Experimental methods for measuring accurate high-amplitude phases and their importance in isomorphous replacement...

Conventional experimental phasing methods are most accurate for moderate-resolution reflections, with progressively greater ambiguity in the phases of reflections away from this optimal point. Frequently, very strong (usually low-resolution) reflections are either poorly phased or altogether unrecorded. While the spatial frequency of these reflections is predominantly too low to dramatically affect the calculated electron density at an atomic level, they have a dominant impact on the determination of the large-scale distribution of matter in the unit cell. Consequently, while these few strong reflections play only a peripheral role in the latter stages of a structure-determination project, they are crucial to the success of initial phasing and model-building efforts. Here, the pivotal importance of a limited number of strong/low-resolution reflection phases is shown and a procedure to derive these phases is described. The improvement in map correlation coefficients after density modification of a marginal ;starting' MAD data set (obtained from two Zn atoms at special positions in rhombohedral insulin crystals) was compared with the improvement in map correlation coefficients observed after density modification of an ;expanded' data set obtained by combining a limited number of highly accurate phases measured using three-beam diffraction with the ;starting' MAD data. It is concluded that a small number of high-amplitude/low-resolution reflections contribute disproportionately to generating an initial structure and it is suggested that a small number of triplet phases could be measured quickly and combined with experimental isomorphous replacement phases in order to move stubborn structures for novel proteins down the structure-solution pathway.