Macromolecular Crystal Annealing - Rigaku · macromolecular crystal annealing. Macromolecular...

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Introduction A macromolecular crystal usually represents an extensive commitment of time and energy, from the direct cost of material production to the intangible cost of learning how to grow a crystal. With so many resources devoted to crystal production and often so much dependent on the outcome of data collection, it is small wonder that most crystallographers develop an emotional attachment to the outcomes of their crystallization experiments. Equally unsurprising is the frustration generated when a flash-cooled crystal proves unacceptable for data collection, either because of icing or excessive mosaicity. Low tem- perature data collection has become the accepted standard within the crystallographic community, but it is understandable why some would be reticent to accept macromolecular cryocrystallography without better guarantees of successful outcomes when crystals are flash-cooled. We have found a way of restoring crystals that have been adversely affected by the flash-cooling process, most significantly those with levels of mosaicity that prevent successful data collection. While not the panacea for all crystals harmed by cryogenic preparation, the technique is sufficiently successful to quiet the trepidations of most crystallo- graphers for flash-cooling. The method, called macromolecular crystal annealing (MCA), has even resulted in improved resolution in some crystals. In the following sections we will describe how to conduct MCA, when it might be useful to apply the protocol and then detail some of the successes of MCA. Our research on MCA continues, so future research directions will be described, as well as our request that you let us know of your successes and failures using MCA. What is Macromolecular Crystal Annealing? The solvent in a flash-cooled crystal so rapidly reaches cold-stream temperature that it becomes vitrified: locked in the disordered liquid phase [2]. By preventing ordering of the solidified solvent, the integrity of the unit cell and crystal structure is maintained. However, stresses from this rapid cooling process can manifest themselves in the crystal, usually as increased mosaicity. These stresses can include deformation of mosaic blocks due to changed solvent interactions, or multiple patches of perfect crystal blocks, each locked in different unit cell dimensions [11]. On rewarming, the vitrified solvent in a crystal can undergo a phase change into ice, a process assumed to further exacerbate the lattice disruption within the crystal. It is easy to understand why the general thinking was that a rewarmed crystal was a lost crystal. However, such perceived wisdom is not always valid. The first instance of rewarming/annealing in a macromolecular crystal occurred during data collec- tion on a nucleosome core particle crystal [5]. These crystals are sensitive to radiation damage at ambient temperature, making cryogenic data collection essential. Flash-cooling is possible using MPD as a cryoprotectant but the mosaicity of the crystal generally increases by a factor of 2 to 4. During a survey of cryoprotectants, a crystal was found to exhibit unacceptably high mosaicity and was removed from the cold nitrogen gas stream. It was placed in a large drop of the cryoprotectant for examination under a light microscope. The crystal remained intact and appeared more transparent than before flash-cooling. Therefore, it was flash-cooled a 6 The Rigaku Journal The Rigaku Journal Vol. 15/ number 2/ 1998 CONTRIBUTED PAPERS MACROMOLECULAR CRYSTAL ANNEALING: TECHNIQUES AND CASE STUDIES GERARD BUNICK, JOEL HARP, DAVID TIMM* AND LEIF HANSON 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, [email protected]

Transcript of Macromolecular Crystal Annealing - Rigaku · macromolecular crystal annealing. Macromolecular...

Page 1: Macromolecular Crystal Annealing - Rigaku · macromolecular crystal annealing. Macromolecular crystal annealing is the process by which a flash-cooled crystal, displaying unfavor-able

Introduction

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

CONTRIBUTED PAPERS

MACROMOLECULAR CRYSTAL ANNEALING:TECHNIQUES AND CASE STUDIES

GERARD BUNICK, JOEL HARP, DAVID TIMM* AND LEIF HANSON

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, [email protected]

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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 µl 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

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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 µl 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.

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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 crystals provide adequate diffraction,however crystals of con A have proven refractory toany annealing technique we have tried. Ourexperiments with a variety of cryoprotectants havealso been largely unsuccessful, our best results being2 minutes of stability in a cryoprotectant solutioncontaining 30% glycerol or 25% MPD in the well

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Molecule Space group Unit cell (C) Crystal growth buffer Cryoprotectant Author

Alcoholdehydrogenase

P21a=55.7 b=100.2 c=69.0 β=104.9°

PEG6000, 0.1m ACES, pH 7.2

PEG200 S. Hurley

Aldehydedehydrogenase

P21a=102 b=177 c=101 β=94.6°

0.1M ACES, pH6.5, 0.1Mguanidine HCL, 2 mMNAD+, 8mM MnCl2

Ethylene glycol S. Hurley

Crotonase P31a=76.5 b=76.5 c=214.6

MPD, potassiumphosphate, pH7.4

MPD S. Hurley

Pyruvatedehydrogenase kinase

P2a=72 b=109 c=73 β=102°

PEG, NaCL Paratone N N. Steussy

Purine operonrepressor

P1a=65.4 b=72.5 c=83.4 α=84.7° β=84.6°γ=67.5°

S. Sinha

Table 1.

