Microcrystallization of HA

Purified recombinant HA protein was crystallized in 100 mM Tris-HCl (pH 8.0), 30% PEG 400, and 200 mM MgCl 2 in 2 days, resulting in occasional formation of granular aggregates, frequent growth to form macrocrystals and the production of showers of tiny micron-sized crystals (Fig. S1, upper panel). To assess the size of nano- and microcrystals that are not clearly visible by light microscopy, a hemocytometer was used to show variable-sized microcrystals ranging in size from approximately 10 to 30 μm (Fig. 1A). However, microcrystallization of the HA protein was not reproducible, particularly in large-volume drops, and yielded microcrystals with different sizes. To improve the reproducibility of crystallization initially, rapid evaporation was applied to hanging drops; a crystallization solution containing a protein solution was dispensed on coverslips to produce hanging or sitting drops in a 24-well or 96-well plate. It was air-dried until white precipitates start to appear on the first coverslip, at which time the remaining coverslips were flipped over to seal the wells. Rapid evaporation produced microcrystals more reproducibly (Fig. S1, lower panel), with the yield reaching about 50%, compared to typical yields of less than 5%, based on the area occupied by microcrystals on the hemocytometer.

Figure 1 Microcrystallization. (A) HA microcrystals observed in a vapor diffusion drop by light microscope (upper-left panel), and transferred to hemocytometer (upper-right panel). The Neubauer chamber on the microscope stage of the hemocytometer showed a square ruled into 9 small squares, which were further divided into 16 smaller squares having sides of length 200 and 250 μm. Grid images in the dotted box are magnified in the lower panel. Scale bars: 100 μm. (B) Images of lysozyme (upper panel) and ferritin (lower panel) microcrystals in hanging drops obtained by supersaturation-controlled microcrystallization. Microcrystals were obtained at the highest density between 16 and 20 min for lysozyme and between 15 and 21 min for ferritin. Full size image

Supersaturation-controlled microcrystallization

This rapid evaporation was further developed to test several microcrystallization conditions, where serial evaporation was controlled with a time delay (e.g., 2 min intervals) at the onset of microcrystallization. Lysozyme from chicken egg white was prepared at 55–75 mg/ml and the single crystallization condition of 100 mM sodium acetate (pH 4.8), 18% NaCl and 6–10% PEG 400 was used. Purified recombinant ferritin from Escherichia coli was prepared at 10 mg/mL and the single crystallization condition of 100 mM sodium acetate (pH 5.0) and 1 M NaCl with an additive buffer of 100 mM Tris-HCl (pH 8.0), 30% PEG 400, and 200 mM MgCl 2 was used23. The protein and precipitant solutions were dispensed on coverslips in a drop size of 1.8 μL in a 24-well plate, air-dried to induce rapid evaporation of drops sequentially from 0 to 22 min at 2 min intervals for lysozyme (from 0 to 33 min at 3 min intervals for ferritin): the first drop containing lysozyme was air-dried for 2 min and sealed, the second one which had been dried for the same time was air-dried for additional 2 min and sealed, and the third one for additional 2 min and sealed, and so forth (Fig. S2). The effects of this time delay in the microcrystallization of the proteins were examined; microcrystals were usually obtained in 6–12 hrs at room temperature, and these typical microcrystals are shown in the figures. Strikingly, single large crystals of lysozyme appeared on the coverslips during the evaporation period of 0–14 min, whereas microcrystals were obtained at high density from 16 min to 20 min, when the lysozyme concentration was 55 mg/ml (Fig. 1B, upper panel). Smaller microcrystals of lysozyme at higher density were produced with higher concentrations of the lysozyme protein (55–75 mg/mL) (Fig. S3). As the concentration of lysozyme increased, the time delay required for microcrystallization also decreased. For ferritin, no single crystals were observed; instead, microcrystals with constant sizes were most typically obtained during the observed periods (Fig. 1B, lower panel). Supersaturation control was also found to produce a higher yield of relatively homogeneous microcrystals of ferritin, and microcrystals at the highest density were obtained at the evaporation period of 15–21 min. Then, supesaturation-controlled microcrystallization was tested for application to HA in sitting drops for the total duration of 11 min with a time delay of 30 sec. The highest yields of microcrystals of HA were observed at the duration of 14–15 min (Fig. S4). Microcrystals of ferritin and HA were again obtained in 6–12 hrs at room temperature. Taken together, these results strongly suggest that the protein microcrystallization conditions can be optimized to produce high-density microcrystals readily and rapidly by the supersaturation-controlled method, particularly when the single crystallization conditions are known.

