Detection of microcrystals for CryoEM

Abstract

Here we present a strategy to identify microcrystals from initial protein crystallization screen experiments and to optimize diffraction quality of those crystals using negative stain transmission electron microscopy (TEM) as a guiding technique. The use of negative stain TEM allowed visualization along the process and thus enabled optimization of crystal diffraction by monitoring the lattice quality of crystallization conditions. Nano crystals bearing perfect lattices were seeded and can be used for MicroED as well as growing larger crystals for X-ray and free electron laser (FEL) data collection.

1. Introduction

Crystallization of protein targets remains the most significant challenge in the process of structure determination by macromolecular crystallography. When setting up crystallization trays experimenters can obtain three different negative outcomes: 1) conditions with amorphous precipitate, 2) few or no conditions with apparent crystals or 3) crystals that do not diffract or diffract to low resolution. To tackle these problems most recent efforts have focused mainly on improving sample “crystallizability” by implementing techniques such as alanine scanning mutagenesis [1–3] or the use of chimeric proteins to promote/improve crystal packing [4–8], which has led to structures of important targets. However, less effort has been applied towards the discovery, evaluation and optimization of crystals and nano-crystals (NCs). Here we present the use of Transmission electron microscopy (TEM) as an easy, reproducible techniques to guide the optimization of protein crystallization [9–12]. We will describe reproducible protocols to use TEM for visualizing lattices from microcrystals as well as fragmented larger crystals and to study details of the crystallization process. Our experiments have shown that for all protein crystals tested, TEM images reveal details of the crystal lattices that prove to be accurate qualitative indicators of the potential diffraction of the crystal. In general, the detection by negative stain TEM methods of well-ordered lattices with third order Bragg spots was a good predictor of X-ray diffraction, while samples with disorganized lattices yielded no diffraction [12]. Additionally, we can show that TEM can play an important role to identify crystal pathologies that contribute to poor X-ray diffraction data [11]. Among them are: 1) crystal lattice defects; 2) anisotropic diffraction; and 3) crystal “polluting” by heavy protein aggregates and micro-crystal nuclei. Detection of lattice defects in some crystals could point to the presence of samples containing protein contaminants, aggregates or partially proteolyzed protein as well as discrepancies in the stoichiometry of the sample. Negative stain TEM analysis was able to identify crystals that possess anisotropic diffraction. In these cases performing steps to improve crystal contacts, such as altering or adding reagents to the crystallization conditions or modification of the protein itself, may be advisable. It is important to note that this information cannot be observed with other techniques before performing X-ray diffraction experiments. The use of TEM has also enabled qualitative estimation of crystal solvent content and allowed the study of lattice dehydration on crystal diffraction [11]. This application was particularly noteworthy since 1) crystal dehydration protocols have proven very useful in the improvement of X-ray diffraction [13–15], and 2) negative staining with uranyl acetate provides very high contrast between solvent channels and biological macromolecules. Overall, information obtained by TEM experiments could provide critical advice to the experimenter about which crystallization conditions to be pursued and would also allow monitoring of crystal optimization protocols.

2. Materials

2.1. Microscopy

1. Brightfield & light microscope (e.g. Jansi, Molecular Dimensions)

2. Ultra violet (UV) microscope (e.g. Jansi, Molecular Dimensions)

3. Electron microscope (e.g. FEI TECNAI T12 operating at 120 kV with a single-tilt specimen holder and 2k × 2k Gatan UltraScan 1000 CCD camera)

2.2. Negative Staining Components

1. 2 % (w/v) uranyl acetate in 15 ml plastic Falcon tube, covered in aluminum foil

2. 0.22 μm sterile filter (e.g. Millex-MP)

3. Carbon coated CF400-CU grids (e.g. Electron Microscopy Sciences)

4. Glow discharge unit (e.g. EmiTech KX 100)

5. Filter paper (e.g. P2 Fisherbrand)

6. Optional: Vortex Mixer (e.g. Fisher Scientific, Cat # 02215365)

7. Optional: glass beads 0.5 mm or 1 mm (Research Products International)

3. Methods

1. Set up hanging drop vapor diffusion crystallization screens of your target protein (see Note 1).

2. Identify conditions with granular aggregate and micro- or nano-crystals by screening the crystallization trays under a Brightfield (BF) microscope to select conditions for the negative stain TEM analysis (see Note 2 and Fig. 1).

3. Confirm the presence of protein in the conditions identified with BF microscopy by taking UV microscope images (see Note 3 and Fig. 1).

