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Trigonal catalase crystals: A new molecular packing assignment obtained from sections preserved with tannic acid
Ultramicroscopy 13 (1984) 103-112 North-Holland, A m s t e r d a m
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TRIGONAL CATALASE CRYSTALS: A NEW MOLECULAR PACKING ASSIGNMENT OBTAINED FROM SECTIONS PRESERVED WITH TANNIC ACID Christopher W. AKEY, Margot SZALAY and Stuart J. EDELSTEIN Section of Biochemistry, Molecular and Cell Biology, Division of Biological Sciences, Wing Hall, Cornell University, Ithaca, New York 14853, USA Received 1 November 1983; presented at EMSA Symposium August 1983
The molecular packing of a trigonal crystal form of catalase initially studied by Longley [l] has been re-evaluated. Sections of crystals fixed and preserved with tannic acid were obtained parallel to the (001) and (100) planes. Specimens prepared by either conventional or low temperature embedding maintained 20 ,~ resolution after sectioning. The space group of the crystals is either P3~21 or P3221 and the observed unit cell parameters for (001) and (100) are a = b = 174 A,, ~, = 119 ° and b = 189 ,~, c = 248 ,~ with a = 89.5 °. Computer-based reconstructions of two principal projections coupled with crystal density measurements allowed the deduction that there is one catalase tetramer per asymmetric unit. The crystal structure consists of 6 molecules packed closely about a c o m m o n triad screw axis. This interpretation differs from that proposed by Longley [J. Mol. Biol. 30 (1967) 323], because thin sections of embedded crystals were assumed a priori to be positively stained in the early work; in actuality the sections were negatively stained. We also demonstrate that tannic acid fixation can lead to well preserved, positively stained crystal sections under certain conditions.
1. Introduction
Three methods of obtaining 20 ,~ resolution in sectioned protein crystals have recently been described: tannic acid fixation [2], low temperature embedding [3] and grid sectioning of negatively stained crystals [4]. Sectioning of protein crystals can provide a valuable adjunct to conventional methods of electron microscopy of thin crystals. The information obtained can be employed to determine the three-dimensional packing of a protein crystal [2,4-6] or to partially supplement the cone of missing data in a three-dimensional data set [7,8]. The ultimate goal in improving the structural preservation maintained in the embedded and sectioned specimen centers on the potential use of thin sectioned crystals for three-dimensional image reconstruction. To be useful for three-dimensional work, thin sections must possess suitable resolution, structural fidelity, and a known contrast. Tannic acid fixation appears to satisfy the above criteria based on studies of two-dimensional projections of crystal
sections of Pseudomonas cytochrome oxidase [2], orthorhombic beef liver catalase [5] and beef heart F1-ATPase [6]. However, a rigorous evaluation of a known structure in three dimensions is needed to define completely the fidelity of this method of sample preservation. Furthermore, a suitable model system would be beneficial in allowing an experimental evaluation of possible problems inherent in three-dimensional reconstruction of sections. Potential problems include non-integral numbers of unit cells, phase origin refinement in specimens with multiple unit cells along c, and limitations on specimen thickness as a result of multiple scattering. In order to develop methods of general applicability and to evaluate the potential usefulness of sections for three-dimensional analysis, we have focused on a trigonal crystal form of catalase initially characterized by Longley [1]. Catalase provides an excellent model system as 5-6 crystal forms are known [9] including at least 2 orthorhombic modifications suitable for conventional three-dimensional electron microscopy of nega-
0304-3991/84/$03.00 © Elsevier Science Publishers B.V. (North-Holland Physics Publishing Division)
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C 14". Akey et al. / Trigonal catalase cD,stals
tively stained specimen [4,5,10]. The space group parameters of the trigonal form employed in this study were obtained by X-ray diffraction [1], and crystals can be grown reproducibly to sizes suitable for direct manipulation and embedding. Furthermore, the X-ray structure has recently been solved at 2.5 A resolution [11], thereby allowing the determination of the fidelity of various microscopy derived structures by comparison at low resolution. In this report we verify that the space group of the crystals is P3121 (or P3221 ) and demonstrate that there are 6 molecules per unit cell packed tightly about a common triad screw axis. Based on crystal density measurements, there is one tetramer per asymmetric unit; therefore the 222 point group symmetry of the molecule is not expressed in the crystallographic packing. Hence, three-dimensional analysis will be aided by the presence of non-crystallographic symmetry. It is also demonstrated that crystal sections prepared by tannic acid can be positively stained, and under optimal conditions sections obtained by either conventional or low temperature embedding are structurally ordered to a resolution of at least 20 A.
