The solution structure of an N-terminally truncated version of the yeast CDC24p PB1 domain shows a different beta-sheet topology.

Phox and Bem1 (PB1) domains mediate protein-protein interactions via the formation of homo- or hetero-dimers. The C-terminal PB1 domain of yeast cell division cycle 24 (CDC24p), a guanine-nucleotide exchange factor involved in cell polarity establishment, is known to interact with the PB1 domain occurring in bud emergence MSB1 interacting 1 (BEM1p) during the regulation of the yeast budding process via its OPR/PC/AID (OPCA) motif. Here, we present the structure of an N-terminally truncated version of the Sc CDC24p PB1 domain. It shows a different topology of the beta-sheet than the long form. However, the C-terminal part of the structure shows the conserved PB1 domain features including the OPCA motif with a slight rearrangement of helix alpha1. Residues which are important for the heterodimerization with BEM1p are structurally preserved.

Quantitative study of the effects of chemical shift tolerances and rates of SA cooling on structure calculation from automatically assigned NOE data.

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.

Influence of chemical shift tolerances on NMR structure calculations using ARIA protocols for assigning NOE data.

Large-scale protein structure determination by NMR via automatic assignment of NOESY spectra requires the adjustment of several parameters for optimal performance. Among those are the chemical shift tolerance windows (delta), which allow for the compensation of badly matching chemical shifts in the assignment-list and peak-lists, and the maximum number of assignment possibilities allowed per peak (n(max)). Here, we test the influence of different values for Delta and n(max) on the performance of automated assignment of NOESY spectra by ARIA. Using Cesta.py (a Python script available from http://pasteur.fr/binfs/), we analyse the number of rejected peaks and the average number of assignments as a function of Delta and derive criteria for optimising delta and n(max) prior to structure calculation. The analysis also makes it possible to detect inconsistencies in the dataset, e.g., badly matching frequencies in the NOESY peak-lists and in the provided assignment-list. We show that ARIA can deal with a large number of assignment possibilities for each peak, provided the correct option is present, and that consequently narrow tolerances should be avoided.