Soaking, protein crystals

Isomorphous replacement is now employed in the determination of the structures of biological macromolecules. These molecules crystallize with 50% or more of the crystal volume filled with solvent molecules. Murray Vernon King, working with David Harker, conceived the idea of soaking protein crystals in solutions of compounds containing a heavy atom. These heavy-atom compounds are diffused into the crystals through the solvent channels and settle on preferred sites on protein molecules. The diffraction patterns of the unperturbed crystal (described as "native ) and the heavy-atom derivative are then compared in such a way that an electron-density map for the protein results. The method of isomorphous replacement, and the manner by which it is used to derive relative phases, are described in detail in Chapter 8. 

Fortunately, once a parent macromolecular structure has been solved, new structures of the macromolecule complexed with different ligands can often be solved very quickly (within a few days in some cases (57)). These new structures are determined by cocrystallization of the ligand-macromolecule complex or by soaking protein crystals in a solution of the ligand and allowing the ligand to diffuse into the binding site. 

Once a suitable crystal is obtained and the X-ray diffraction data are collected, the calculation of the electron density map from the data has to overcome a hurdle inherent to X-ray analysis. The X-rays scattered by the electrons in the protein crystal are defined by their amplitudes and phases, but only the amplitude can be calculated from the intensity of the diffraction spot. Different methods have been developed in order to obtain the phase information. Two approaches, commonly applied in protein crystallography, should be mentioned here. In case the structure of a homologous protein or of a major component in a protein complex is already known, the phases can be obtained by molecular replacement. The other possibility requires further experimentation, since crystals and diffraction data of heavy atom derivatives of the native crystals are also needed. Heavy atoms may be introduced by covalent attachment to cystein residues of the protein prior to crystallization, by soaking of heavy metal salts into the crystal, or by incorporation of heavy atoms in amino acids (e.g., Se-methionine) prior to bacterial synthesis of the recombinant protein. Determination of the phases corresponding to the strongly scattering heavy atoms allows successive determination of all phases. This method is called isomorphous replacement. 

Protein crystals suitable for soaking. Preferable if known inhibitor(s) have already verified the suitability of the crystals for soaking. If co-crystallization with the fragments is to be used instead of soaking, sufficient protein to grid around a robust crystallization condition for all fragments of interest is required. 

The fastest way to obtain co-structures with a protein and fragments is to soak the fragments into existing crystals. Since each protein is unique, trial and error will be necessary to deduce the conditions where your protein crystals are stable and the fragments are suitably soluble (referred to as the protein stabilization buffer) (rrrNote 9). 

A suitable starting concentration for DMSO in a soaking experiment is 5%. Combine 9.5 xL of protein stabilization buffer with 0.5 xL fragment solution on a cover slip and mix thoroughly. Transfer protein crystal(s) to this solution and invert over a crystallization well (rrrNote 10). 

If the phasing model and the new protein are isomorphous, as may be the case when a small ligand is soaked into protein crystals, then the phases from the free protein can be used directly to compute p(x,y,z) from native intensities of the new protein [Eq. (6.15)]. 

This method of isomorphous replacement (Figure 8.27), together with anomalous dispersion data collection (see Chapter 14) is, to date, the principal method that has been successful for phase determination of macromolecules.Unfortunately, it is common to find that, although a heavy-atom solution has been soaked into a protein crystal, no regular (ordered) substitution has occurred, and solutions of other heavy-atom compounds must be tried. 

Extend the relative phases to include the higher resolution data. Soak in substrate or inhibitor molecules into the protein crystals. Measure data on the crystal with bound small molecules. 

The first step is to introduce heavy atoms into the protein crystal. This is usually done by soaking the crystals in a solution containing 0.1—10 mmol 1 1 of the heavy atom compound (Hg, Pt, Au, U compounds are often used) but sometimes the macromolecule is also co-crystallized with the heavy atom compound. As discussed in Section 9.03.4, protein crystals contain large solvent channels, which allow the diffusion of small molecules within the crystal. An important caveat is that the binding of the heavy atom compound must not distort the crystal appreciably neither the overall unit cell dimensions nor the conformation of the macromolecule. If it does, the underlying assumption that we can subtract away the protein component is false. In other words, the native (no heavy atom) and derivative (with heavy atom) must be isomorphous, and the techniques are called in general isomorphous replacement. 

For those proteins containing metal ions or metal clusters, such as Fe, Ni, and Cu, the MAD experiment can be carried out directly on the native protein crystals without the need of seleno-derivatives. Crystals containing heavy atoms incorporated via chemical modification or by soaking (McPherson, 1982) can also be used for MAD experiments. Finally, Dauter et al. (2000) has shown that ordered bromine in the protein s solvation shell could provide MAD phasing information. 

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