Determining the molecular-packing arrangements on protein crystal faces by atomic force microscopy

Abstract

Previous atomic force microscopy (AFM) studies and periodic bond-chain (PBC) analyses of tetragonal lysozyme crystals have suggested that the (110) face consists of chains of molecules related to one another by 43 axes parallel to the crystal face. In this study, high-resolution AFM images of the (110) face were obtained and analyzed in order to verify this prediction. A computer program was employed which constructs the theoretical AFM image corresponding to a specific crystallographic molecular-packing arrangement and AFM tip shape. The packing arrangement and tip shape were varied in order to obtain the maximum possible correlation between experimental and theoretical images. The prediction from PBC analysis of an arrangement involving 43 helices was confirmed in this manner, while the alternate arrangement, consisting of molecules related to one another by 21 axes, was not observed. However, the surface structure was found to differ significantly even from this crystallographic arrangement. The molecules were found to pack slightly closer about what will become the 43 axes within the interior of the crystal, suggesting the occurrence of surface reconstruction or rearrangement on the tetragonal lysozyme (110) face. This study represents a new approach for more precise determination of the molecular-packing arrangements on protein crystal faces employing AFM.

Macromolecular crystal growth as revealed by atomic force microscopy

“…From such images, it may be possible to deduce packing arrangements or even molecular orientations. Li et al (1999) and Kuznetsov et al (1999a), for example, used AFM to analyze packing arrangements on the faces of lysozyme and thaumatin crystals, respectively, and from these deduced the ordered pathways for molecule incorporation. Because height information is preserved, enantiomorphic space groups can be resolved.…”

Crystallographic Structure of the T=1 Particle of Brome Mosaic Virus

“…A distinctly different process occurred which involved the restructuring of the surface lattice of a transition particle. Restructuring of ordered, or partially ordered, surfaces is known to occur in other systems, most prominently, the surfaces of growing crystals [39][40][41] and the surfaces of micelles. 42,43 If such restructuring can take place in the transformation of the virions and particles studied here, then it may very well occur in natural virus assembly processes as well.…”

Growth of (101) faces of tetragonal lysozyme crystals: determination of the growth mechanism

“…It was also shown that the 4 3 helix is formed by strong bonds and that crystal growth is likely to proceed in a manner which preserves this unit's structure. These predictions were con®rmed by AFM scans which showed that the (110) faces are formed by planes corresponding to this construction (Konnert et al, 1994;Li, Perozzo et al, 1999). Further con®rmation was provided by a recent study of the crystal-packing arrangement employing a somewhat different approach (Strom & Bennema, 1997a,b).…”

“…Growth units of at least tetramer size are needed to preserve the completeness of the 4 3 helices in the crystal, which is a requirement for both the (110) and (101) faces (Nadarajah & Pusey, 1996;Strom & Bennema, 1997a,b). As mentioned previously, only complete 4 3 helices were found on the (110) face in AFM investigations (Konnert et al, 1994;Li, Perozzo et al, 1999). This requirement also rules out larger crystallizing units for this face, as this would have resulted in the growth steps being multi-layered.…”

“…This relationship then allowed the molecular-growth mechanism of the crystal face to be deduced and the results to be con®rmed from atomic force microscopy (AFM) and electron-microscopy studies. These analyses revealed that tetragonal lysozyme crystals were constructed by helices centered around the 4 3 axes, consisting of four molecules in a single turn (Konnert et al, 1994;Li, Perozzo et al, 1999). The growth mechanism was shown to proceed by the addition to the (110) face of a variety of growth units corresponding to these helices .…”