Abstract
Lanthanoid ions exhibit extremely large anomalous X-ray scattering at their L(III) absorption edge. They are thus well suited for anomalous diffraction experiments. A novel class of lanthanoid complexes has been developed that combines the physical properties of lanthanoid atoms with functional chemical groups that allow non-covalent binding to proteins. Two structures of large multimeric proteins have already been determined by using such complexes. Here the use of the luminescent europium tris-dipicolinate complex [Eu(DPA)(3)](3-) to solve the low-resolution structure of a 444 kDa homododecameric aminopeptidase, called PhTET1-12s from the archaea Pyrococcus horikoshii, is reported. Surprisingly, considering the low resolution of the data, the experimental electron density map is very well defined. Experimental phases obtained by using the lanthanoid complex lead to maps displaying particular structural features usually observed in higher-resolution maps. Such complexes open a new way for solving the structure of large molecular assemblies, even with low-resolution data.
Highlights
Even though most of the newly deposited structures in the Protein Data Bank (PDB) were solved by molecular replacement, experimental phasing remains essential for determining three-dimensional protein structures if only for solving structures with new folds or which significantly differ from any known model structure
Over the last ten years, methods based on anomalous scattering, namely the single-wavelength anomalous diffraction (SAD) and multiple-wavelength anomalous diffraction (MAD) methods, have replaced the traditional methods based on isomorphous replacement, becoming the methods of choice for solving de novo protein structures
The addition of the tris-dipicolinate complex led to the initial F4132 crystal form diffracting at low resolution, that was used for the initial structure determination of PhTET1-12s at 3.09 Aresolution (Porciero et al, 2005; Schoehn et al, 2006)
Summary
Even though most of the newly deposited structures in the Protein Data Bank (PDB) were solved by molecular replacement, experimental phasing remains essential for determining three-dimensional protein structures if only for solving structures with new folds or which significantly differ from any known model structure. Over the last ten years, methods based on anomalous scattering, namely the single-wavelength anomalous diffraction (SAD) and multiple-wavelength anomalous diffraction (MAD) methods, have replaced the traditional methods based on isomorphous replacement, becoming the methods of choice for solving de novo protein structures. The preparation of effective heavyatom derivatives displaying anomalous scattering has become a key point for de novo crystal structure determination. With the incorporation of selenium through the substitution of methionine residues by seleno-methionine (Hendrickson et al, 1990; Doublie, 1997), and with the developments at thirdgeneration synchrotron radiation sources, which allow weak anomalous signals from intrinsic scatterers to be used, the time-consuming preparation of heavy-atom derivatives has been facilitated. The use of such procedures is not always possible, which revives the problem of incorporating effective anomalous scatterers into protein crystals. Lanthanoid ions, Ln3+, are well suited to anomalous diffraction experiments since they all exhibit a strong white line in their LIII absorption edge leading to extremely large anomalous contributions of almost 30 eÀ for both f 0 and f 00
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