Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

Hello and welcome to the Membrane Structural and Functional Biology Group. My name is Martin Caffery. This is another in a series of JO of articles on crystallizing membrane proteins for structured determination using the in meso or lipic cubic phase method.

In the first article, Jo of 1 7 1 2, we demonstrated how the protein laden cubic phase is prepared and how the viscous and sticky misa phase is dispensed manually into glass sandwich crystallization plates for crystallization trials. This video article will describe the method of harvesting and cryo cooling crystals grown by the in meso method. The materials and equipment needed to harvest and cryo cool crystals grown in meso are shown here.

They include a laboratory notebook in which to record information on crystals and harvesting details for future reference safe goggles, glass and metal waste containers, the glass sandwich crystallization plate from which crystals will be harvested. Well containing crystals are marked legibly, a glass cutting tool for opening crystallization wells tissue and a water bottle curved tweezers precipitant screen solution, A micropipet and tips cryo loops for harvesting crystals, a magnetic wand, a pok for storing cryo cooled loops containing crystals. A foam doer filled with liquid nitrogen for cryo cooling, the crystals tongs, a transport or storage doer and a harvesting microscope.

In preparation for harvesting, the foam doer should be filled with liquid nitrogen and placed beside the microscope where harvesting is to take place ahead of time. The storage puck with its open end up should be submerged in the liquid nitrogen in the doer and allowed to cool a cryo loop of a size that matches the crystal to be harvested should be secured on a micro mount on the magnetic wand with all of the materials and equipment in place. Our next task is to identify plates and wells that contain crystals.

We can do this in one of two ways. The simplest and most direct method is to inspect wells by hand using a microscope with normal and with polarized light. The second me method makes use of an imager where plates are screened automatically with normal and polarized light.

Digital images are recorded by the imager and are evaluated on a computer monitor. Wells with crystals for harvesting should be labeled clearly. Comments on the size, quality, and location of the crystals in the misage should be recorded in the notebook or on a computer.

This will help at the harvesting stage. Here, a plate containing crystals for harvesting is removed from the imager opening the well the plates in which crystals grow by the end meso method are glass sandwich plates of the type shown here. In order to access the mease and the crystals they're in, it is necessary to open the well.

This is done by using a glass cutting tool to cut the upper cover glass that seals the well, which can then be removed. There are several approaches for doing this. The one to use is dictated by the type of mease in which the crystal is found growing.

This can be the very viscous and sticky cubic phase, or it's more fluid variant referred to as the sponge phase. In this video article, we show how to open wells and to harvest crystals from both of these hosting materials. In this video sequence, we demonstrate two ways of scoring the cover glass on a well containing the viscous cubic phase.

The scoring and cutting is shown in detail by viewing it with a light microscope. We begin by examining a single crystallization well and identifying its different parts. The first red arrow points to the bolus of mease that contains crystals.

Incidentally, crystals are not visible in this particular sequence. The second arrow points to the precipitant solution that surrounds the mease. The third arrow points to the perimeter of the well.

The fourth arrow points to the double stick spacer upon which the cover glass sits using the glass cutter. The cover glass is scored lightly with two concentric circles situated just outside the perimeter of the well. The glass cutter is then used to break up the glass in the space between the two scored circles.

For the purpose of releasing the inner cover glass, this generates lots of glass shards and dust, which can be cleared away with a moist and paper towel. The freed cover glass rests on the spacer along its circular edge. The cover glass is removed by gripping it with a fine tipped tweezers and tilting it away from and off of the well.

In this case, the cubic phase remains stuck and in place at the base of the well. Some of the precipitant is removed with the cover glass, but a sizable fraction of precipitant remains in place around the mease bolus. Zooming in, we get a clearer view of the cubic mease, which is now ready for use in crystal harvesting.

In the next sequence, we show a second way to open a crystallization well containing the viscous cubic phase. In this case, additional straight score and cup lines are made in the cover glass to one side of the well that extends across the well itself. This enables easier tweezers access to and removal of the cover glass.

In this particular demonstration, the cover glass cracks and in freeing the cover glass from the sticky space or surface, the cover glass itself moves and the precipitant separates from the cubic phase. When the cover glass is lifted, some of the precipitant goes with it. The insert shows a zoomed out view of the process.

Now we are left with an exposed bolus of mease without any surrounding precipitant to prevent the mease from drying out and undergoing a phase change, which may damage the crystals. Fresh precipitant is added on top of the bolus using a micro prepared. The bolus is now ready to be used in crystal harvesting.

The sponge phase is a little less forgiving to work with because of its ability to flow. If that flow results in the sponge phase, contacting the perimeter of the well popularity will draw it out and the crystals will be lost. An example of this happening is shown in the next video clip.