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buffer (0.1 M NaNO3, 50 mM Tris-acetate, pH 6.5).When using either of these solutions in the applicationof MCA, the crystals become opaque during the roomtemperature incubation, and show an irreversible lossof diffraction after flash-cooling. Annealing on theloop was also unsuccessful with con A.

Successful ImplementationSperm Whale Myoglobin

Sperm whale myoglobin crystals grown usingammonium sulfate as a precipitant were initially flash-cooled using 30% glycerol in the reservoir solution(0.1 M Phosphate, pH 6.9, 2.8 M Ammonium Sulfate). The crystals (P21) grew as plates with a minimumdimension of 0.1-0.15mm. Myoglobin does notappear to suffer from increased mosaicity after initialflash-cooling. The very low solvent content of themyoglobin crystals, 34% [8], may account for thisease of handling and flash-cooling. Crystals ofmyoglobin can be annealed using MCA or on the loop, but to the limit of resolution for our detector we havebeen unable to detect increases in diffractionresolution using either method from the initial datacollected after flash-cooling.Chicken Egg Lysozyme

Lysozyme and lysozyme bound with N-acetylglucosamine (NAG) crystals (P43212) grown using 0.9 M NaCl as a precipitant were flash-cooled using 30%glycerol in the reservoir solution (50 mM NaOAc, pH4.5, 0.9 M NaCl). The solvent content of lysozymecrystals was calculated as 37%. As with myoglobinthere is little or no increase in mosaicity associatedwith the initial flash-cooling of either lysozyme orlysozyme/NAG crystals. In a comparative studycrystals of lysozyme had a mosaicity of 0.180°,0.449°, and 0.348° for data collected from a crystal at277K, initially flash-cooled and after annealing,respectively. Refinement of data from both a flash-cooled and annealed crystal are essentially identicalbased on the R factor obtained from processing thedata separately and from processing the merged data.

MCA routinely enables us to save crystals for data collection that have become iced during to flash-cooling, even using liquid He as the cryogen. During a series of experiments to determine the utility formacromolecular crystallography of an open flowhelium cryostat developed by Alan Pinkerton's groupat the University of Toledo [4], a single crystal ofmyoglobin accidentally contacted the cold streamdiverter. This resulted in a badly iced crystal that

showed rings in the diffraction pattern. After onecycle of MCA, the crystal was reflash-cooled anduseful diffraction data collected [3]. This experimentwas notable in several respects. Crystals can be flash-cooled and annealed at the lower He temperature(–100K cooler than our N2 cryostat). In ourexperiments, rewarming from such a cold tempera-ture (<33K within this coldstream) does not adversely affect the lattice. Also, this crystal was fairly large(0.8mm x 0.6mm x 0.6mm), suggesting thatannealing is a possibility for nearly any sizemacromolecular crystal used in X-ray diffraction.The advantages of He temperature data collection(increased diffraction due to decreased thermalmotion) will be better understood as He cryostatsbecome more available.Nucleosome Core Particle

The nucleosome core particle is composed ofhistone octamer wrapped with double stranded DNA.The intermolecular contacts comprise a complexassortment of interactions between protein and DNA.The crystals grow as hexagonal rods and regularlyreach dimensions of 0.4 mm x 4 mm. Thecryoprotectant for the initial flash-cooling was 22.5%MPD, 30 mM MnCl2, 30 mM KCl, 20 mM K-cacodylate pH 6.0. The solvent content of nucleo-some crystals is approximately 51%. The crystalsexhibit significant increase in mosaicity on initialflash-cooling. Annealing produced a greater thantwofold decrease in the mosaicity of the crystal from0.825° after flash-cooling to 0.345° after annealing.This value compares to a mosaicity of 0.275° from asimilarly grown crystal measured at 277K. We havefound that for initial flash-cooling, minimization ofthe time between removing the crystals from thecryoprotectant solution and flash-cooling is critical.Annealing on the loop is generally unsuccessful forthe nucleosome core particle. Successful MCA ofnucleosome crystals enabled us to predict that thistechnique could be adapted to other macromolecularcrystals. Figure 2 has images of diffraction before and after MCA for a crystal of the nucleosome coreparticle.Chicken Histone Octamer

Chicken histone octamer (CHO) crystals grow ashexagonal bipyramids and are routinely 0.5 mm orgreater in diameter. Cryoprotection is achieved bydialysis into 15% glycerol in crystal storage buffer(71% saturated ammonium sulfate, 20 mM pyro-phosphate, 5 mM EDTA, 10 mM β-mercapto-

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ethanol). The solvent content of the chicken histoneoctamer is 65%. In our initial paper, we described acomparison between two halves of the same histoneoctamer crystal, one half flash-cooled, the otherprocessed with the MCA protocol. The flash-cooledportion of the crystal had a mosaicity of 0.338°, theannealed portion was 0.217°, a reduction of 36%. Theannealed value compares with 0.175°0 mosaicitymeasured at 277K from a similar crystal.

