The first three-dimensional protein structure calculated exclusively on the basis of information obtained in living cells was solved by in-cell NMR for the putative heavy metal-binding protein TTHA1718 from Thermus thermophilus HB8 overexpressed in E. coli cells. A major hurdle for determining in-cell NMR structures is the limited lifetime of the cells inside the NMR sample tube. Standard NMR experiments usually require 1–2 days of data collection, which is an unacceptably long time for live cells. This time could be shortened to 2–3 hours by preparing a fresh sample for each experiment and by applying a nonlinear sampling scheme in combination with maximum entropy processing for the indirectly acquired dimensions.
- Sakakibara, D., Sasaki, A., Ikeya, T., Hamatsu, J., Hanashima, T., Mishima, M., Yoshimasu, M., Hayashi, N., Mikawa, T., Wälchli, M., Smith, B. O., Shirakawa, M., Güntert, P. & Ito, Y. Protein structure determination in living cells by in-cell NMR spectroscopy.Nature 458, 102-105 (2009)