Understanding Membrane Protein Relevant in Human Diseases

While membrane proteins only make up around 20% of the proteome, they constitute around 60% of drug targets. However, high resolution structural information is severely lacking for them, with only about 2% of the structures deposited in the public protein database (PDB) being annotated as membrane proteins.

The Tan lab strives to fill in this gap by targeting membrane proteins involved in human diseases such as cancer and diabetes. Our forte is using state-of-the-art single-particle cryogenic electron microscopy (cryo-EM) to obtain the high resolution atomic coordinates of these membrane proteins. With structural information, we would be able to understand how various mutations cause protein dysfunction. We also use other biochemical tools to complement cryo-EM in understanding the function of the protein such as activity assays.

Artistic renderings of mycobacterial EmbB, the target of front-line tuberculosis drug ethambutol (Tan et al., 2020)

Artistic renderings of mycobacterial EmbB, the target of front-line tuberculosis drug ethambutol (Tan et al., 2020)

Using Cryo-EM

Imaging a grid with vitrified T20S proteasome using cryo-EM at different magnifications (Tan et al., 2015)

Imaging a grid with vitrified T20S proteasome using cryo-EM at different magnifications (Tan et al., 2015)

Cryo-EM is a structural biological technique in which electrons are used to image macromolecules. Due to the small wavelength of electrons of picometres, it is possible to resolve atomic details in the sub-nanometres range. However, these macromolecules are highly sensitive to radiation damage, hence the overall electron dose has to be kept low. An additional complication is that the amplitude contrast of the atoms that make up biological macromolecules (carbon, hydrogen, oxygen, nitrogen) are only slightly more than the background solvents. Hence biological cryo-EM images have low signal-to-noise per image. In order to overcome these limitations, computational averaging of multiple individual macromolecule particles increases the signal, and with enough particles it is possible to reconstruct a high resolution density map of the macromolecule.

In the Tan lab, we not only use cryo-EM to unravel biological questions; we also sought to develop various methods to push the envelope of cryo-EM.