Histone octamer crystals can undergo MCAwithout problems but are constantly damaged by theannealing on the loop protocol. During the annealingon the loop protocol crystals were observed to clearduring the first warming period, but became cloudyupon recooling. Diffraction after recooling wassignificantly degraded. In one example, data werecollected from a 0.4mm x 0.4mm x 1.0mm crystal.The initial diffraction was adequate but, neverthelesswas improved by MCA. MCA can be consistentlyapplied with success to these crystals: this crystaldisplayed good diffraction after 3 rounds of theannealing protocol. The same crystal lost diffractionquality after annealing on the loop, and diffractioncould not be restored by subsequent MCA. Toinvestigate the effect of size on the response of CHOcrystals to annealing on the loop, another histonecrystal was cut to produce a fragment with smallestdimension of about 0.1 mm. Annealing on the loopwas not successful on the small crystal fragment.

Fumarylacetoacetate Hydrolase

A single SeMet substituted crystal of fumary-lacetoacetate hydrolase (FAH) was initially flash-cooled in a nitrogen cold stream using 30% PEG400,300 mM NaOAc, l00 mM Cacodylate pH 6.5 ascryoprotectant. The flash-cooled crystal initiallydiffracted beyond 2.0C resolution at home, butdeveloped ice during cryogenic storage or transporta-tion to NSLS at the Brookhaven National Laboratory.The initial image taken at X12C showed diffractionless than 6 C resolution and strong ice rings. Thecrystal was restored using MCA, with the time of theincubation in cryoprotectant reduced to 30 sec. Theannealed crystal showed no signs of ice and diffracted to 1.7 C resolution. The structure of this protein wasultimately derived from this annealed crystal [12].Figure 3 shows before and after images of diffractionsimilar to those seen at Brookhaven, however thesewere collected on an in house Rigaku system atIndiana University School of Medicine.CIpP Protease

The annealing process also has been applied to ahighly mosaic crystal of CIpP protease exhibitingsevere spot overlap [1]. A single crystal of CIPP, a300 kDa tetradecameric protease from E colicomplexed with an inhibitor N-Succ-LLL-al wasgrown in 45% MPD, 100 mM MES, pH 6.5. Thecrystallization conditions provide a cryogenicsolution in which to flash-cool the crystals. Thecrystal was prepared by mounting it in a loop and

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Fig. 2. Diffraction images from nucleosome core particle crystal. (a) Diffraction image from flash-cooledcrystal. (b) Diffraction image from same crystal after a cycle of MCA.

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Fig. 3. Diffraction images from fumarylacetoacetate hydrolase (FAH) crystal. (a) Image ofdiffraction showing effects of cryogen failure during storage after initial flash cooling. (b) Samecrystal after MCA as described in the text.

Fig. 4. Diffraction images of ClpP protease kindly provided by John Flanagan. (a) Crystaldiffraction image with inset close-up displaying mosaicity of diffraction. (b) Same crystal afterMCA. Note improvement in spot quality. This crystal also showed improved data resolution after MCA.

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plunging it into a 1 L dewar containing liquidnitrogen. The flash-cooled crystal was transferred to agoniostat which was maintained at a temperature of99K by an Oxford cryosystem unit. The HamptonResearch Crystalcap system was used throughout thecryogenic experiment.

Ten degrees of data were collected at NSLS onstation X12b using a Mar imaging plate and processed using the HKL package. The crystal was removedfrom the cryostream, transferred back to a fresh dropcomprised of mother liquor containing the cryogenand left for 3 minutes. The crystal was then reflash-cooled as before and 10 degrees of data collected inapproximately the same region of the crystal. Imagesof the diffraction are shown in Figure 4. With thiscrystal diffraction resolution improved after an-nealing.

Subsequently a full dataset was collected with areannealed crystal of this complex. The dataprocessed well and the inhibitor was clearly visible inFo-Fc and 2Fo-Fc electron density maps (JamesHartling, Maria Bewley & John Flanagan, pers.comm.).

