Crystallization of the actin nuclear export complex

We used the purified actin export complex (Figure 3-13) for crystallization trials. The sample was concentrated to 15 mg/mL, with good polydispersity values (12%). As a control we have used Xpo6RanGTP complex at 15 mg/mL concentration, purified in the same buffer. Using the robotic system we have screened a broad range of conditions using commercially available sparse matrix screens (6.1.6). For each screen we have prepared plates for two different temperatures (4ºC and 20ºC). We identified several conditions, where promising crystalline growth was observed. Figure 3-29 shows images of some of the initial hits. Interestingly, for the actin export complex all hits were from plates at 20ºC.

in their counterparts at 4ºC, no crystals have been observed. For the conditions termed as initial hits, Xpo6RanGTP controls did not show any crystalline growth.

Figure 3-29 Initial hits for actin nuclear export complex crystallization

The crystallization conditions and temperature are indicated. A) needle crystals appear at day one and continue to grow until day 30. The crystals are indicated with the red boxes. B) numerous small needles appear on day 3, but do not grow further. C) Dark precipitates appear on day one and grow outwards as needles.

It was an interesting observation that all the initial hits were conditions that did not contain any salt. Considering the ionic sensitivity of the actin export complex, this was a positive indication. Two of the initial hits were especially promising. In one condition (10% PEG 6000, 10 mM MgCl2) a small needle crystal appeared in day 1, and continued growing as a bunch of needles until day 30. In another very minimalistic condition (15% PEG 1500), numerous small needles appeared at day 3, but they did not grow further. Another possible hit is an alcohol-based condition, which also contains a high concentration of heavy metal salt (40% MPD, 200mM CdCl2). In this condition, dark precipitates appeared on day 1, and continued growing radially in a way that resembles crystalline growth. It is possible, however, that those are crystals of the heavy metal salt.

We prepared refinement screens for conditions based on PEG 6000 and PEG 1500. For the first hit, we kept the 10 mM MgCl2 fixed, whereas we varied PEG 6000 concentration from 0-25% over 96 wells of the screening plate. For the other refinement screen we scanned PEG 1500 concentrations of 0-20%. Unfortunately, we could not identify a better crystallization condition, nor we could reproduce the initial observations.

Nucleation is a rare event, and it is possible that it can not be reproduced. But nucleation step could be by-passed with a technique called seeding. In seeding method, sub-optimal crystals are crushed to form microcrystals, which act as nuclei for the next round of crystallization. We wanted to use the needles obtained in the initial screen (10% PEG 6000, 10 mM MgCl2) as seeds for our refinements. The needle crystals were taken out from the drop and a seed stock solution was prepared (see 6.2.13.4). While preparing new reservoir solution for the manual seeding experiments, we realized that the pH of the manually prepared PEG 6000 solution was very acidic (pH 4.3). Although the commercial screen did not have any buffer in the condition, they might have pH adjusted the PEG 6000 solution, which was not explicitly described in the manuals. Considering that the pH might have been the reason why we did not see any crystals in our refinements, we added 100 mM Tris pH 7.5 to the solution. The pH was then manually confirmed. Using the seed stock dilutions and the newly prepared reservoir solution (10% PEG 6000, 10 mM MgCl2, 100 mM Tris pH 7.5) we prepared our seeding drops. Serial dilutions of the seed stock with reservoir were mixed with protein (15 mg/mL) in 1:1, 1:2 ratios and sealed in small chambers with reservoir at room temperature. Bunches of needles appeared in almost all dilutions of the seed stock within 1-2 days. On the third day, needles appeared also in the control drops without seed stock, but only reservoir. A characteristic bowtie shape was

observed: needles from one nucleus usually grew towards both directions radially. Figure 3-30 shows the crystals obtained in manual drops.

Figure 3-30 Reproduction of the actin nuclear export complex crystals

The needles from the initial hit (10% PEG 6000, 10 mM MgCl2) were used to prepare seeds, which were used in the manual crystallization drops with the following crystallization solution: 100 mM Tris pH 7.5, 10% PEG 6000, 10 mM MgCl2. In drops with various seed concentrations (A, C, D), as well as in drops without seeds (B), needle shaped crystals could be reproduced in several days at room temperature.

The crystals were reproducible with and without seeding, and there were no visible improvements in the crystal shape due to seeding. We therefore wanted to know what the crystals were made of: was it the tetrameric export complex or any of the sub-complexes?

In order to investigate the content of the crystals, we removed them from the crystallization drop with a nylon loop, and washed them in a fresh drop of reservoir in order to prevent carry over of soluble protein from the solution. Finally the crystals were placed in 5 µL of fresh reservoir in a tube. Two drops were harvested as described and several crystals from each drop were combined and analyzed by SDS PAGE. As a control, 1µL of protein sample was loaded to the gel. Figure 3-31 shows the SDS PAGE analysis of the crystals from two separate drops. As can be seen, all four proteins were present in both crystals.

We have managed to crystallize the full actin nuclear export complex from human, and that in a very minimalistic condition: 10% PEG 6000, 10 mM MgCl2, 100 mM Tris pH 7.5. For sure the crystals are still not optimal for diffraction analysis and require further optimizations. But it was very important to show that the full-length human actin nuclear export complex can be crystallized, despite its ionic sensitivity, the low affinity between its sub-complexes and even the possible inhomogeneity in the crystallization sample. In the end the crystallization process itself has been the ultimate purification in a mixed population between the full complex and sub-complexes.

Figure 3-31 SDS PAGE analysis of the actin nuclear export complex crystals

The needle crystals from the manual drops were fished, washed in fresh reservoir and resuspended in reservoir solution with SDS buffer. The content of the crystals were analyzed with SDS-PAGE, and a microliter of the protein sample was loaded as a reference. Crystals from two different drops are shown here to contain all four proteins of the export complex.

The strange running behavior of the proteins is probably a result of the PEG 6000 in the loaded sample.

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