Abstract
Adequate digital resolution and signal sensitivity are two critical factors for protein structure determinations by solution NMR spectroscopy. The prime objective for obtaining high digital resolution is to resolve peak overlap, especially in NOESY spectra with thousands of signals where the signal analysis needs to be performed on a large scale. Achieving maximum digital resolution is usually limited by the practically available measurement time. We developed a method utilizing non-uniform sampling for balancing digital resolution and signal sensitivity, and performed a large-scale analysis of the effect of the digital resolution on the accuracy of the resulting protein structures. Structure calculations were performed as a function of digital resolution for about 400 proteins with molecular sizes ranging between 5 and 33 kDa. The structural accuracy was assessed by atomic coordinate RMSD values from the reference structures of the proteins. In addition, we monitored also the number of assigned NOESY cross peaks, the average signal sensitivity, and the chemical shift spectral overlap. We show that high resolution is equally important for proteins of every molecular size. The chemical shift spectral overlap depends strongly on the corresponding spectral digital resolution. Thus, knowing the extent of overlap can be a predictor of the resulting structural accuracy. Our results show that for every molecular size a minimal digital resolution, corresponding to the natural linewidth, needs to be achieved for obtaining the highest accuracy possible for the given protein size using state-of-the-art automated NOESY assignment and structure calculation methods.
Highlights
Determining three-dimensional structures of biomolecules experimentally by nuclear magnetic resonance (NMR) spectroscopy has become an invaluable tool for structural and functional studies of proteins at atomic resolution in solution [1]
We study computationally a set of about 400 protein molecules obtained from the Protein Data Bank at all digital resolutions, aiming to understand the adequate number of points required for any particular protein molecule to obtain an accurate structure
In order to achieve similar digital resolutions in both 13C- and 15N-resolved NOESY spectra, the same set of numbers of points being studied was used in 1H indirect dimensions
Summary
Determining three-dimensional structures of biomolecules experimentally by nuclear magnetic resonance (NMR) spectroscopy has become an invaluable tool for structural and functional studies of proteins at atomic resolution in solution [1]. The number and types of experiments necessary for the resonance assignment and structure calculation can vary depending on the complexity and behavior of protein molecules [3,4,5]. The availability and stability of NMR spectrometers, as well as short lifetimes, molecular stability, and proteolytic degradation of protein molecules limit the total measurement time, and the number of experiments [6]. If the multidimensional experiments are sampled uniformly, there is often not enough time to measure an adequate number of sampled points, which results in poor spectral resolution and low-quality of the calculated protein structures. It is common practice to use the maximal resolution limited either by the allocated spectrometer time or the sample conditions
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