4. Prepare a 2 % (w/v) uranyl acetate solution in dH₂O, dissolving the UA by rocking it in a 15 ml Falcon tube covered with aluminum foil at RT for at least half an hour before filtering with a 0.22 μm filter immediately before use (see Note 4).

5. Negatively glow discharge the TEM grids (e.g. CF400-CU, Electron Microscopy Sciences) for 1 min at 25 mV and 0.2 mbar (EmiTech KX 100) with the carbon side up, not more than 20 minutes before loading your sample (see Note 5).

6. Using a razor blade, remove the coverslip of a well, which bears UV-positive granular aggregates from the crystallization tray. Place it upside down on the transparent cover of the crystallization tray in the path of the microscope. Then carefully add about 2 μl of mother liquor (or stabilizing solution; see Note 6 & 7) to the crystal drop and gently mix by pipetting up and down several times (see Note 6).

7. Optional: Fragmentation of larger crystals, i.e. bigger than about 2 μm for the smallest side. First add approximately 10 μl of glass beads (Research Products International) to a 1.5 ml Eppendorf tube and wash them with 500 μl of 20% (v/v) ethanol, followed by washing with 500 μl dH₂O. Then the beads are equilibrated with about 30 μl of stabilizing buffer (e.g. reservoir buffer). Pipette the collected crystals into the Eppendorf tube with the glass beads. The crystals are fragmented by vortexing (about 2 seconds to 2 min) until UV microscopy images reveal a homogeneous slurry of high density crystal fragments with edge lengths of around 1–5 μm (see Note 7 and Fig. 2).

8. Pipette approximately 2 μl of the granular mix or the fragmented crystals onto the carbon film side of the electron microscopy grid and incubate for 1 min. Then remove the excess liquid by blotting the grid with P2 filter paper (Fisherbrand) from the side (see Note 7 and Fig. 3).

9. Immediately after blotting place the grid (carbon side down) on top of a drop of 200 μl of a 2 % (w/v) uranyl acetate solution for 40 seconds. Transfer the grid to a second drop of 200 μl of a 2 % (w/v) uranyl acetate solution and stain for another 40 seconds without blotting in between the two staining steps. After staining, the grid is blotted from the back with filter paper in order to remove the staining solution and dried on air for at least 20 minutes prior to further use (see Note 8).

10. Inspect the nature of the granular aggregates and lattice quality of the (fragmented) microcrystals by collecting TEM images (see Note 9 and Fig. 4).

Fig. 1:

The “shiny” effect of the granular aggregates under the BF Microscope may not be visible in regular settings (A) but when the polarization and contrast are carefully adjusted the aggregates are clearly identified (B). UV image of the “shiny” sample (C). The scale bar in (A) and (B) represents 50 μm and in (C) 30 μm.

Fig. 2:

Fragmentation of microcrystals for negative stain TEM analysis. (A) BF image of non-fragmented crystals, (B) BF image of crystals after vortexing with 0.5 mm beads, (C) UV image of crystals after vortexing with 0.5 mm beads, (D) neg. stain TEM image of crystals after vortexing with 0.5 mm beads and (E) negative stain TEM image of crystals after vortexing with 1.0 mm beads. (F) Comparison of 5 mm Teflon ball (white, left) and glass beads of 1 mm (center) or 0.5 mm (right) used for microcrystals fragmentation. Panels A-C, D reproduced from Lin et al. (2019) [16] with permission from Elsevier. Panels D, E, reproduced from Stevenson et al. (2016) [12] with permission of the International Union of Crystallography.

Fig. 3:

Schematic representation of the blotting process.

Fig. 4:

Negative stain images and corresponding FFT calculations of (A) and (B) amorphous granular aggregate, (C) crystal without lattice, (D) crystal with disordered lattice, (E) and (F) anisotropic crystals, (G) well-ordered crystals and (H) fragmented well-ordered crystal.

4. Notes

1. The final volume of the crystallization drop should be around 1 μl, therefore about 1.0 μl protein stock solution and 1.0 μl reservoir buffer should be used to set up each initial crystallization drop due to evaporation process during the crystallization experiment. The exact values and ratio between protein and reservoir solution will depend on the specific “crystallizability” of each protein of interest.

2. The initial screening of the crystallization tray should identify conditions displaying a significant amount of “shiny” aggregate (see Fig. 1 for examples). This screening is best performed at magnifications of 30 – 100 X. In order to obtain the best possible image quality and differentiate between shiny and non-shiny aggregate the experimenter might need to change the polarization and contrast settings of the microscope. It is advised to especially look for any type of shape (e.g. needle like or other symmetric structures), which are likely indicators for the presence of nano-crystals.

3. Crystal drops containing granular aggregates under BF microscopy are selected for further evaluation using ultra violet (UV) microscopy (Jansi, Molecular Dimensions). UV positive granular aggregates can be easily discerned with the use of a 10 X objective (see Fig. 1). As with the BF microscopy, it is essential to adjust the focus planes to obtain the most contrasting UV-images possible. For very fine granular aggregates use of a 40 X objective might be required. False UV-positive granular aggregates can be observed in crystallization conditions that include calcium salts.

4. The 2 % (w/v) uranyl acetate solution can be used for up to 1 month but should be filtered with a 0.22 μm filter on the day of grid preparation to obtain best crystal staining results.

5. We routinely use Carbon coated grids with a 400-mesh. These grids result in less broken areas compared to larger mesh grids (e.g. 300-mesh, 200-mesh) when blotting by hand from the back with the P2 filter paper.

6. When preparing the sample to load onto the grid be sure to visually check that the microcrystals are soaked into the pipette. In some cases the sample might stick to the cover slid. In those circumstances use the pipette tip or a crystallization tool to carefully perform slow stirring motions to detach the microcrystals or aggregate from the cover slid. If the reservoir buffer has a very high viscosity (e.g. a PEG-10000 concentration above 10 % (w/v) or other similar precipitants), it is recommended to use a stabilizing solution containing methyl pentanediol (MPD) instead of the highly viscous reservoir solution (see also Note 7).

7. The size of glass beads (0.5 mm or 1.0 mm diameter) should be selected depending on the stability of the protein crystal; smaller beads result in harsher crystal fragmentation (see Fig. 2) [11]. For fragile crystals (e.g. needles and thin plates) we recommend using 1.0 mm beads; for sturdier and “chunkier” crystals, 0.5 mm beads are generally needed. Similarly the vortexing time has to be chosen depending on the crystal sturdiness. In case of temperature sensitive protein crystals the fragmentation process has been carried out at 4°C while cooling the sample on ice in between vortex intervals to prevent protein denaturation. For scarce material containing fewer granular aggregates, it is recommended to use stainless steel beads that can be removed from the tube using a strong magnet. Concentration of the slurry can be achieved by removing the magnetic beads, followed by low speed centrifugation (1500 rpm) to pellet down crystal fragments followed by removal of excess stabilizing solution. Crystallization conditions that have highly viscous solutions (for example 25% polyethylene glycol 8000 (peg8K)) are difficult to handle or visualize on EM grids. For such conditions it is convenient to find a stabilizing solution with low viscosity where aggregates do not dissolve. A solution containing 25% peg8K could be exchanged with a stabilizing solution containing 30–40% methyl pentanediol (MPD) during several rounds of concentrating, removing excess liquid and slowly adding the new stabilizing solution.

8. Hold the grid at a perpendicular angle relative to the filter paper and slowly move it along the filter paper with only its edge touching the filter paper. Make sure to visually check the filter paper during to blotting process for signs of the blotted liquid (see Fig. 3). When instead blotting from the front, the crystals might be inadvertently removed from the grid as well, while blotting from the back might not be possible with a non-holey grid due to the high buffer viscosity. Generally the more viscous the reservoir solution, the slower the blotting process will be and it will take about 2 to 10 s to fully blot away the excess liquid.

9. Within a couple of seconds after blotting, place the blotted grid sample side down on top of the first drop of uranyl acetate solution in order to keep the sample from completely drying out before the staining. It is important to fully blot away excess liquid but not to let the crystals crack due to dehydration when trying to optimize staining results, therefore a thorough blotting and fast transfer are essential.

10. For the TEM screening we have predominantly used a FEI TECNAI T12 electron microscope operating at 120 kV with a single-tilt specimen holder. Images are acquired with a 2k × 2k Gatan UltraScan 1000 CCD camera, typically at magnifications between 11000 and 52000 x. Fast Fourier Transform (FFT) calculations of the lattices are used to determine the crystal quality. The experimenter should be prepared to spend up to 2 hours looking for crystal lattices on each grid. When looking for crystals it is important to focus on objects with sharp symmetric edges, which show a dark fringe and a brighter color in its central parts. Some crystals are radiation sensitive and the lattice might “melt” during the focus adjustment. In these cases it might be necessary to use the “Low Dose” mode of the microscope for imaging purposes.