Test case: a Notch1 transcription complex containing the RAM region. (a) The first 4.2–7 Å low-resolution structure of a human Notch1 complex consisting of the Notch ankyrin-repeat domain, the CSL transcription factor and the Mastermind-like 1 (MAML-1) co-activator was determined by combining molecular replacement and selenomethionine scanning. Single Leu-to-Met or Val-to-Met mutations (labeled with the MAML-1 residue number) were introduced into the MAML-1 polypeptide for incorporation of selenomethionine. Anomalous Fourier difference maps were calculated for each of five mutants (the high-resolution limit for each data set was between 6 and 7.5 Å) using the anomalous signal from selenomethionine and the phase calculated by molecular replacement. Each map shows a clear peak at the predicted location of the mutated residue, indicated by the matching colors. The gray mesh represents the density for the MAML-1 as part of a 2Fo − Fc density map calculated without any atoms modeled for the MAML-1 helix. Adapted from the supplementary information in Nam et al. (2006). (b) An Rfree heat map of results from the Notch protein DEN optimization using an initial starting temperature of 1000 K. The minimal Rfree values for each parameter pair (ωDEN and γDEN) over multiple refinement repeats are shown. (c) The corresponding Ramachandran statistics (percentage of disallowed backbone angles). For each parameter pair, the structure with the lowest Rfree value was usen to calculate the Ramachandran statistics. (d) A histogram showing all calculated Rfree for a complete portal DEN optimization of the Notch1 complex. The dashed line is the best that could be achieved without DEN (i.e., the lowest Rfree of the all the `no DEN' repeat refinements); the lowest (or best) Rfree chosen is the far left tail of the histogram.