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Journal of Structural Biology 132, 106–111 (2000)doi:10.1006/jsbi.2000.4312, available online at http://www.idealibrary.com on

Crystallization of the F41 Fragment of Flagellin and Data Collectionfrom Extremely Thin Crystals

Fadel A. Samatey,* Katsumi Imada,* Ferenc Vonderviszt,†,‡ Yasuo Shirakihara,§ and Keiichi Namba*,†

*Protonic NanoMachine Project, ERATO, JST, 3-4 Hikaridai, Seika 619-0237, Japan; †International Institute for Advanced Research,Matsushita Electric Industrial Co., Ltd., 3-4 Hikaridai, Seika 619-0237, Japan; ‡Department of Physics, University of Veszprem,

Egyetem, u.10 H-8201, Hungary; and §Structural Biology Center, National Institute of Genetics, Mishima 411-8540, Japan

Received August 7, 2000, and in revised form September 22, 2000

1smis1

Flagellin, which constructs supercoiled filamentsof the bacterial flagellum, is very difficult to crys-tallize because of its strong tendency to polymerize.We therefore crystallized the F41 fragment of flagel-lin, which does not polymerize because terminalregions that play important roles in polymerizationare cleaved off. F41 was crystallized by the hangingdrop vapor diffusion method in a mixture of poly-ethylene glycol, glycerol, and isopropanol, with areservoir solution covered with silicon oil. The twokey factors for success in growing sufficiently largecrystals were isopropanol and silicon oil, whichworked well to reduce the otherwise very high nu-cleation rate that resulted in hundreds of tiny crys-tals. The crystals were grown to very thin plateswith thickness less than 10 mm, which made theollection of diffraction data very difficult. Freezingnd annealing of the crystals and irradiation at syn-hrotron beamlines had to be carried out by specificethods and under specific conditions for its struc-

ure analysis at 2.0-Å resolution. © 2000 Academic Press

Key Words: bacterial flagellum; flagellin frag-ment; crystallization; thin crystal; X-ray data collec-tion; Salmonella; cryocrystallography.

INTRODUCTION

Bacteria move in liquid environments by rotatingflagellar filaments of a helical form. The rotary mo-tor at the base of the filament drives the rotation ofthe filament (Berg and Anderson, 1973; Silvermanand Simon, 1974). The helical form is normally left-handed, but is switched into right-handed formsupon quick reversal of the motor rotation, whichmakes the cell tumble and change direction of swim-ming during taxis (Macnab and Ornston, 1977). Theflagellar filament is a helical assembly of single pro-tein flagellin. Therefore, the helical form of the fila-ment is produced by supercoiling, the mechanism for

which was proposed by Asakura (1970), assuming a

1061047-8477/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

conformational switching of the subunits to be intwo distinct states and with nonequivalent intersub-unit interactions.

Structural studies by electron cryomicroscopy andX-ray fiber diffraction at around 10-Å resolution re-vealed the shape and packing of the subunits in thetwo distinct types of straight filaments (Mimori etal., 1995; Morgan et al., 1995; Mimori-Kiyosue et al.,996, 1997, 1998; Yamashita et al., 1998). Thesetructures led us to propose a mechanism for poly-orphic supercoiling, which involves a fine mechan-

cal switch that produces the two distinct states withubangstrom accuracy (Namba and Vonderviszt,997; Yamashita et al., 1998). However, much

higher resolution is necessary to understand thestructural basis of supercoiling in atomic detail. Af-ter many trials, we have not had any success incrystallization of flagellin because of its strong ten-dency to polymerize into filaments. Therefore, wetried to crystallize fragments of flagellin that haveno or an extensively reduced ability to polymerize.