General Comments and Future Plans

The evidence continues to mount that MCAworks. In future experiments we hope to provide amore complete explanation as to why this is the case.It is likely that the interactions involved in annealingoccur at the mosaic block level, especially since themajority of crystals demonstrate a decreased mosa-icity without a concomitant increase in resolution.The absence of improved order at the atomic levelsuggests that the ordering improvements occur atlower resolution, and with larger clusters ofmolecules. However, improved atomic order is notprecluded from occurring during annealing, espe-cially if disordered regions are rearranged during theannealing process. We have received an additionalreport of successful MCA that resulted in improvedresolution in treated crystals (Marilyn Yoder, pers.comm.).

There is a possible connection between crystaldefects and mosaicity, with increasing numbers ofdefects associated with greater mosaicity and lessresistance to the insult of cryopreservation. One wayto better understand the mosaic block structure ofcrystals is to compare those grown in unit gravity andmicrogravity. These crystals could then be evaluatedfor indications of increased mosaicity before flash-cooling, after flash-cooling, and after annealing. The

mosaic spread of all reflections would be examined toevaluate these experimental effects on resolution andisotropy.

We have noted the solvent content of several ofthe protein crystals used in MCA and annealing on the loop studies. It is possible that water structure is amajor contributing factor to the experimental obser-vations of mosaicity. Properties of crystals, charac-terized by resolution limit and mosaicity can varysubstantially without significantly affecting finalmolecular parameters such as mean atomic displace-ments and temperature factors (Don Caspar, pers.comm.). These observations suggest that thediffraction resolution limit in crystals is not deter-mined solely by the macromolecule and other factorsare involved such as the solvent structure. It ispossible that flash-cooling induced changes inmacromolecular crystal mosaicity and subsequentchanges brought about by annealing both act on thesolvent structure in defining the mosaic properties ofcryo-treated macromolecular crystals. Other chemi-cals in macromolecules may also affect the solventstructure.

One method of evaluating low resolutionstructure is to collect very low resolution diffractiondata. There are several ways available for measuringthe very low resolution diffraction data that containsinformation on the solvent structure within the unitcell of the crystal. On any in house system, thestandard beam stop can be replaced by a precisionadjustable beam stop positioned just ahead of thedetector window. With the use of focusing mirrors itis possible to collect low resolution diffractioninformation including primary Bragg reflections andsurrounding halos of diffuse scatter to d-spacingaround 100 C. Alternatively, synchrotron stationshave the capability of making the necessarymeasurements. Finally, it may be possible to use theORNL 10-meter Small-Angle X-ray Scatteringinstrument with a few modifications. The specificchange in the mosaic spread of low resolutionreflections and analysis of the diffuse scatter shouldfurnish the most information of how annealing ischanging the mosaic block structure within a crystalat the level of the solvent structure.

We are currently collecting information onsuccesses and failures of MCA in an effort tostandardize protocols and provide guidance to thoseusing these techniques with new crystal systems. Werequest your assistance in this project, by letting us

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know of your experiences with MCA. Our e-mailaddress is appended to the author list. We plan onmaking the data web accessible so that in the future,information can be quickly accessed for those seekingannealing conditions for their crystals. With yourhelp, annealed crystals will make the future of lowtemperature data collection bright indeed.

References[1] Flanagan, J. M., J. A. Hartling, R. Sweet and J. Wang.

1997 American Crystallographic Association AnnualMeeting Abstracts, July 19-25, St. Louis, Missouri.

[2] Garman, E. F. and T. R. Schneider. 1997. J. Appl. Cryst.30, 211-237.

[3] Hanson, B. L., A. Martin, J. M. Harp, D. A. Parrish, K.Kirschbaum, C. B. Bunick, A. A. Pinkerton and G. J.Bunick. J. Appl. Cryst. (submitted).

[4] Hardie, M. J., K. Kirschbaum, A. Martin and A. A.Pinkerton. 1998. J. Appl. Cryst. 31: 815-817.

[5] Harp, J. M., Timm. D. E., and Bunick, G. J. 1997.American Crystallographic Association Annual MeetingAbstracts, July 19-25, St. Louis, Missouri.

[6] Harp, J. M., D. E. Timm and G. J. Bunick. 1998. ActaCryst. D54: 622-628.

[7] Harp, J. M., B. L. Hanson, D. E. Timm and G. J. Bunick.Acta Cryst. D (submitted).

[8] Matthews, B. W. 1968. J. Mol. Biol. 33: 491-497.

[9] Rodgers, D. W. 1994. Structure 2, 1135-1140.

[10] Sauer, U. H. and T. A. Ceska. 1997. J. Appl. Cryst. 30,71-72.

[11] Teng, T.-Y. and K. Moffat. 1998. J. Appl. Cryst. 31: 252-257.

[12] Timm, D. E., H. A. Mueller, J. M. Harp and G. J. Bunick.Structure (submitted).

[13] Yeh, J. I. and W. G. J. Hol. 1998 Acta Cryst. D54: 479-480.

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