Molecular replacement

When a protein has more than one molecule or subunit in the asymmetric unit, then redundancies exist in the intensity data that can be used to generate phase information 

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Molecular replacement is where the phases of a known structure are used to determine the structure of a protein that may be identical but crystallized in a different space group or may adopt essentially the same structure (e.g., a homologous protein). Essentially, the calculations find the rotation and translation of the molecule that work with the phases to produce an interpretable electron density map. 

The computational requirements of these calculations are relatively modest on modern computers. However, some groups have exploited computer power to design ambitious molecular replacement protocols in which many models are assessed in parallel (see Section 12.6). 

Out of the work with HT-XPIPE it became clear to the consortium that there was a need to expand the automated molecular replacement protocol embodied in HT-XPIPE to handle scenarios in which either the target was a new project or ligand-binding caused a packing change in the protein that... 

Navaza J. AMORE—an automated package for molecular replacement. Acta Cryst. 1994 A50 157-63. 

Stability implies a resistance to change, and may be defined qualitatively in those terms. In the specific case at hand, stability is defined as resistance to molecular or chemical disturbance. This requirement recognizes that a flocculated dispersion may be more stable than a peptized dispersion from the standpoint of its future behavior. A physically stable dispersion is one which will not undergo molecular replacements at the interface between the dispersed solid and the continuous phase. 

The molecular replacement method assumes similarity of the unknown structure to a known one. This is the most rapid method but requires the availability of a homologous protein s structure. The method relies on the observation that proteins which are similar in their amino acid sequence (homologous) will have very similar folding of their polypeptide chains. This method also relies on the use of Patterson functions. As the number of protein structure determinations increases rapidly, the molecular replacement method becomes extremely useful for determining protein phase angles. 

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. 

Molecular replacement (MR) A similar, known structure is used as a model for the unknown protein. 

The model protein is used to search the crystal space until an approximate location is found. This is, in a simplistic way, analogous to the child s game of blocks of differing shapes and matching holes. Classical molecular replacement does this in two steps. The first step is a rotation search. Simplistically, the orientation of a molecule can be described by the vectors between the points in the molecule this is known as a Patterson function or map. The vector lengths and directions will be unique to a given orientation, and will be independent of physical location. The rotation search tries to match the vectors of the search model to the vectors of the unknown protein. Once the proper orientation is determined, the second step, the translational search, can be carried out. The translation search moves the properly oriented model through all the 3-D space until it finds the proper hole to fit in. 

Several steps were needed to determine the structure of the core particle to higher resolution (Fig. Id). The X-ray phases of the low-resolution models were insufficient to extend the structure to higher resolution, since the resolution of the early models of the NCP was severely limited by disorder in the crystals. The disorder was presumed to derive from both the random sequences of the DNA and from heterogeneity of the histone proteins caused by variability in post-translational modification of the native proteins. One strategy for developing an atomic position model of the NCP was to develop a high-resolution structure of the histone core. This structure could then be used with molecular replacement techniques to determine the histone core within the NCP and subsequently identify the DNA in difference Fourier electron density maps. 

Fig. 2. The histone octamer. The 3.1 A X-ray diffraction data model of Arents et al. [20] is shown in secondary structure cartoon format. The core of the histone octamer is well defined, but more than 30% of the histone sequence is in regions without secondary structure. These are unfortunately the most interesting regions in terms of epigenetic signaling. 25% of the molecule located in the N-terminal tails (and the C-termini of H2A) in the 3.1 A octamer structure has no interpretable electron density. Despite these limitations, this structure is sufficient to use as a starting model for molecular replacement phasing of the NCP. (Image courtesy of E. Moudrianakis.)...

One major problem crystallographers have to deal with is the so-called phase problem, which states that of the two components of an irrational Figure (magnitude and phase) only the magnitude can be measured. A technique called molecular replacement is an approach to deal with this problem [131]. 

Rossmann, M.G. The Molecular Replacement Method. Gordon Breach, New York, 1972. 

Molecular replacement techniques for high-throughput structure determination... 

Figure 7.1 Histogram of the number of articles in Acta Crystallographica D containing Molecular Replacement in the title or abstract, year by year. The score for 2004 is a projection based on the first 6 months.

The rule of thumb for a successful application of molecular replacement is that the model should have a root-mean-square deviation (RMSD) on C-alpha coordinates 2.0-2.5 Angstroms with the target structure, corresponding to a sequence identity with the target of 25-35%. In practice, however, there are many more structures solved by MR in the PDB using models with sequence identity of 60% or higher than otherwise. 

The possibility and feasibility of molecular replacement was demonstrated by Rossmann and colleagues in the 1960s, as part of an effort to use non-crystallographic synnmetry to solve the phase problem for macromolecules (Rossmann, 1990). 

Figure 7.2 Definition of the molecular replacement problem and the six degrees of freedom needed to describe it.

Run your favourite molecular replacement program using (i) the unrotated model as a search model and (ii) calculated data as experimental data. 

Protocol 7.3 A typical molecular replacement session using AMoRe (Navaza, 2001) ... 

Sometimes, it is not so easy to convince oneself that the solution of the molecular replacement problem has, in fact, been found, even after rigid-body refinement indeed, the first solution is not always well detached and different scores may produce different rankings. The most commonly used scores are correlation coefficients on either intensities or structure-factor amplitudes, and R-factors. Even though these criteria are formally related (Jamrog et ah, 2004), they can produce different rankings, especially if no solution is clearly detached. Some other criterion is then needed to discriminate between the potential solutions. 

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