Flagellin from a wild-type strain of Salmonellatyphimurium, SJW1103, is composed of 494 aminoacid residues and its molecular mass is 51.5 kDa(Kanto et al., 1991). Earlier studies showed that aproteolytic fragment of flagellin named F41, missing52 N-terminal residues and 44 C-terminal residues,does not form any filament structures but gives nee-dle-like crystals of a few micrometers in size in thepresence of ammonium sulfate (Vonderviszt et al.,1991). F41 was named after its apparent molecularmass of 41 kDa. F41 was therefore chosen for crys-tallization.

We obtained very thin but well-ordered crystalsthat diffracted beyond 2-Å resolution after survey-ing and devising crystallization conditions exten-sively. The mechanical flexibility of the thin crystalswith a thickness of 10 mm or less forced us to tryvarious methods of freezing to obtain high-resolu-

tion diffraction data by cryocrystallography. We de-

107CRYSTALLIZATION OF A FRAGMENT OF FLAGELLIN

scribe in this report our experiences in those meth-odological developments, which finally led to thesolution of the F41 structure at 2.0 Å.

MATERIALS AND METHODS

Sample preparation. Isolation of flagellin from S. typhi-murium was performed as previously described (Vonderviszt etal., 1990). Limited proteolysis of flagellin was carried out at roomtemperature in a solution containing flagellin at a protein con-centration of 5 mg/ml and 20 mM Tris–HCl, pH 7.8. A solutioncontaining clostripain (from Sigma), which specifically cleavespeptide bonds at the C-terminal side of arginine residues, and 2.5mM dithiothreitol (DTT) was added to the flagellin solution. Theratio of the protease to flagellin was 1:500 (w/w). The digestionwas monitored every 15 min by anion-exchange column chroma-tography using a Mono-Q column with a column volume of 1 ml(Pharmacia), and the clostripain activity was stopped by addingNa-p-tosyl-L-lysine-chloromethyl ketone and ethylenediaminetet-raacetic acid (both from Sigma) to the solution. Proteolytic frag-ments were separated by using a larger Mono-Q anion-exchangecolumn with a column volume of 10 ml. Elution was done with alinear gradient from 75 to 200 mM NaCl in 20 mM Tris–HCl, pH7.8. Protein fragments were monitored in the effluent at 280 nm.The quality of the purified sample was checked by sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and alsoby matrix-assisted laser desorption ionization time-of-flight(MALDI-TOF) mass spectrometry using a Voyager-DE/PRO spec-trometer (PE Biosystems).

Crystallization. Purified F41 was concentrated to 50 mg/ml in20 mM Tris–HCl buffer at pH 7.8, kept as a stock solution, anddiluted to appropriate protein concentrations for crystallizationscreening. Crystallization conditions were screened by vapor dif-fusion using the hanging drop method. Droplets were made bymixing an equal volume of a protein solution and a precipitantsolution in the reservoir.

Data collection. The cryocrystallographic method was used tocollect diffraction data. Crystals were picked up in a nylon loop(Hampton) and frozen by various methods, including flash coolingin a nitrogen gas flow at 2170°C and plunging into liquid pro-pane, ethane, or nitrogen. The quality of diffraction from frozencrystals was checked by taking a few frames of diffraction pat-terns with our laboratory X-ray source, optics, and detector. TheX-ray source is a rotating anode X-ray generator with a coppertarget, type FR-D (Rigaku), which was operated at 50 kV and 70mA. The effective focal size of the X-ray source is 0.1 mm 3 0.1mm. The X-ray beam was collimated using the Yale-type double-mirror optics (Rigaku), which produced a beam size of approxi-mately 0.15 mm 3 0.25 mm at the crystal as well as on thedetector surface at about 200 mm downstream. The relativelyhigh intensity and the small beam size compared to those avail-able from conventional laboratory sources and optics were essen-tial in collecting useful diffraction patterns from extremely thincrystals of F41 for screening well-diffracting crystals for actualdata collection at synchrotron beamlines. An RAXIS-IV imageplate detector (Rigaku) was used to record diffraction patterns. ARigaku-made cryostreamer (GN2 low-temperature device) wasused to keep crystals at 2170°C during the data collection.