2. Materials and methods
2.1. Crystals and density measurements Beef liver catalase was obtained as a twice-recrystallized suspension in 0.1% thymol from Sigma. The crystal form in this commercial preparation is predominantly trigonal. For thin section experiments, the trigonal prisms were recrystallized to obtain larger crystals free of inherent twinning found in the preparation. The trigonal crystals were grown by the procedure of Tauber and Petit [12], using 0.05 M potassium phosphate, pH 7.3, after the removal of thymol. Crystals grown by this procedure often exceeded 1 mm in length after 1-6 months and appeared as elongated, untwinned prisms with hexagonal cross-sections. Crystal buoyant density was measured using a linear gradient of water-saturated bromobenzene and xylene [13] with standardized CsC1 solutions for calibration.
2.2. Tannic acid fixation Over a period of 5 years, the chemical characteristics of a batch of tannin from Merck changed as judged by the predominant contrast present in crystal sections under standard embedding conditions. Therefore, we recently have screened 5 batches of commercially available tannic acid from 4 suppliers, in an attempt to optimize our results with crystal sections. Optimal results were obtained with a purified tannic acid from Mallinckrodt (lot ZCN). Crystals are now routinely fixed in 8% tannic acid, 2% glutaraldehyde in 50 mM monobasic potassium phosphate, pH 7.3, overnight at room temperature. For this choice of buffer, the fixative solution must be brought to pH with N a O H to avoid precipitation. Following fixation, crystals were washed twice with 50 mM phosphate, 50 mM glycine for 5 min, then rinsed twice with 50 mM phosphate buffer without glycine. The crystals were pre-stained with 0.5% OsO 4 in 50 mM phosphate at pH 7.3 for 1 h at room temperature. This was followed by 3 rinses in phosphate buffer. The crystals were then processed for conventional embedding using a graded dehydration series of water-ethanol, followed by infiltration with a low viscosity resin [14]. After a final infiltration in 100% resin for 12 24 h, the crystals were aligned in flatbed molds and polymerized at 60°C. 2.3. Low temperature embedding The method of low temperature embedding [3] has been combined with tannic acid fixation. The Lowicryl kit was obtained from Balzers Union. Specimens were fixed and pre-stained as described above; however, the ethanol dehydration and subsequent infiltration steps were carried out at low temperatures, essentially as described in the Balzers Union instruction manual. Some steps were modified; the initial dehydration step was done at 4°C and all subsequent steps were carried out in a sample block thermally equilibrated with dry ice-ethanol. To achieve the requisite conditions of low temperature and dryness for successful embedding, all manipulations were carried out in a glovebag under N 2 atmosphere. The samples were
c.w. Akey et al. / Trigonal catalase crystals
kept in white plastic vials which were seated in an aluminum block which fit snugly into the top of a styrofoam box (20 × 10 x 20 cm). All manipulations of the crystals were monitored directly with a stereomicroscope which was fitted to the glovebag. The resin mixture was kept dry and consisted of K 4 M A 1 g, K4MB 6.5 g, initiator C 76 mg and 35 mg dithiothrettol. Crystals were aligned in flatbed molds and the samples were polymerized by UV irradiation in a sealed Thermos cooler box. The samples were kept cold by contact with a layer of dry ice. Samples prepared without D T T did not yield sufficiently hard blocks for sectioning. The conditions described herein must be considered approximate; refinements are needed to reproducibly obtain blocks with good thin sectioning quality. 2.4. Thin sections, electron microscopy and image processing
All blocks were sectioned with a diamond knife as described previously [2]. Sections (600-1200 ,~
105
thick) were picked up on either 400 mesh or Formvar-backed slot grids. Post-staining was carried out with either 2% uranyl acetate in 35% ethanol ( v / v ) and 12.5% methanol ( v / v ) [15], or methanolic 2% uranyl acetate. Sections were also post-stained with lead citrate [16]. Images were recorded on Kodak electron image film using a Philips EM 301 and minimum beam methods [17,18,2]. Micrographs were digitized at 30 micron steps on a Syntex AD-1 densitometer. The resultant arrays were processed on our laboratory computer system using Fourier [2] or a combination of real space-Fourier space analysis [19,20].
3. Results and discussion
A trigonal modification of catalase has been chosen for development of general methods for three-dimensional analysis of thin sections. This crystal form can be grown at p H 7.3 and was analyzed by Longley [1] using X-ray diffraction and thin section methods. The space group was
C
b Fig. 1. Micrographs of thin sections along (001) and (100). (a) Thin section ( - 600 ,~ thick) cut parallel to the (001) planes. The contrast is identical to that observed by Longley [1]. (b) Thin sections cut parallel to the (100) planes. The scale bar is 500 A.
