Probing toward atomic resolution in molecular topography.

Work representing an important milepost in the development of atomic force microscopy (AFM) of biological macromolecules, just published in these Proceedings (1), deserves special note and comment, both for its advanced level of sophistication and for its unique degree of accomplishment. What distinguishes this work, in terms of its sophistication, is the quantitative comparison made between high-resolution surface topographs obtained from identical protein specimens by two completely independent methods. The specimen used in this work was a twodimensional, crystalline protein array isolated from the surface of a bacterium, Deinococcus radiodurans. The surface topography of this sheet-like protein crystal was first imaged by AFM; more about this shortly. In addition, however, a high-resolution, three-dimensional reconstruction of the surface of the same specimen was produced by state-of-theart, low-dose electron microscopy. Both of the molecular topographs exhibit a lateral resolution close to 1 nm. What is most significant about the resulting comparison between the two topographs is the fact that, when superimposed, they show an average deviation in height of less than 0.5 nm. One is hard pressed to say which is more important: Does electron microscopy, the gold standard of resolution and accepted methodology, validate the AFM image, which might otherwise be thought to be limited by a host of effects that are still being discussed in the literature? Or does the AFM image, obtained in the wet state and with incomparable resolution in the third dimension, establish that electron crystallographic reconstructions of macromolecular surfaces are perhaps better than might have been supposed, given the relatively poor resolution in the third dimension that is an unavoidable consequence of access to only a restricted range of specimen tilt angles (2, 3)? The fact that AFM could even be considered as being a suitable tool to validate structural information obtained by electron microscopy shows just how far this new technique has come in the short time, less than 8 years, since it was first introduced (4). Even more could be said by Karrasch et al. (1) about the reproducibility and validity of the current AFM images, however, by using methods developed previously in electron microscopy to align and superimpose images of individual molecules. Some variations in the height of the AFM surface topograph were found at equivalent positions in different unit cells, of course. What is so impressive in the final result is that the height variations were found to be less than 0.1 nm in some parts of the unit cell, which in retrospect can be described as areas of exceptional rigidity and crystallographic perfection, while other parts of the unit cell showed height variations approaching 0.5 nm. Since the variation in height is not uniform over the unit cell, it is natural to attribute the observed pattern to differences in flexibility of different parts of the structure itself. A state-of-the-art innovation in specimen immobilization employed by Karrasch et al. is yet another novelty that distinguishes this work, and no doubt has been an essential ingredient of its success. In work described at greater length elsewhere (5), Karrasch et al. have shown that glass surfaces can be covalently modified with an aminotriethoxysilane, which can then be derivatized with N-5-azido-2nitrobenzoyloxysuccinimide, a photosensitive crosslinker. Sample materal is applied to this modified glass surface, where it becomes attached by covalent bonds when illuminated with light in the near ultraviolet. It is becoming increasingly well established that firm attachment of the specimen to the substrate is a prerequisite for high-resolution applications of AFM with biological molecules. Firm attachment of the specimen to the substrate, and rigid connections within the plane of the specimen, would seem to be required by the fact that the force used during acquisition of the AFM image is orders of magnitude greater than the force needed to move individual domains within biological macromolecules. The smallest force reported for successful operation with biological specimens has been as little as 0.1 nN, yet even this tiny force is overwhelming in comparison to the force of -1 pN exerted by myosin molecules during the individual power strokes that underlie muscle contraction (6). It is not so surprising, then, that elastic deformation and outright plastic deformation of the specimen are easily observed during AFM imaging. A very important study in this regard was published by Butt et al. (7), using the purple membrane of Halobacterium halobium as a test object. This specialized patch of cell membrane, made up of a twodimensional crystalline array of a single protein (bacteriorhodopsin) and lipid, would seem to be ideal for AFM studies because it has very little variation in surface height, ensuring that resolution will not be degraded by "convolution" with the large radius of the probe tip. Even with forces kept below 1 nN, however, it was evident that the hexagonal symmetry of the lattice was markedly distorted by the frictional (transverse) force exerted when recording the AFM image. As a result, the resolution in the direction parallel to the scan was worse than the nominal 1.1-nm resolution observed in the direction perpendicular to the scan direction (7). Another example relevant to this point is found in recent images of cholera toxin, a protein of 85 kDa, stabilized within a bilayer of polymerizable phospholipid (8). In specimens prepared with just the B subunit, a high percentage of molecules show the pentagonal subunit structure and the central hole expected of this molecule when the probe force is below 1 nN, but any relationship to known molecular structure is lost at probe forces close to 1 nN. Many other accounts of image distortions (9, 10) and even structural "dissection" or cutting by the probe tip (11, 12) have been reported in the recent literature. It is encouraging that none of these deformations and distortions seemed to be apparent in the study by Karrasch et al. (1) of the bacterial surface-layer protein. When the biological specimen structure is simple and the topography is almost flat, even some earlier results of AFM imaging have come surprisingly close to achieving atomic resolution. Resolution of individual methyl groups was seen on the surface of a cadmium arachidate monolayer supported on a silicon oxide substrate by polar-group interactions (13). Nearly as high resolution was also achieved in imaging individual phospholipid head groups on samples prepared by transfer of a lipid monolayer onto a hydrophobic surface prepared by covalent reaction of mica with octadecyl-