Section 9.1.13. Relating data collection to the problem in hand

The phasing of proteins by isomorphous replacement requires the collection of data from crystals of one or more heavy-atom derivatives of the protein that are isomorphous to the parent native crystal. Preparation of derivatives involves either soaking of native crystals in the heavy-atom solution or co-crystallization with the heavy-atom reagent (Part 12 ). Data collection can be split into two parts. The first step is to establish whether a potential derivative is isomorphous and contains the expected heavy atoms. The second is to collect the data on this derivative to provide the necessary phase information for the native structure factors. The problems of how to utilize the phase information are addressed in Part 12 . Here, strategies applicable to the two steps are described.

Screening of derivatives can be carried out by collecting data to the resolution limits of the crystals. This can consume substantial data-collection resources and lead to irrelevant data that are not from isomorphous crystals or do not contain the anticipated heavy-atom signal. It is preferable to record the minimum data sufficient to identify a potential derivative in order to save time and resources, as many samples may need to be screened. A minimal strategy can exploit some or all of the following protocols:

Some practical points are highly relevant here. The ability to store and reuse frozen crystals means that potential derivatives can first be screened at the lowest possible resolution, and the crystal preserved and used later only if the derivative proves to provide useful phase information. The final resolution for data collection will then depend on the degree of isomorphism. The wavelength, if tunable, should be set to a value just below the absorption edge in order to maximize the anomalous signal. The redundancy can also play an important role, as it is useful to have a large number of independent measurements so that outliers in the native or derivative data can be excluded, as these can cause major problems in either the Patterson or direct-methods approaches for locating the heavy atom (Part 12 ).

The requirements for collecting data with an intrinsically weak anomalous signal are several. As with the isomorphous measurements in the previous section, the highest possible resolution may not be the primary consideration. Here the emphasis lies in data quality, as the measurement of very small differences in macromolecular amplitudes, which are already in themselves relatively weak, is required. Important considerations include the following.

For MAD experiments (Hendrickson, 1991; Smith, 1991), which can only be carried out at SR sites, the optimum number of wavelengths at which data should be recorded remains unclear. The minimum is one (SAD) and the conventional wisdom is that four are optimal. Given finite beam time, the trade-off is between measuring with limited redundancy at several wavelengths as against higher redundancy at a smaller number of wavelengths. The jury is still out on this one.

Single-wavelength anomalous dispersion (SAD) represents the limiting case. All data are recorded at one wavelength, reducing the requirement for fine monochromatization and for fine tunability and stability. Now quality, especially in the form of redundancy, is the dominating factor since all phasing is based purely on a single anomalous difference for each reflection.

For the initial data required for molecular replacement (MR), high resolution is not essential. Firstly, the method depends on homologous models that are usually only an imperfect representation of the structure under investigation and hence high-resolution data cannot be accurately modelled, and will only introduce noise into the analysis. Secondly, the rotation function, the first step in MR, is based on the representation of the Patterson function in terms of spherical harmonics, which is limited in its accuracy.

In contrast, it is essential for MR applications that the most intense low-resolution terms are measured. The lack of such reflections strongly affects the rotation- and translation-function computations, as the functions are based on Patterson syntheses involving the square of the structure-factor amplitudes, and are dominated by the largest terms. Elimination of the strongest few per cent of the low-resolution data may well prevent a successful solution by MR.

However, for refinement of structures solved by MR, it is essential that data be recorded to a resolution sufficient to allow escape from the phase bias introduced by the model.

Here it is intended to include all structures that benefit from the highest accuracy in their atomic coordinates to shed light on the details of their biological function. These may include substrate or inhibitor complexes and mutants if the analysis requires the full potential of X-ray crystallography. Many of these will not diffract to atomic resolution; nevertheless, all steps in a detailed crystal structure analysis are made simpler as the resolution and quality of the data are increased. This includes the solution of the phase problem, interpretation of the electron-density maps and the refinement of the model.

The most appropriate strategy for data collection involves decisions based on a complex and mutually dependent set of parameters including:

Whatever the resource, it is good to define a strategy that will provide high completeness of the unique amplitudes at the highest resolution, with the realization that there is some conflict between these two requirements.

The detailed geometry of the molecule is already known and the rather general effects of ligand binding or mutation can be initially identified at a relatively modest resolution and completeness. As with heavy-atom screening, it is often advisable to check that the desired complex or structural modification has been achieved by first recording data at low resolution.

However, if the analysis then proves to be of real chemical interest, with a need for accurate definition of structural features, the data should be subsequently extended in resolution and quality. As with the identification of isomorphous derivatives, this approach has benefited greatly from cryogenic freezing, where the sample can be screened at low resolution and then preserved for subsequent use.

As for MAD data, the needs for atomic resolution data are extreme, but rather different in nature. Atomic resolution refinement is addressed in Chapter 18.4 . Suffice it to say that by atomic resolution it is meant that meaningful experimental data extend close to 1 Å resolution. There are two principal reasons for recording such data. Firstly, they allow the refinement of a full anisotropic atomic model, leading to a more complete description of subtle structural features. Secondly, direct methods of phasing are largely dependent upon the principle of atomicity.

The problems likely to be faced include: