological molecules in a more native way as it reflects the intrinsic flexibility and dynamics that most molecular machines possess. 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 that cannot be visualized by any other means is therefore cryo-ET. 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 algorithms can be employed (i.e. subtomogram averaging). In this process, proteins are extracted into smaller boxes (called subtomograms) that then are iteratively aligned to generate a higher resolved structure.
Click on the links to get more info on cryo-ET data acquisition, tomogram reconstruction and subtomogram averaging
|Cryo-ET data acquisition||Tomographic reconstruction||Subtomogram averaging|