Characterization of microcrystals

To monitor the size, density, and quality of the obtained microcrystals, bright-field high-resolution microscopy, intrinsic UV fluorescence imaging, SONICC, X-ray powder diffraction, and TEM were used. The HA microcrystals showed positive ultraviolet two-photon excited fluorescence (UV-TPEF) and second-harmonic generation (SHG) signals. As the size of the HA microcrystals depended on the pH, those obtained at pH 7.0–7.25 were not visible in the SHG images, possibly due to their small sizes, in contrast to those obtained at pH 7.5–7.75 (Fig. 2A and Fig. S5). The lysozyme microcrystals also produced strong UV signals when they were monitored during the crystallization period (Fig. 2B). Microcrystals were clearly obtained at high density at the serial evaporation from 16 min to 20 min, consistent with the results from light microscopy. The ferritin microcrystals did not produce UV or SHG signals, possibly due to high symmetry packing (data not shown). The microcrystals of lysozyme and ferritin were shown to have sizes of 15–30 µm. As lysozyme and ferritin microcrystals are well known and monitored readily, the HA microcrystals obtained at pH 7.0–7.25 were further validated by powder diffraction. They produced powder diffraction rings to 6 Å (Fig. S6). Larger numbers of microcrystals produced higher resolution diffraction rings. Similar results were also reported in a recent powder diffraction study of lysozyme microcrystals18. Further, XFEL experiments were conducted to examine the diffraction quality of the microcrystals obtained from supersaturation-controlled microcrystallization. Representative images of HA and lysozyme microcrystals were found to diffract up to resolutions of 3.5 Å and 1.25 Å, respectively (Fig. S7). The poor quality of the HA diffraction data (low resolution limit) was mainly due to the sensitivity of HA microcrystals to physical mixing with agarose prior to loading into the LCP injector. Dissolved lysozyme microcrystals showed enzyme activity at pH 5.5, which was used for pH-dependent conformational change studies, and the refined structure also showed the tertiary structure very similar to that determined by synchrotron X-ray diffraction (Fig. S8).

Visualization and analysis tools for microcrystals

As shown in Fig. 2 and Figs S4–S5, the size, quality, and crystalline order of microcrystals can be monitored using bright-field high-resolution microscopy, UV fluorescence imaging, SONICC, and powder diffraction. Because the equipment for these measurements is not usually available in many ordinary laboratories, there is a genuine need to develop visualization tools to estimate the crystal size and density distributions. With progress in supersaturation-controlled microcrystallization, we developed a visualization tool to process the drop images by light microscopy; the original drop image had a spatially varying nonuniform bias near the boundary, from which a bias map was created by removing microcrystals and aggregates of proteins (Fig. 3A, top left and middle panels, respectively). The image size was 2592 \(\times \) 1944 pixels in this case, and the image background was subtracted (Fig. 3A, top right). The image then showed a strong position-dependent bias in the drop region, which was also removed and filled with a background consisting of the average of the edges of the bias-corrected image (Fig. 3A, bottom left). The proposed method (the localized fuzzy c-mean clustering algorithm) was applied to segment the drop, generating approximately 20 divisions, each consisting of a small square region 200 \(\times \) 200 pixels in size (Fig. 3A, bottom middle). These segmented images were used to analyze the number of microcrystals (Fig. 3A, bottom right). The numbers of microcrystals counted by the proposed method in six randomly selected regions were found to be very close to those counted manually (Fig. 3B, upper panel). For instance, the mean numbers of microcrystals counted manually in regions 1 to 6 in one image were 46.8, 167.4, 188.9, 16.2, 63.2, and 15.3 with standard deviations of 5.3, 12.3, 16.8, 1.8, 11.0, and 2.5 and those obtained by the proposed method, which was observer-independent and counted them automatically, were 39.0, 170.0, 189.0, 21.0, 79.0, and 16.0, respectively. For validation, three experts were selected to manually count the microcrystals four times, and the results were compared with those of the proposed method (Table S1). In addition, the proposed method can be used to monitor the size of crystals during supersaturation-controlled microcrystallization (Figs S9 and S10). The size distribution as a function of the evaporation time delay could reveal heterogeneous or homogeneous crystal sizes in different crystallization conditions of proteins, which is critical for optimization of microcrystal growth.

