Abstract
The structure of canavalin, the vicilin-class storage protein from jack bean, was refined to 1.7 A resolution in a highly twinned rhombohedral crystal of space group R3 and unit-cell parameters a = b = c = 83.0 A, alpha = beta = gamma = 111.1 degrees. The resulting R and R(free) were 0.176 and 0.245, respectively. The orthorhombic crystal structure (space group C222(1), unit-cell parameters a = 136.5, b = 150.3, c = 133.4 A) was also refined with threefold non-crystallographic symmetry restraints. R and R(free) were 0.181 and 0.226, respectively, for 2.6 A resolution data. No significant difference in the protein structure was seen between these two crystal forms, nor between these two and the hexagonal and cubic crystal forms reported elsewhere [Ko et al. (1993), Acta Cryst. D49, 478-489; Ko et al. (1993), Plant Physiol. 101, 729-744]. A phosphate ion was identified in the lumen of the C-terminal beta-barrel. Lattice interactions showed that the trimeric molecule could be well accommodated in both 'top-up' and 'bottom-up' orientations in a rhombohedral unit cell of the R3 crystal and explained the presence of a high twin fraction. The large inter-trimer stacking interface of the C222(1) crystal may account for its relative stability. Atomic force microscopy (AFM) investigations of the growth of three crystal forms of canavalin indicate the rhombohedral form to be unique. Unlike the other two crystal forms, it contains at least an order of magnitude more screw dislocations and stacking faults than any other macromolecular crystal yet studied, and it alone grows principally by generation of steps from the screw dislocations. The unusually high occurrence of the screw dislocations and stacking faults is attributed to mechanical stress produced by the alternate molecular orientations in the rhombohedral crystals and their organization into discrete domains or blocks. At boundaries of alternate domains, lattice strain is relieved by the formation of the screw dislocations.
Highlights
Crystal twinning is problematic in the structure determination of proteins, especially when phasing is by multiple isomorphous replacement (Yeates, 1997)
In parallel with our X-ray diffraction studies, we have investigated, by in situ Atomic force microscopy (AFM), the growth of three different crystal forms of canavalin: the rhombohedral, hexagonal and orthorhombic crystals
The total interface area on a canavalin trimer was larger in the orthorhombic crystal (2 580 AÊ 2) than in the rhombohedral (2 043 AÊ 2) and the hexagonal (2 340 AÊ 2) crystals, but smaller than in the cubic crystal (3 282 AÊ 2)
Summary
Crystal twinning is problematic in the structure determination of proteins, especially when phasing is by multiple isomorphous replacement (Yeates, 1997). For merohedrally twinned crystals with low fractions of twinning, it is possible to directly recover the true diffraction intensities. When the twin fraction approaches 0.5, the observed intensities Iobs(h1) and Iobs(h2) related by hemihedral twinning will become nearly equal and additional symmetry appears in the diffraction pattern. For these nearly perfect twin crystals, direct detwinning of the data is not reliable. Structure determination and re®nement of twinned protein crystals have become of increasing interest as seen, for
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