C. I'lL Akey et al. / Trigonal catalase crystals
106
,
,,
(~
F
,j
b
~.
,
d
(001)
.,
---..5
c
(100)
Cl
Fig. 2. Reconstructions of projections of the (00l) and (100) planes of trigonal catalase. Heavy contours correspond to protein. (a) Unenforced reconstruction of a thin section of (001). The unit cell is indicated. The symmetry elements in this projection are shown in fig. 3a. The most prominent features of the m a p are the large morphological units connected by 6 crossbridges. (b) Symmetry-enforced reconstruction from the same area used for fig. 2a. The crossbridges are indicated (CB). Also note that each morphological unit is surrounded by 6 channels with 3-fold symmetry. (c) Symmetry-enforced reconstruction of the (100) planes from 3 areas. The true crystallographic unit cell is indicated. The major features of the m a p correspond to the morphological units designated A, B and C which are stacked along the c-axis, parallel to the triad screw axis.
determined to be either P3~21 or P3221 with a and b equal to 173 .~ and c = 237 A, 7 = 120°. However, the initial interpretation of the crystal packing was incorrect because the assumption was made that the thin sections were positively stained. In fact, we conclude that the sections were negatively stained for reasons discussed in more detail below.
3.1. The (001) projection The (001) projection is of interest since it gives a view along the unique c-axis. Sections from crystals prepared by either the method described by Longley [1] or with tannic acid-fixation have identical contrast; however, the resolution is improved at least two-fold in the latter. A representative micrograph of the (001) projection is presented in fig. la. This crystal form has a c-axis repeat of 248 A, therefore sections 600-1200 ,~ thick are about 2.5-5 unit cells thick. Variability was encountered in reconstructions from 8 sections ( - 6 0 0 ,~ thick), undoubtedly caused by non-integral numbers of unit cells. A reconstruc-
tion from a section judged to be either 2 or 3 unit cells thick, based on apparent section thickness and symmetry preservation, is presented in fig. 2a. The symmetry-enforced reconstruction is shown in fig. 2b. The unit cell consists of a large triangular unit with interconnecting crossbridges (CB) which link the morphological units together. The thick contours have been identified as protein, based on the following considerations. First, tannic acid fixation has consistently yielded negatively stained sections of a number of macromolecular arrays including fibers of tubulin [12] and hemoglobin S [22] and crystals of Pseudomonas cytochrome oxidase [2], orthorhombic catalase [6] and beef heart F1-ATPase [6]. Second, a series of embeddings were carried out comparing both trigonal and thin orthorhombic catalase crystals under identical fixation conditions. Sections of the trigonal form demonstrated the same contrast as in fig. l a while sections of the orthorhombic form were negatively stained as deduced elsewhere [5]. The two crystal forms of catalase are expected to stain similarly, hence the trigonal crystals must be negatively stained. Further evidence is presented later,
C.w. Akey et al. / Trigonal catalase crystals in the section on positively stained sections. T h e unit cell p a r a m e t e r s f r o m 8 m i c r o g r a p h s were obt a i n e d a n d are a = b = 184 ,~ with ~ = 119 °.
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excluded. T h e m e a s u r e d crystal d e n s i t y was 1.078 + 0.012 g / c m 3 for crosslinked crystals in water (n = 8). This c o r r e s p o n d s to a c a l c u l a t e d molecular weight of 223 k d + 34 kd, b y the m e t h o d of M a t t h e w s [23]. T h e t e t r a m e r m o l e c u l a r weight is 228 k d [11], therefore the correct value is 1 molecule p e r a s y m m e t r i c unit with 6 molecules p e r unit cell. This crystal form has a Vm of 5.3 (,~3/dalton) a n d a solvent c o n t e n t of 77%. A p a c k i n g m o d e l for the trigonal crystals with 6 molecules p e r unit cell in space g r o u p P3121 is shown schematically in fig. 3a. T h e catalase t e t r a m e r s are d r a w n as spheres for ease of representation, a n d are p a c k e d tightly a b o u t a triad axis to generate the large m o r p h o l o g i c a l unit o b s e r v e d in the (001) projection. The molecules are labelled b y height along c ( 1 - 6 ) a n d the 2 sets of 31-screw axis related molecules are 1, 3, 5 a n d 2, 4, 6. In space group P3z21, details of the p a c k i n g w o u l d differ b e c a u s e the s y m m e t r y axes l o c a t e d at 1 / 6 (see fig. 3a) w o u l d be l o c a t e d at 1 / 3 , while those shown at 1 / 3 in the d i a g r a m w o u l d be at 1 / 6 . N o t e that the (100) a n d (001) p r o j e c t i o n s in space g r o u p P3t21 are indistinguishable. T h e o n l y difference concerns the p o s i t i o n o f the 21-screw a n d 2-fold axes which
3.2. The (100) projection A m i c r o g r a p h of a section parallel to (100) is p r e s e n t e d in fig. l b . A s y m m e t r y - e n f o r c e d reconstruction f r o m 3 areas is p r e s e n t e d in fig. 2c. The true unit cell b-vector is twice the o b s e r v e d r e p e a t in this p r o j e c t i o n due to the s y m m e t r y of this space group which has a 21-screw axis a n d a 2-fold axis o v e r l a p p i n g in a direction parallel to b. The m e a s u r e d unit cell c o n s t a n t s are b = 179 ,~, c = 248 with a = 89.5 °. T h e r e are 3 m a j o r m o r p h o l o g i cal units labelled A, B a n d C stacked along the c-axis in this projection.
3.3. Packing model T h e possible n u m b e r of catalase t e t r a m e r s p e r unit cell in the trigonal space g r o u p is either 3, 6 or 12. However, b a s e d on c o n s i d e r a t i o n of the possible values of the crystal p r o t e i n specific v o l u m e [23], the value of 3 molecules p e r unit cell can be
tt
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.
.
.
.
,. . . .
.
. . . . . . . .
,. . . .
~
~
,
~
;
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Fig. 3. Packing diagram for trigonal crystals viewed along [001] and reconstructions of (001) from 1200 A thick sections. (a) Schematic diagram of crystal packing in trigonal modification of catalase, space group P3121. Tetramers are represented by spheres. Triad-screw-axis-related molecules are shaded in a similar manner and are 1, 3, 5 and 2, 4, 6. The molecules are numbered 1-6 in order of increasing height along c. (b) Reconstruction of (001) from section in fig. 4a. The expected 32 symmetry is strongly preserved in the unenforced reconstruction. The section was approximately 4-5 unit cells thick. (c) Reconstruction of (001) from section in fig. 4b. The 32 symmetry is again strongly preserved in the unenforced reconstruction. The molecular edges (M) of the disk-shaped catalase tetramer appear to be resolved near the periphery of the morphological units. Note that there are now 12 channels of 15 ,~, diameter surrounding a given morphological unit. The horn-like (H) features which occur on alternate molecules around the periphery of the morphological unit may be an artifact caused by multiple scattering or may represent a real feature of this projection,
108
C. 144 Akev et al. / Trigonal catalase crystals
overlap in projection. In the (100) projection this overlap occurs at c = + 1 / 3 and + 5 / 6 (see fig. 2c). The three morphological units in the (100) projection probably arise from superposition of two catalase tetramers, viewed in projection. A complete three-dimensional analysis should reveal the correct space group and the structure of the catalase tetramer preserved by tannic acid fixation. 3.4. Improved resolution in sections
Two approaches were investigated in an attempt to improve the structural order maintained in crystal sections. The two methods consisted of determining the best source of tannic acid for fixation and combining tannic acid fixation with low temperature embedding. Initially, tannic acid
8
Fig. 4. Micrographs of 1200 ~, thick sections of trigonal catalase. (a) Section cut parallel to (001). Crystal was fixed with tannic acid and embedded by conventional methods. The section is negatively contrasted and appears similar to those obtained by Longley [1]. (b) Section cut parallel to (001), Crystal was fixed with tannic acid and embedded using low temperature methods. The section is positively stained. The clarity of the structural preservation is good enough to count the 12 "'channels" which encircle a morphological unit. The mean diameter of the channels is 15 ,~ and is a measure of the resolution. Scale bar is 1000 ~,.