Figure 2 Characterization of microcrystals. (A) HA crystals detected by light microscopy, UV-TPEF, and SHG (left to right). Scale bars: 50 μm. (B) Lysozyme microcrystals detected by UV-TPEF and light microscopy in the crystallization buffer, 0.1 M sodium acetate (pH 4.8), 18% NaCl, and 6% PEG 400. Supersaturation-controlled microcrystallization was examined by using evaporation times of 0 to 22 min in 2 min delay steps. Full size image

Figure 3 Images of protein microcrystallization drops. (A) Microcrystallization images processed by the proposed segmentation method: original drop image obtained by an ordinary light microscope, image bias map, and image after removal of the background (upper panel, left to right). The image after removal of the image bias and filling of the image background with the average of the edges of the bias corrected image, image after application of the localized fuzzy c-mean clustering algorithm, and segmented images were used to analyze the number of microcrystals (lower panel, left to right). The original image size was 2592 \(\times \) 1944 pixels, and the localized fuzzy c-mean algorithm was applied to a small region of 200 \(\times \) 200 pixels. (B) Comparison of manual and proposed microcrystal counting methods for selected regions of an image (upper panel): selected regions 1–6 of the original image (top row), manually marked microcrystals in each region (middle row), and microcrystals in each region marked by the proposed method (bottom row). The number of microcrystals counted manually was plotted against that of microcrystals counted by the proposed method (lower panel). The microcrystals were counted by three experts four times and by the proposed method three times. The data represent the means ± SD. (C) Original high-resolution (left) and ordinary light (right) microscopy images (upper panel) and the histogram analysis results of the high-resolution (left) and ordinary light (right) microscopy images (lower panel). Full size image

To quantitatively evaluate the performance of the proposed counting method, first, the manual microcrystal counts were plotted against those obtained by the proposed method (Fig. 3B, lower panel). The slope of the line is 0.9964, and the correlation coefficient between the results of the manual and proposed methods was 0.9866. Second, we compared the proposed results with those obtained using high-resolution microscopy images (Fig. 3C). The nonuniformity was more pronounced in the high-resolution microscopy image than in the ordinary light microscopy. Nevertheless, histogram analysis showed that the mean sizes of microcrystals in the high- and low-resolution microscopy images were 22.5 and 25.6 μm with standard deviations of 0.8 and 2.5 μm, respectively, which were very similar. Particularly, the number of microcrystals in the high-resolution and ordinary light microscopy images were 4,086 and 3,556, respectively. Third, when the number of microcrystals was quantified as a function of the evaporation period at the onset of microcrystallization, the microcrystal counts by the manual and proposed methods showed consistent results, with tendency to increase at higher protein concentrations of lysozyme (Fig. 4A). As the evaporation period increased, the microcrystal densities of lysozyme and ferritin increased, albeit relatively slower increase in ferritin than lysozyme (Fig. S11). Notably, the proposed method could show that as the evaporation period increased further, the density of microcrystals started to decrease, when proteins are likely to form amorphous aggregates. The results suggest that the proposed method can distinguish between amorphous aggregates and crystalline protein samples, which is not easy for the manual task. Taken together, our proposed visualization and analysis methods provided an observer-independent tool to analyze the number and size of microcrystals automatically and accurately using light microscopy images. The proposed segmentation method was implemented using an Intel Xeon CPU E5-2690 v2 (3 GHz) with 128 GB of memory.