Atomic Force Microscopy Data

We employed three types of atomic force microscopy (AFM) data collection. The first type consisted of images of 200 nm to 1 mm, such as the one in Fig. 2, on which the protein molecules, 13 nm in size were clearly detectable. Calibration of the microscope was verified by determining the spacing of molecules along a densely, packed direction in the lattice and the thickness of the top crystalline layer. As Fig. 2 reveals, the respective numbers for one of the systems, used for the studies summarized here, the protein apoferritin, are 13.1 and 10.5 Á. These are in good agreement with the evaluations from the X-ray structure of the crystal. Using the crystal lattice parameter of a = 18.4 nm for the crystallographic group F432 (4,5), the intermolecular spacing is (1/2)(2a2)1/2 = 13.0 nm, and the layer thickness is (1/3)(3a2)1/2 = 10.6 nm.

To evaluate the maximum resolution of molecular-scale imaging attainable with ferritin/apoferritin in situ, during crystallization, we scanned a 200 x 200 nm square on the surface of a ferritin crystal (see Fig. 3A). The two-dimensional Fourier transform of the image in Fig. 3B has the expected hexagonal symmetry, with the distance between the peaks in the first hexagon and the center of the plot corresponding to resolution equal to the molecular size of 13 nm. The maximum resolution, determined from the location of the most distant

Fig. 2. Accuracy of AFM imaging. (A) View of a (111) apoferritin crystal face; (B) height profile along line in (A) allowing determination of layer thickness and molecular spacing. (From ref. 10.)

peak, is 1.6 nm. This resolution allows us to distinguish some of the submolecular-level details of the molecules on the crystal surface in Fig. 3C. Comparison with the structure of the molecules coming from an X-ray determination in Fig. 3D (5) shows that the triangular formation in Fig. 3C likely corresponds to bundles of a-helices.

Fig. 3. Resolution of AFM imaging. (A) Real-space high-resolution image of a (111) apoferritin crystal face. (From ref. 10.) (B) Fourier transform of A: circles highlight high-resolution peaks; eighth-order peaks at the top and bottom of the image correspond to a resolution of approx 1.6 nm. (From ref. 10.) (C) Real-space image in which each molecule is replaced by average of all molecules in frame; processed with SEMPER Software package (N. Braun, S. Weinkauf, personal communication). (D) Ribbon presentation of X-ray structure of apoferritin molecule viewed along (111) direction. Images of molecules in C appear to have similar triangular features.

Fig. 3. Resolution of AFM imaging. (A) Real-space high-resolution image of a (111) apoferritin crystal face. (From ref. 10.) (B) Fourier transform of A: circles highlight high-resolution peaks; eighth-order peaks at the top and bottom of the image correspond to a resolution of approx 1.6 nm. (From ref. 10.) (C) Real-space image in which each molecule is replaced by average of all molecules in frame; processed with SEMPER Software package (N. Braun, S. Weinkauf, personal communication). (D) Ribbon presentation of X-ray structure of apoferritin molecule viewed along (111) direction. Images of molecules in C appear to have similar triangular features.

The second type of AFM data consisted of images on the mesoscopic length scales from several tens of nanometers to several micrometers, as in many previous AFM studies of crystallization from solution (13-24). These images, similar to the one in Fig. 4, with a view-field width between 2 and 40 ^m, allow characterization of growth steps and step patterns.

For the third type of AFM data, we employed scans with a disabled y-axis (25,26), as in previous scanning tunneling microscopy (STM) work on metals and semiconductors in ultrahigh vacuum (27-30). The AFM tip is drawn over a single line of the crystal surface. In the collected pseudoimages, the vertical

Fig. 4. AFM images of surface of growing apoferritin crystals taken near center of respective facet. Facet size is approx 90 |im, ensuring near-uniform supersaturation over the facet. New crystal layers are generated at random locations by surface nucle-ation and spread to merge with other islands and cover the whole facet.

Fig. 4. AFM images of surface of growing apoferritin crystals taken near center of respective facet. Facet size is approx 90 |im, ensuring near-uniform supersaturation over the facet. New crystal layers are generated at random locations by surface nucle-ation and spread to merge with other islands and cover the whole facet.

axis represents time. The technique allows monitoring of processes with characteristic times of fractions of a second.

If the scan widths were 600 nm and less, the displacement of a single site on the step could be monitored with molecular resolution; that is, we could trace the attachment and detachment of single molecules to and from steps (10,25,31). Data collection lasted typically 2 to 3 min. Immediately after such scans, area scans included the line along which the tip was drawn. In about 80% of cases, these tests revealed that the tip impacted over the same location had delayed the growth. These data were discarded, and only data sets that did not show tip impact are discussed herein.

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