Multiple isomorphous replacement MIR

The problem of phase determination is the fundamental one in any crystal structure analysis. Classically protein crystallography has depended on the method of multiple isomorphous replacement (MIR) in structure determination. However lack of strict isomorphism between the native and derivative crystals and the existence of multiple or disordered sites limit the resolution to which useful phases may be calculated. 

In order to resolve the phase ambiguity from the first heavy-atom derivative, the second heavy atom must bind at a different site from the first. If two heavy atoms bind at the same site, the phases of will be the same in both cases, and both phase determinations will provide the same information. This is true because the phase of an atomic structure factor depends only on the location of the atom in the unit cell, and not on its identity (Chapter 5, Section III.A). In practice, it sometimes takes three or more heavy-atom derivatives to produce enough phase estimates to make the needed initial dent in the phase problem. Obtaining phases with two or more derivatives is called the method of multiple isomorphous replacement (MIR). This is the method by which most protein structures have been determined. 

In addition to the MAD and SAD methods, there are the traditional isomorphous replacement methods that include multiple isomorphous replacement (MIR), which uses several heavy atom derivatives, and single isomorphous replacement (SIR), which uses only one heavy atom derivative. The underlying principle to all these methods is the phase-triangle relationship. To understand this relationship we shall begin our discussions with the isomorphous replacement method. 

The value of heavy atom derivative is made, so that now FH1, FH2, FHpi, FHp2 and FP are to be determined. This is called multiple isomorphous replacement (MIR) and is generally used rather than the single isomorphous replacement technique. The values for FHi and Fh2 can be determined using Patterson techniques. Two equations similar to equation 6.6 now exist ... 

Figure 6.28 Representation in the Gaussian plane of the phase relationships derived by multiple isomorphous replacement, MIR, in a protein. The structure factor of the protein vector, FP, lies on a circle of radius FP centred at O. The structure factor of the heavy metal derivatives, FPHi and FPH2, lie on circles of radii FPHi and Fph2 with centres at the tips of the vectors -FHi and -Fje. The intersection of the three circles represents a unique solution to the position of FP, corresponding to a single phase angle...

In the recent studies, the enzyme shows that the overall polypeptide fold of chymotrypsin-like serine protease possesses essential SI specificity determinants characteristic of elastase using the multiple isomorphous replacement (MIR) method and refined to 2.3 A resolution Fig. (5). Structure-based inhibitor modeling demonstrated that EFEa s SI specificity pocket is preferable for elastase-specific small hydrophobic PI residues, while its accommodation of long and/or bulky PI residues is also feasible if enhanced binding of the substrate and induced fit of the SI pocket are achieved [Fig. (6) shows the active sites of serine protease]. EFEa is thereby endowed with relatively broad substrate specificity, including the dual fibrinolysis. This structure is the first report of an earthworm fibrinolytic enzyme component, a serine protease originated from annelid worm [17]. 

Metal clusters have also been considered as heavy metal labels for determining the phasing in crystallography of large biological molecules.Heavy atom labeling by a tetrairidium cluster was used to determine the X-ray crystal structure of the small subunit from Thermus thermophilus. Attachment of the cluster was made to the surface-exposed SH groups prior to crystallization. The positions of these sulfhydryls were localized in difference Fourier maps that were constructed with the multiple isomorphous replacement (MIR). ... 

Multiple isomorphous replacement (MIR). Crystals are soaked in solutions with heavy atoms (Hg, Pt, Au salts, etc.) under such conditions that a few of these heavy atoms attach themselves to well-defined spots on the protein molecule. The heavy atom positions are found by... 

The structure of Escherichia coli FDHh, as solved by multiple isomorphous replacement (MIR) and multiwavelength anomalous dispersion (MAD) methods, consists of four ap domains [82]. It has been prepared with MOLSCRIPT and RASTER3D [83-85]. 

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