Crystallography molecular replacement

Two other common methods are used to estimate phases in protein crystallography molecular replacement (MR), which uses the... 

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. 

The most demanding element of macromolecular crystallography (except, perhaps, for dealing with macromolecules that resist crystallization) is the so-called phase problem, that of determining the phase angle ahkl for each reflection. In the remainder of this chapter, I will discuss some of the common methods for overcoming this obstacle. These include the heavy-atom method (also called isomorphous replacement), anomalous scattering (also called anomalous dispersion), and molecular replacement. Each of these techniques yield only estimates of phases, which must be improved before an interpretable electron-density map can be obtained. In addition, these techniques usually yield estimates for a limited number of the phases, so phase determination must be extended to include as many reflections as possible. In Chapter 7,1 will discuss methods of phase improvement and phase extension, which ultimately result in accurate phases and an interpretable electron-density map. 

Previously, the structure of MGP-1 /GGL2 was solved by NMR and subsequently by X-ray crystallography using the NMR structure as a starting model for molecular replacement (Lubkowski et al, 1997). As described in Section II.B, the solution structure revealed a dimer similar to dimerization motifs observed for other GC chemokines. In the case of the X-ray structure, two different crystal forms were isolated from the same droplet. One form contained a dimer similar to the solution structure (Fig. 2B), while the other form contained a tetramer as shown in Fig. 8. In the tetramer, there is a primary CC dimer interface that consists of the... 

As with any protein simulation, the nature and limitations of the structural solutions for proteins provided by X-ray crystallography should always be borne in mind [125]. One obvious point is that hydrogen atoms are generally not observed because of their low electron density (neutron diffraction experiments can be useful to overcome this problem), and so it can be difficult to assign protonation states unambiguously, and to decide between possible rotamers or tautomers. This, and other factors such as model bias (for example in a molecular replacement solution), or simple error in construction of a model, may lead to the structural model being incomplete or incorrect in some places. 

Fitzgerald, P. M. D. Molecular replacement. In Crystallographic Computing 5. From Chemistry to Biology. (Eds., Moras, D., Podjarny, A. D., and Thierry J. C.) International Union of Crystallography/Oxford University Press Oxford (1991). 

As more macromolecular structures become known through X-ray crystallography, then this form of molecular replacement will see ever greater application. With sequence information to guide us, we may eventually be able to accurately and confidently predict what known model structure should be chosen to determine the approximate phases for any new but still unknown macromolecular crystal. 

Tel. 44-925-603528, fax 44-925-603100, e-mail ccp4 daresbury.ac.uk Suite of almost 100 protein crystallography programs for data processing, scaling, Patterson search and refinement, isomorphous and molecular replacement, structure refinement, such as PROLSQ, phase improvement (solvent flattening and symmetry averaging), and presentation of results, such as SURFACE for accessible surface area. Available via ftp from anonymous ... 

Figure 1.3 shows the procedure for structure determination by X-ray crystallography and Table 1.6 lists software used in structural analysis (data processing, molecular replacement, heavy atom site identification, model building, refinement and validation). 

For macromolecules such as proteins, the numbers of atoms that compose molecules are huge, therefore the crystal cells contain large numbers of atoms. It is not possible to apply the methods for small molecules, such as the direct method or Patterson map searching, in the structure determinations of proteins. The methods for retrieving the phases of protein crystal diffractions are molecular replacement, isomorphous replacement and anomalous scattering. In recent years, the direct method, which has been widely and successfully used in the determination of small-molecule structures, has also been applied in protein crystallography. 

Deeper structural studies by crystallography, molecular modeling, and SAR were performed until recently to modulate the potency and the selectivity of synthetic harmine (1) derivatives (see e.g., [37]). These smdies showed that lipophilic substituents, replacing the methyl group of the methoxy moiety at C-7, increased the potency for MAO-A inhibition in comparison with 1. Additionally, it was found that synthetic compounds containing a cyclohexyl group as substituent at C-7 were more potent MAO-B inhibitors than 1. Docking simulations demonstrated that this cyclohexyl chain points to a lipophihc pocket in the entrance cavity of the human MAO-B active site. 

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