Multiple Isomorphism

In small-molecule crystallography the phase problem was solved by so-called direct methods (recognized by the award of a Nobel Prize in chemistry to Jerome Karle, US Naval Research Laboratory, Washington, DC, and Herbert Hauptman, the Medical Foundation, Buffalo). For larger molecules, protein aystallographers have stayed at the laboratory bench using a method pioneered by Max Perutz and John Kendrew and their co-workers to circumvent the phase problem. This method, called multiple isomorphous replacement... 

The intensity differences obtained in the diffraction pattern by illuminating such a crystal by x-rays of different wavelengths can be used in a way similar to the method of multiple isomorphous replacement to obtain the phases of the diffracted beams. This method of phase determination which is called Multiwavelength Anomalous Diffraction, MAD, and which was pioneered by Wayne Hendrickson at Columbia University, US, is now increasingly used by protein cystallographers. 

The structure was solved by the multiple isomorphous replacement technique using four heavy atom derivatives uranyl acetate, plati-nous chloride, tetramethyllead acetate, and p-chloromercury benzoate. All four derivatives gave interpretable heavy atom Patterson syntheses. The heavy atom sites could be correlated between the de-... 

Multiple isomorphous replacement allows the ab initio determination of the phases for a new protein structure. Diffraction data are collected for crystals soaked with different heavy atoms. The scattering from these atoms dominates the diffraction pattern, and a direct calculation of the relative position of the heavy atoms is possible by a direct method known as the Patterson synthesis. If a number of heavy atom derivatives are available, and... 

The crystal structure of the MoeB-MoaD complex (Figure 3.4) was determined by multiple isomorphous replacement in its apo-state at 1.7-A resolution, with bound ATP at 2.9-A resolution and after formation of the covalent MoaD-adenylate at 2.1-A resolution [38]. The latter two structures were obtained by soaking either ATP or Mg-ATP into crystals of the apo-complex. 

La Fortelle, E. D. and Bricogne, G. (1997). Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. Method Enzymol. 276, 472 94. 

Ramakrishnan, V. and Biou, V. (1997). Treatment of multiwavelength anomalous diffraction data as a special case of multiple isomorphous replacement. Method Enzymol. 276, 538-557. 

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 understand the interactions between these bis-intercalating drugs and IMA more fully, we have crystallized several complexes of them and undertaken the structure determination by x-ray diffraction technique. One of the crystal forms diffracts to 1.6 A resolution with a space group of F222. The crystal structure was determined by the multiple isomorphous replacement method using three different heavy atom derivatives. The structure was refined to an R-factor of 19% and there were moderate number of solvent molecules clearly visible. The crystal... 

The three-dimensional structure was determined by multiple isomorphous replacement techniques using synchrotron radiation [17]. The native and guanine-PNP complex structures have been refined to 2.8 A resolution [18,19]. 

Crystals of pronase-released heads of the N2 human strains of A/Tokyo/3/67 [44] and A/RI/5+/57 were used for an x-ray structure determination. The x-ray 3-dimensional molecular structure of neuraminidase heads was determined [45] for these two N2 subtypes by a novel technique of molecular electron density averaging from two different crystal systems, using a combination of multiple isomorphous replacement and noncrystallographic symmetry averaging. The structure of A/Tokyo/3/67 N2 has been refined [46] to 2.2 A as has the structures of two avian N9 subtypes [47-49]. Three influenza type structures [50] have also been determined and found to have an identical fold with 60 residues (including 16 conserved cysteine residues) being invariant. Bacterial sialidases from salmonella [51] and cholera [52] have homologous structures to influenza neuraminidase, but few of the residues are structurally invariant. 

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. 

A preliminary x-ray structure of T. thermophilus manganese catalase (oxidized state) at 3 A resolution has been reported [80], The original solution was obtained by multiple isomorphous replacement followed by phase improvement [80], Recently both the reduced (MnnMnn) (Figure 10) and the oxidized... 

If two heavy-atom derivatives can be crystallized which preserve the space group and unit cell size of a large protein, then the structure can be solved directly this method of multiple isomorphous replacement was used by Perutz152 and Kendrew153 to solve the first two protein structures by laborious, decade-long film methods hemoglobin and myoglobin. 

Early crystallographic studies of TMADH provided data from two derivatives at 6 resolution that revealed the domain structure and certain elements of secondary structure (Lim et al., 1982 Lim et al., 1984). Higher resolution data at 2.4 resolution have been collected and the structure solved by the multiple isomorphous replacement method with anomolous scattering (Lim et al., 1986). Analysis of the diffraction pattern lead to the identification of ADP as the third cofactor in TMADH. At the time the 2.4 data set was analysed, there was no sequence information available for TMADH (Lim et al., 1986), except for a 12 residue peptide which contained the covalently bound flavin (Kenney et al., 1978). Gas-phase sequencing of isolated peptides initially provided 80% of the primary sequence of... 

GOase from D. dendroides has been crystallised from acetate buffer at pH 4.5 using ammonium sulphate as precipitant. The structure has been solved by the multiple isomorphous replacement method using three heavy atom derivatives and the model refined to 1.7 resolution to allow detailed structural analysis (Ito et al., 1991 Ito et ah, 1994). 

Of course, I have just emphasized the so-called direct methods, which determine molecular structures ab initio from the observed intensities, and I have not even touched upon much larger macromolecular systems, such as proteins, which consist of thousands of atoms in the molecule. These structures also can be determined by special techniques. These include such techniques as multiple isomorphous replacement and anomalous dispersion, which take advantage of our ability to modify a given crystal containing molecules consisting of thousands of atoms by diffusing into the crystal a small number of really heavy atoms without disturbing the crystal structure. One then does the diffraction experiment on these so-called derivatives as well as on the native protein that one is interested in. 

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