Integrated NMR and cryo-EM atomic-resolution structure determination of a half-megadalton enzyme complex
From Nature Communication (2019)
“Unity is strength”, as the old saying goes. This is true also in structural biology. The determination of the atomic-resolution structure of a protein is fundamental for understanding its function. NMR spectroscopy and, more recently, cryo-EM, have become powerful tools for protein structural determination. However, when it comes to large protein complexes, the successful application of NMR or EM alone for structure determination is often hampered by the lack of resolution. To address this challenge, Gauto et al. used an elegant integrated approach, combining simultaneously NMR and EM data to overcome the limits of each of these techniques, and determine the high-resolution structure of a large dodecameric enzyme.
In their approach, relevant structural features (like such as α-helices) are initially identified from EM map. Then, these structural elements are unambiguously assigned to primary sequence stretches using NMR-derived information. Finally, the protein structure is jointly refined against NMR data and EM map. The structure is determined with a near-atomic-resolution, considerably higher compared to the initial EM data, providing also insights into regions likely related to the catalytic activity of the enzyme that are not visible in previously determined X-ray structures.
This integrated approach will likely play an important role in the study of challenging large biological complexes.
Gauto, D.F., Estrozi, L.F., Schwieters, C.D. et al., “Integrated NMR and cryo-EM atomic-resolution structure determination of a half-megadalton enzyme complex“, Nat Commun 10, 2697, 2019. doi:10.1038/s41467-019-10490-9.
Protein NMR resonance assignment without spectral analysis: 5D Solid-state Automated Projection Spectroscopy (SO-APSY)
From Angewandte Chemie International Edition (2019)
The wonderful functional variety of proteins is determined by their structure and their dynamics. Magic-Angle Spinning (MAS) NMR emerged in the recent years as a powerful technique for the characterization of structure and dynamics in large proteins. Resonance assignment, i.e. the procedure by means of which NMR resonances are assigned to specific atoms in the primary sequence, is the fundamental step for the successful characterization of a protein by NMR. For MAS NMR this is usually still achieved manually from the joint analysis of several 2-dimensional and 3-dimensional NMR experiments, each providing a different kind of nuclear connectivity. The amount of time required for the acquisition and the unambiguous manual analysis of the data derived from these experiments can become prohibitive for large proteins. Orton, Stanek et al. developed a 5-dimensional solid-state NMR experiment that encode all the information needed for resonance assignment in a single experiment that can be acquired in only few days, thus dramatically reducing the experimental time required. Moreover, thanks to the high resolution and sensitivity provided by fast (100-111 kHz) MAS, the obtained datasets are suitable to automated analysis, yielding rapid and unbiased resonance assignment for proteins of different sizes and aggregation states, including challenging systems featuring limited dispersion of chemical shifts.
Orton, H. ., Stanek, J. , Schubeis, T. , Foucaudeau, D. , Ollier, C. , Draney, A. ., Le Marchand, T. , De Paepe, D. ., Felli, I. ., Pierattelli, R. , Hiller, S. , Bermel, W. and Pintacuda, G., “Protein NMR resonance assignment without spectral analysis: 5D SOlid‐state Automated Projection SpectroscopY (SO‐APSY)“, Angew. Chem. Int. Ed., 2019. doi:10.1002/anie.201912211.