We then brought those potentially well-diffracting crystals tobeamlines at synchrotron radiation facilities, such as beamlineID14 at ESRF and beamlines BL41XU, BL45XU, and BL40B2 atSPring-8, to collect high-resolution diffraction data sets. The datawere reduced by DENZO and SCALEPACK (Otwinowski andMinor, 1997) or MOSFLM (Leslie, 1992) and SCALA (CCP4,

1994).

RESULTS

Crystallization

In initial crystallization trials we used ammoniumsulfate as precipitant, but we only obtained manysmall needle-like crystals similar to those shown inFig. 1a. Obviously the nucleation rate was too high.We did not see much improvement by lowering the

FIG. 1. The improvement process of F41 crystallization. Crys-tals of F41 grown in 20 mM Tris–HCl buffer, pH 7.8, undervarious protein and precipitant concentrations: (a) 9 mg/ml pro-tein, 9% PEG 6000, 50 mM NaCl, at 16°C; (b) 6 mg/ml protein, 9%PEG 6000, 50 mM NaCl, at 4°C; (c) 3 mg/ml protein, 5% PEG6000, 25 mM NaCl, at 16°C; (d) 6 mg/ml protein, 6% PEG 6000, 50mM NaCl, 12% glycerol, 6% isopropanol, at 16°C. The bar at thebottom right represents 0.3 mm.

protein or precipitant concentration. Temperature

fgmm

F

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108 SAMATEY ET AL.

and pH changes did not bring any significantchanges in the crystal size, either.

We then used polyethylene glycol (PEG) of variousmolecular weights for crystallization screening. Theresults showed that crystals appeared only whenPEG 4000 or PEG 6000 was used. A combination ofPEG 6000 and NaCl gave many small needle-likecrystals after 5 days at 16°C, and these crystals wereslightly larger than those obtained with ammoniumsulfate (Fig. 1a). We saw some improvements in thesize of crystals at 4°C when the protein concentra-tion was slightly lowered (Fig. 1b). However, thecrystals obtained at 4°C were found to be unstable atroom temperature. We made further improvementsat 16°C by lowering the protein and the precipitantconcentrations. The crystals became significantlylarger, although these plate-like crystals were verythin and had many small crystals stuck on them(Fig. 1c). It was clear at least that slowing down thenucleation worked well to grow the crystals larger.

To further slow down the nucleation we added thefollowing two features in the crystallization method.The first was the use of glycerol as additive. Afterscreening various glycerol concentrations, we ob-tained the best result with 12% glycerol in the drop.The crystals were already large enough to pick upfor a diffraction test. This relatively high concentra-tion of glycerol in the drop enabled us to directlyfreeze the crystals and cool to liquid nitrogen tem-perature for cryodiffraction experiments. However,the crystals showed rather poor diffraction. The res-olution was limited to around 3.0 Å even by 5-hexposure with our laboratory X-ray source. As thesecond modification, we used silicon oil to cover thereservoir solution to slow down the vapor exchangerate (Chayen, 1997). The crystals grew larger andslightly thicker as well, but these crystals stillshowed poor diffraction. The crystal quality was im-proved by adding isopropanol in the range between 4and 6%. The nucleation rate was dramatically low-ered by the addition of isopropanol. Crystals ap-peared in 10 days with much better crystal qualityand reproducibility. All the crystals were plate-like,and the largest crystal size was 2.0 3 0.4 3 0.005mm3. The best crystallization conditions we foundor a drop solution were with 6% PEG 6000, 12%lycerol, 3–6% isopropanol, 50–75 mM NaCl, 20M Tris–HCl, pH 7.8, a protein concentration of 8g/ml, and a temperature of 16°C (Fig. 1d).

