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A macromolecular crystal usually represents anextensive commitment of time and energy, from thedirect cost of material production to the intangiblecost of learning how to grow a crystal. With so manyresources devoted to crystal production and often somuch dependent on the outcome of data collection, itis small wonder that most crystallographers developan emotional attachment to the outcomes of theircrystallization experiments. Equally unsurprising isthe frustration generated when a flash-cooled crystalproves unacceptable for data collection, eitherbecause of icing or excessive mosaicity. Low tem-perature data collection has become the acceptedstandard within the crystallographic community, butit is understandable why some would be reticent toaccept macromolecular cryocrystallography withoutbetter guarantees of successful outcomes whencrystals are flash-cooled.

We have found a way of restoring crystals thathave been adversely affected by the flash-coolingprocess, most significantly those with levels ofmosaicity that prevent successful data collection.While not the panacea for all crystals harmed bycryogenic preparation, the technique is sufficientlysuccessful to quiet the trepidations of most crystallo-graphers for flash-cooling. The method, calledmacromolecular crystal annealing (MCA), has evenresulted in improved resolution in some crystals. Inthe following sections we will describe how toconduct MCA, when it might be useful to apply theprotocol and then detail some of the successes ofMCA. Our research on MCA continues, so futureresearch directions will be described, as well as ourrequest that you let us know of your successes andfailures using MCA.

What is Macromolecular Crystal Annealing?

The solvent in a flash-cooled crystal so rapidlyreaches cold-stream temperature that it becomesvitrified: locked in the disordered liquid phase [2]. Bypreventing ordering of the solidified solvent, theintegrity of the unit cell and crystal structure ismaintained. However, stresses from this rapid cooling process can manifest themselves in the crystal,usually as increased mosaicity. These stresses caninclude deformation of mosaic blocks due to changedsolvent interactions, or multiple patches of perfectcrystal blocks, each locked in different unit celldimensions [11]. On rewarming, the vitrified solventin a crystal can undergo a phase change into ice, aprocess assumed to further exacerbate the latticedisruption within the crystal. It is easy to understandwhy the general thinking was that a rewarmed crystalwas a lost crystal. However, such perceived wisdomis not always valid.

The first instance of rewarming/annealing in amacromolecular crystal occurred during data collec-tion on a nucleosome core particle crystal [5]. Thesecrystals are sensitive to radiation damage at ambienttemperature, making cryogenic data collectionessential. Flash-cooling is possible using MPD as acryoprotectant but the mosaicity of the crystalgenerally increases by a factor of 2 to 4. During asurvey of cryoprotectants, a crystal was found toexhibit unacceptably high mosaicity and wasremoved from the cold nitrogen gas stream. It wasplaced in a large drop of the cryoprotectant forexamination under a light microscope. The crystalremained intact and appeared more transparent thanbefore flash-cooling. Therefore, it was flash-cooled a

6 The Rigaku Journal

The Rigaku Journal

Vol. 15/ number 2/ 1998




Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831

*Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis ID 46202

Corresponding author: Gerard Bunick, phone 423-576-2685, e-mail address,

second time. Subsequent diffraction images showedthat the quality of the crystal had dramaticallyimproved. The reduction in mosaicity of the crystalwas evident in the decreased width of the lunes and the more sharply formed Bragg reflections in photographs taken over the same rotation angle. After experi-mentation with other crystal systems, we were able todevelop a protocol to take advantage of the improve-ments seen after crystal rewarming which we callmacromolecular crystal annealing.

Macromolecular crystal annealing is the processby which a flash-cooled crystal, displaying unfavor-able diffraction qualities, is brought to roomtemperature and then reflash-cooled to overcomethese deficiencies. We prescribe a standard cryo-crystallographic process: the crystal must be suitablytreated with a cryoprotectant solution prior to flash-cooling. A frame or image of diffraction is collectedand an assessment made of the crystal quality. If thecrystal diffraction is unsatisfactory the crystal isquickly transferred from the cold stream into 300 l of the crystal's cryoprotectant solution. It is allowed toincubate there for 3 minutes (we use an egg timer tomeasure this interval), then remounted on thecryoloop and reflash-cooled in the N2 cold stream.During the incubation the crystal needs to be

completely submerged in the cryoprotectant solution,and the droplet holding the crystal is covered toprevent excess drying or other buffer modificationsduring the annealing process. The length ofincubation has been successfully varied from ourstandard methodology, but overall we feel theincubation period should not be changed unlessshown to be necessary. Figure 1 details our method.