It begins with a zooming in on the sponge phase and then switching back and forth between normal and crossed polarized light. The crystals which can be spotted with some difficulty in normal light are clearly visible between crossed polarizers as bright flex on a black background. In preparation for opening the well, the cover glass is scored and cut as described above.

In the process however, the cover glass cracks where it should not. Some manipulations are performed with a view to con continuing the process of opening the well, but the precipitant shifts in the direction of the crack and eventually it comes in contact with the spacer, and as luck would have it with the precipitant goes some of the sponge phase, and of course its cargo. Of course, crystals in this particular sequence, the polarizers on the microscope are not completely crossed, and the crystals can be seen as bright objects at the same time that the well and its contents remain visible.

In the next clip, we show how the problem is solved by first removing excess precipitant. We begin by zooming in on the sponge phase and identify a crystal, both a normal light and between crossed polarizers, a segment of the cover glasses gored and cut, and then removed carefully from over. The well Incompletely polarized light is used once again to keep an eye on the crystal, which lights up nicely.

A piece of dry tissue paper is introduced through the opening in the cover glass and into the well until it just touches the precipitant solution. The solution is wicked away carefully until it is almost all gone and the sponge phase begins to move in the direction of the wick. Removing the tissue causes the remaining precipitant and the sponge phase with the crystals still in place to retract under the cover glass.

A slow motion repeat of this last sequence is shown here for clarity. The rest of the cover glass is gored and cut. As described earlier, the cover glass is now lifted off with the tweezers.

The insert shows a zoomed out view of the process. In this particular case, the sponge phase splits some remains in the well and some sticks to the cover glass. It turns out that the crystal is in the bolus on the cover glass with the cross polarizers.

The crystal is clearly visible in the sponge phase, but because there is very little precipitant present, it begins to undergo a phase transition likely due to drying out. This can be seen as a ring of bio fringes that migrates towards the center of the bolus. By adding precipitant to the bolus, the process can be stopped and reversed in preparation for the next step, which is to harvest crystals.

Having opened the well and exposed the hosting Misa phase, the next step in the process is to harvest crystals. In the following clips, we show examples of harvesting from the cubic phase and from the sponge phase. The mea phase in the freshly opened well is examined under the microscope to locate suitable crystals.

Going back and forth between normal and polarized light can greatly assist this process. Precipitant solutions should be at hand and available to add to the bolus to slow or to stop any drying. A mounted cryo loop is then used to probe the freshly exposed visa phase for crystals to fish out crystals, and then to plunge them in the cryo loop into liquid nitrogen in the dur immediately upon harvesting.

In what follows, we show harvesting first from the viscous and sticky cubic phase in this harvesting sequence up to four Biore fringe, and crystals can be seen in the cubic phase bolus using polarized light. The cryo loop comes in from the right scoops up the crystal with as little adhering mease as possible is immediately cryo cooled and the loop is placed in the storage puck under liquid nitrogen. The harvesting process is repeated three times in this clip.

Each time the polarizer is used to check on the location of the crystal. Since it is not possible to look for the crystal in the cryo loop after harvesting, it is a good idea to check the mesa phase from which the crystal was harvested to verify that it is no longer there suggesting that it was harvested successfully. In the next sequence, harvesting from the more fluid sponge phase is shown, as with the cubic phase, as little of the sponge phase should be harvested and remain with the crystal.

The more there is in the cryo loop surrounding the crystal, the more difficult it will be to locate the crystal by eye. For diffraction data collection and the more background scatter, it will contribute during diffraction measurement. In the next sequence, we show the cryo cooling process, which should happen immediately.

The crystal is harvested with as little time as possible elapsing between the actual harvesting event and plunging it into liquid nitrogen. Having plunged the mounted loop into liquid nitrogen, it is positioned in one of the holding slots of the storage puck in the door, position number one in this instance, and released from the magnetic wand. At this point in time, the location and details of the harvested and cryo cooled crystals should be recorded in the notebook and or the computer.

When the puck in the foam drawer is full, or you have finished harvesting for the day, transfer the puck into a shelved puck holder in a storage or a transport J filled with liquid nitrogen. The next two images are of cryo cooled crystals in cryo loops that have been harvested. Following the above protocol here, the crystals can be seen clearly within the meza phase in the loop.

In this case, however, the crystal is not visible and would have to be located by diffraction rasing. The next step in the overall process of structured determination using macromolecular crystallography is to collect a fraction data on crystals harvested as demonstrated in this article. This is usually done using synchrotron X radiation and is the focus of a separate j ove article in this series.