reezing and Annealing Crystals for DataCollection at Cryotemperature

The crystals were frozen prior to the X-ray diffrac-ion experiments, and diffraction data were collectedt 2170°C. We found that flash cooling, namely,

ooling the crystals by nitrogen gas flow at 2170°C e

rom the cryostream nose or plunging crystals di-ectly into liquid nitrogen, gave rather poor diffrac-ion. The freezing method that worked best for the41 crystals was to cool them to liquid nitrogenemperature quickly by plunging them into liquidropane in a small bath equilibrated in a larger bathf liquid nitrogen. Because of the extremely thinlate-like shape of the crystals, most crystals wereomewhat bent at the time of freezing and gaveather poor diffraction with elongated spots (Fig.a). Annealing the frozen crystals proved to be very

FIG. 2. Improvement of diffraction quality by annealing thecrystal. X-ray diffraction patterns from a F41 crystal recorded at2170°C using the laboratory X-ray equipment: (a) before anneal-ing; (b) after annealing. The quadrant at the bottom right ismagnified 1.5 times to show more details.

ffective in improving the molecular packing order

109CRYSTALLIZATION OF A FRAGMENT OF FLAGELLIN

within the crystal, in both the long and the shortrange, by removing the bend and any other mechan-ical perturbations caused to the crystals and there-fore improving the diffracting power as well (Fig.2b). Annealing was done while keeping the crystalon the goniometer head (Yeh and Hol, 1998; Harp etal., 1999). First, the frozen crystals were mounted inthe cooled nitrogen gas flow from the cryostreamnose and kept at 2170°C. The gas flow was thenshut off, until the crystals were completely thawed,by inserting a piece of paper between the nose andthe crystal. The crystals were frozen once again byputting back the cooled gas flow. It is important tomake sure that the crystals are completely thawedin this process. Therefore, the process was moni-tored with a microscope attached to the crystalmount and repeated a few times. All the crystalswere systematically annealed before diffraction datawere collected.

Preparation of Heavy Atom Derivatives

Fiber diffraction studies of the flagellar filamentsin well-oriented liquid crystalline states showed pre-viously that dysprosium (Dy) and lead (Pb) bind atspecific sites in flagellin from strain SJW1655,which forms the R-type straight filament, and mer-cury (Hg) binds to a cysteine that was introduced bysite-directed mutagenesis (Yamashita et al., 1995).Therefore, these heavy atoms were chosen for prep-aration of heavy atom derivatives of the F41 crystal.The lead and dysprosium derivatives were preparedby soaking the crystals in a solution containing 25%PEG 6000, 24% glycerol, 0.1 M NaCl, and either 0.5mM lead acetate or 0.5 mM dysprosium chloride, for3 days. The mercury derivative was prepared bycocrystallization of the G365C mutant of F41. Toavoid oxidation of the introduced cysteine sidechain, only nitrogenated buffers were used for puri-fication of the cysteine mutant. The purified proteinwas treated with DTT. Mercury compounds wereadded to the protein solution at least 24 h prior to

TASummary of C

Crystal

Crystal parameters

a (Å) b (Å) c (Å) b (°)

W14 51.9 36.5 119.1 91.2W77 51.9 36.8 119.0 91.3L57 51.9 36.6 118.9 90.9C77 51.8 36.4 118.7 90.9

Note. Crystals are as follows: W14, native; W77, Dy derivativetable were collected on beamline ID14-3 at ESRF.

the crystallization setting up. Before the mercury

compounds were added, the protein solution wasdialyzed to remove the DTT. Two mercury com-pounds were used: HgI2-2KI and p-hydroxymercuri-phenylsulfonic acid sodium salt (both from Sigma).The concentration of the mercury compounds incocrystallization drops was 0.5 mM, with other con-ditions the same as for the native crystal. To removeunbound heavy atoms, all the crystals treated withheavy atom compounds were back-soaked in a solu-tion containing 25% PEG 6000, 24% glycerol, and0.1 M NaCl. This is in part to increase the cryo-protectant concentration as well. To maximize theisomorphism between the native and the derivativecrystals, the native crystals were also soaked in thesame solution before they were frozen.