During our development of this technique weexperimented with other possible methods ofannealing. Sauer and Ceska [10] indicated that someprotein crystals could be rewarmed and cooled on theloop without loss of diffraction. We experimentedwith this methodology, which we refer to as annealing on the loop, during our development of MCA. In thisprocedure, the cold stream is diverted from the crystal and it is allowed to warm to ambient temperature.Once the crystal is warmed, i.e., clear when viewedeither through a telemicroscope or video camera, thecold stream diverter is removed and the crystalreflash-cooled. Although a recent paper promulgateda variation this technique [13] we have found that theresults of this in situ crystal annealing to be generallyless satisfactory than the application of MCA. Themass and solvent content of a crystal becomesignificant variables with annealing on the loop, with

Vol. 15 No. 2 1998 7

Fig. 1. How to implement Macromolecular Crystal Annealing in 4 easy steps. 1a. Flash-cooled crystal is removed from thecold stream. 1b. Crystal from cold stream is quickly placed in a 300 l droplet of the cryoprotectant solution. In thisdiagram, a well plate is used to hold the droplet. Both the plate and the glass cover slip should be silanized. 1c. Crystallodged in droplet within well is covered with a glass cover slip to prevent moisture loss, and allowed to incubate for 3minutes. 1d. Crystal is repositioned on cryoloop and reflash-cooled. Crystal is now ready for data collection.

larger size and higher solvent content crystals lesslikely to have a successful outcome than whensubjected to MCA. The amount of mother liquor onthe loop with the crystal is another important factor.The drier the mount, the more likely a successfuloutcome of annealing on the loop, but if the crystal iswicked to remove excess moisture at the time of theinitial flash-cooling, then during the rewarmingprocess we have sometimes noticed the formation ofinorganic crystallites which complicate the diffrac-tion pattern. The MCA methodology has beendeveloped to maintain close to the same osmoticbalance as exists within the crystal during theincubation process, and to our knowledge representsthe gentlest, most conservative method of trying tosave a flash-cooling damaged crystal.

We first discussed the MCA technique at the 1997 American Crystallographic Association meeting inSt. Louis [5]. Judging from the response at that time, it generated a certain amount of interest, and a good deal of healthy skepticism. We subsequently published our methodology and the results we obtained with 3crystal systems [6]. More recently, we presented newfindings and have expanded our experimental data setwith 3 more proteins (Bunick, Rigaku Users Meeting,1998; [7]). Even more gratifying, others have tried our methods and reported on their success, and these areshown in Table 1. The demonstrable success of MCAsuggests to us that basic information on crystalperfection can be gleaned from understanding theprocess.

When to Anneal

Annealing techniques are useful when themosaicity of a flash-cooled crystal causes difficulty in processing the diffraction data. Not all crystals willexhibit increased mosaicity associated with flash-cooling, and will not be improved by annealing.When a crystal exhibits 0.2 mosaicity and diffracts to 1.7 C after flash-cooling, it is probably unnecessary to anneal. Sadly, such crystals are rare. The scope of theproblem is indicated in a study of 19 crystal systems[9]. Only eight of the 19 crystal types used in thisstudy exhibited little or no mosaicity increase afterflash-cooling, while eight of the remaining 11 werefound to exhibit a doubling of rocking curve widths(mosaicity) after flash-cooling. From our experiencemaking MCA a general procedure during datacollection is warranted, because with few exceptions,any crystal can be annealed. The exceptions to general use of the MCA protocol arise when a crystal isunstable in the cryoprotectant solution.

A cryoprotectant solution in which the crystal isstable is the necessary first step for successful MCA.This was brought home to us in a series of experi-ments with Concanavalin A (con A). On initial flash-cooling the c


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