Fossi Michele, Oschkinat Hartmut, Nilges Michael, Ball Linda J
Forschungsinstitut für Molekulare Pharmakologie, Robert-Rössle-Str. 10, 13125 Berlin, Germany.
J Magn Reson. 2005 Jul;175(1):92-102. doi: 10.1016/j.jmr.2005.03.020.
The calculation of protein structures from nuclear magnetic resonance (NMR) data has been greatly facilitated by improvements in software for the automatic assignment of NOESY spectra. Nevertheless, for larger proteins, resonance overlap may lead to an overwhelming number of assignment options per peak. Although most software for automatic NOESY assignment can deal with a certain level of assignment ambiguity, structure calculations fail when this becomes too high. Reducing the number of assignment options per peak by reducing the chemical shift tolerances can lead to correct assignments being excluded, and thus also to incorrect structures. We have investigated, systematically, for three proteins of different size, the influence of the chemical shift tolerance limits (Delta) and of the number of simulated annealing (SA) cooling steps on the performance of the software ARIA. Large tolerance windows, and the correspondingly high levels of ambiguity, did not cause problems when appropriately slower cooling was used in our SA protocol. In cases where a high percentage of well-converged structures was not achieved, we demonstrate that it is more productive to calculate fewer structures whilst applying slow cooling, than to calculate many structures with fast cooling. In this way, high-quality structures were obtained even for proteins whose NMR spectra showed great degeneracy, and where there was much inconsistency in peak alignment between different samples. The method described herein opens the way to the automated structure determination of larger proteins from NMR data.
用于NOESY谱自动归属的软件的改进极大地推动了从核磁共振（NMR）数据计算蛋白质结构的进程。然而，对于较大的蛋白质，共振重叠可能导致每个峰的归属选项数量过多。尽管大多数用于NOESY自动归属的软件能够处理一定程度的归属模糊性，但当这种模糊性过高时，结构计算就会失败。通过降低化学位移容差来减少每个峰的归属选项数量可能会导致正确的归属被排除，从而也会得出错误的结构。我们系统地研究了不同大小的三种蛋白质的化学位移容差极限（Δ）和模拟退火（SA）冷却步数对ARIA软件性能的影响。当在我们的SA协议中使用适当较慢的冷却时，较大的容差窗口以及相应较高的模糊水平并未造成问题。在未获得高比例收敛良好的结构的情况下，我们证明，与快速冷却计算许多结构相比，在应用慢冷却时计算较少的结构更有成效。通过这种方式，即使对于其NMR谱显示出高度简并性且不同样品之间峰对齐存在很大不一致性的蛋白质，也能获得高质量的结构。本文所述方法为从NMR数据自动确定较大蛋白质的结构开辟了道路。