Crystal Parameters and Anomalous Patterson Map

Diffraction data were collected using synchrotronradiation with wavelengths of around 1 Å. The crys-tal space group is P21, with almost constant cellparameters from crystal to crystal (Table I). Thecompleteness was high and some crystals showed agood Rsym value. Anomalous Patterson maps in Fig.3 show that there is one heavy atom per asymmetricunit in each derivative. We have tried multiple iso-morphous replacement phasing as well as multiple-wavelength anomalous dispersion (MAD) phasing.Although both phasing methods gave electron den-sity maps of good quality, the best map was obtainedfrom MAD data collected using beamline BL45XU atSPring-8 (data not shown). Based on the 2-Å resolu-tion map we were able to build the molecular modelof F41, which we will describe in detail elsewhere.

DISCUSSION

Sample Quality Control by Mass Spectrometry

From time to time we experienced cases wheresome preparations of purified F41 did not producegood crystals, although the preparation of F41 wascarried out every time in the same way and the

Il Parameters

Completeness (%) Rmerge (%) Resolution (Å)

98.4 5.8 2.098.9 9.0 2.599.1 4.6 2.299.8 8.1 2.3

Pb derivative; C77, Hg derivative of G365C. All the data in this

BLErysta

; L57,

samples obtained showed a single band on the SDS–

mwml2tT

110 SAMATEY ET AL.

PAGE gel. We therefore checked the quality of thesample by MALDI-TOF mass spectrometry. Wefound that some of the preparations had minor con-tamination of the F40 fragment, a fragment slightlysmaller than F41 (Vonderviszt et al., 1989). Obvi-ously this minor contamination of fragments of sim-ilar but different sizes disturbed the crystal growth.Therefore, it was essential for reproducible crystal-lization to check the sample purity by mass spec-trometry and to remove all these contaminated frag-ments by going through the column chromatographystep again, if any contamination was found.

Reducing High Nucleation Rate

We occasionally encounter cases where largeenough crystals do not grow because of the highnucleation rate of proteins. It is usually difficult toreduce the nucleation rate by lowering the protein orprecipitant concentration, because the nucleationrate is not a linear function of those concentrations.The rate rises abruptly as these concentrationsreach certain critical values. Therefore, one seeseither no nucleation or so much nucleation by aslight difference in the protein or precipitant con-centration. We found three factors very effective inreducing the nucleation rate of F41 to grow largecrystals: using isopropanol as an additive; usingglycerol as an additive; and covering the surface ofreservoir solutions with silicon oil.

Slowing down the vapor exchange rate betweendroplets and reservoir solutions by covering the sur-

FIG. 3. Harker section of anomalous difference Pattersonaps obtained from heavy atom derivative data. The two mapsere calculated with the data obtained from crystals (a) C77 (aercury derivative of the G365C mutant crystal) and (b) L57 (a

ead derivative of the native crystal) (see also Table I). Data from0 to 5 Å were used for the calculation. Anomalous data wereaken at a wavelength of 0.947 Å on beamline ID14-3 at ESRF.he maps are contoured with a base contour level of 2.0 s and an

increment level of 1.0 s. Peaks corresponding to the heavy atomsites are marked with a plus sign.

face of reservoir solutions with silicon oil (Chayen,

1997) certainly worked well for F41, and this indi-cates that this method appear to have general use-fulness. The method of adding glycerol or isopropa-nol to reduce the nucleation rate may not be asgeneral as the silicon oil method, because it woulddepend on the nature of molecular interactions thatdrives the crystal formation. After the crystal struc-ture of F41 was solved, the molecular interactionswe have found in the crystal are a combination ofsalt bridges, hydrogen bonding, and hydrophobic in-teractions: nothing special except that the interac-tions are more intensive and intimate than usualmolecular contacts found in many crystals. Thismeans that glycerol and isopropanol may be goodcandidates to be tried in reducing the high nucle-ation rate of any protein crystallization, particularlyfor proteins that have a strong tendency to polymer-ize into filament structures.

Annealing Crystals

Among many different freezing methods that wetried on the F41 crystals, the only method thatworked well was freezing them by plunging intoliquid propane and then annealing them in a coolednitrogen gas flow. The freezing speed is highest byplunging into liquid propane. However, since thecrystals were all soaked in a solution containing 25%PEG 6000 and 24% glycerol before being frozen,these high cryoprotectant concentrations are sup-posed to protect the crystal order from perturbationscaused by any type of freezing method, either fast orslow. We do not have a very clear idea why only oneparticular freezing method worked significantly bet-ter than the others. On the other hand, the anneal-ing method was clearly essential to improve thediffraction quality of the crystals. The effects of an-nealing have already been reported in some publi-cations (Harp et al., 1998, 1999; Yeh and Hol, 1998).The annealing method, when appropriately applied,appears to be generally useful in improving the crys-tal qualities such as mosaicity and diffraction limit.What we found particularly in our case is that an-nealing was essential to remove mechanical stressesput on these very thin crystals of F41 at the time offreezing. Before annealing, diffraction spots fromF41 crystals were not only weak but also often hadelongated or distorted profiles. When the thin platecrystal was placed with its face parallel to the X ray,the spot elongation was serious. However, when theedge portion of the crystal was irradiated, nice dif-fraction spots tended to appear. This indicated thatthe freezing procedure by plunging caused signifi-cant distortions of the thin crystals. After annealing,the spot profile was dramatically improved. We triedthe annealing method on the other thin protein crys-

tals showing rather elongated spots and the diffrac-

wpaltbbwltss

A

M

M

M

M

M

N

O

S

V

V

V

Y

Y

Y

111CRYSTALLIZATION OF A FRAGMENT OF FLAGELLIN

tion profile was also improved (data not shown).Very thin crystals easily suffer from mechanical dis-tortion that causes problems in their diffraction pro-files. From this point of view, it is worth trying theannealing method systematically for thin crystals.

Radiation Damage

Some of the beamlines at third-generation syn-chrotron radiation facilities, such as BL41XU atSPring-8, produce an extremely intense X-ray beamat its full capacity. On such beamlines, the exposuretime required to see clear reflections beyond 2-Åresolution from those very thin F41 crystals was asshort as a few seconds. However, under such anintense beam the radiation damage was significant.After a few tens of frames were collected, the circu-lar shape of diffraction spots gradually turned intoelongated or seriously distorted profiles and thenhigh-resolution spots started fading out. This behav-ior suggests that relatively large distortions wereinduced in the crystal by irradiation. The thermalload by such an intense X ray is probably too high tosuppress the local temperature rise inside the crys-tal just by nitrogen gas flow at 2170°C. However,

hen the beam was attenuated to make the appro-riate exposure time longer than 30 s, we did not seeny significant sign of radiation damage. At thisevel of beam intensity, the temperature rise insidehe crystals may have been suppressed to a negligi-le level. Therefore, we always attenuated the X-rayeam by inserting a thin metal plate to a level athich the appropriate exposure time is 30 s or

onger. In this way, most crystals survived over mul-iple sets of data collection or even over multipleessions of data collection at different synchrotronites.

We thank T. Tomizaki, L. Dumon, W. Burmeister, S. Arzt, andS. Wakatsuki at ESRF and M. Kawamoto and N. Kamiya atSPring-8 for technical help in the use of beamlines, and we thankS. Nagashima for help in data collection. We also thank F.Oosawa for continuous support and encouragement. This workwas supported in part by the Hungarian National Science Foun-dation (OTKA) Grant to F.V.

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