obtained from Merck was used in thin section studies of Pseudomonas cytochrome oxidase [2!, and gave negatively stained sections with 20 A resolution. The same batch, when used with trigonal catalase crystals, rarely preserved the structural order !o better than 35 A; hence~ this system was chosen for further studies aimed at improving resolution in sections. As described earlier, various tannin mixtures were screened and a batch which consistently gave 20 A resolution with the trigonal crystals was obtained. The quality of optical transforms of sections was also improved by employing a 100 micron objective aperture during imaging to enhance image contrast. In earlier studies no objective aperture was used (see ref (18]). An optical transform of the section ( - 1200 A thick) in fig. 4a is presented in fig. 5a and demonstrates reflections to 1//20 A resolution. Protein crystals become disordered during preparation of thin sections as a result of three major factors; dehydration damage, thermal denaturation and damage arising from the sectioning process. Diffraction studies with orthorhombic crystals [5] have demonstrated that tannic acid can protect the protein structure from dehydration, in a manner analogous to glucose embedding [18]. It was therefore reasoned that tannic acid might be partially protecting the crystal structure from dehydration during embedding. By combining tannic acid fixation with low temperature embedding, the dual effects of dehydration and thermal denaturation during embedding should be ameliorated. A section of a crystal fixed with tannic acid and embedded using low temperature methods is presented in fig. 4b. The structural order is preserved to better than 1/20 A as judged from optical transforms (see fig. 5b). However, the absolute contrast of the section is reversed when compared with fig. 4a. Sections with positively stained areas were also obtained with Spurr-embedded crystals when certain older batches of tannin were used. In these cases, the sections generally contained both negatively and positively stained areas as well as are was with intermediate characteristics. In these instances, the positive staining was optimized by employing methanolic 2% uranyl acetate as a post-stain.
C.W. ,4key et al. / Trigonal catalase crystals
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.......
/
¢
b
109
/
¢
hi0
Fig. 5. Optical transforms from 1200 ,~ sections fixed with tannic acid. (a) Optical transform from the section shown in fig. 4a. The resolution extends to 7 diffraction lines in some directions. The expected 3 m m symmetry of the pattern is only approximate due to an optical artifact associated with the high contrast in the original image• (b) Optical transform from the section shown in fig. 4b. The resolution extends to 8 diffraction lines in some directions. The optical artifact is more marked in this transform, which displays a strong 3-fold distribution of intensities.
Optical transforms from micrographs of 1200 A thick sections imaged with an objective aperture demonstrated an unusual 3-fold intensity distribution as a result of breakdown in Friedel symmetry. The optical artifact was caused by high image contrast and the resultant variable emulsion thickness associated with extremely dense silver grain development. This results in the micrograph becoming a complex object for visible light scattering. Computed transforms were free of the artifact but maintained resolutions equivalent to those observed in optical transforms. Thicker sections were used at this point because earlier studies suggested that sections 4-5 unit cells thick were virtually free of problems caused by non-integral numbers of unit cells generated during the sectioning process [2,5,6]. Because of the large c-axis repeat of the trigonal crystals, 1000-1200 .~ thick sections were used.
Reconstructions of the (001) plane from relatively thick sections of both conventional and low temperature embedded crystals are presented in figs. 3b and 3c. The reconstructions were obtained using correlation methods and have not been symmetry-enforced. The contrast in fig. 3c was reversed to allow direct comparison with the maps of negatively stained sections. Fig. 3b is similar to the reconstructions in figs. 2a and 2b with large morphological units interconnected by crossbridges. In addition, the expected 32 symmetry is strongly preserved in the thicker sections, with alternate crossbridges about the periphery of the triangular unit demonstrating a weaker connectivity, similar to the enforced reconstruction in fig. 2b. Comparison of the reconstruction of a positively stained section in fig. 3c with the reconstruction in fig. 3b demonstrates both similarities and
110
C. 14d Akev et al. / Trigonal catalase crystals
differences. First, the large morphological units are packed in a similar manner. However, the units are not interconnected by crossbridges. Second, the substructure of the morphological units in fig. 3c is more strongly defined. Third, the 6 putative solvent channels centered on triad screw axes in the reconstruction in fig. 3b have been reduced in size along with the appearance of 6 additional channels occupying positions formerly filled by the apparent crossbridges in fig. 3c. These differences are reproducible and must be a consequence of the different types of contrast in the two sections. It is apparent at this resolution that the positively contrasted specimen is not merely a contrast-reversed image of the negatively stained structure. Further analysis and comparison of both negatively and positively contrasted specimens in three dimensions will be needed to fully evaluate differences in the two reconstructions. There are a number of possible explanations for the observed differences in the two projections. The first possibility is that both structures are faithful copies of the absolute structure in three dimensions, but that the projections differ intrinsically because of the contrast mechanisms at low resolution. In fig. 3b, the contrast is provided by stain versus protein; in fig. 3c, the contrast is provided by stain (protein) versus either plasticfilled or empty solvent channels in the crystal. The second alternative is that both structures are truly different due to intrinsic differences in the staining mechanisms. While the mechanism of negative staining in tannic-acid-fixed sections is reasonably well understood [5], the mechanism of positive staining has not been elucidated. Along these lines, further work is needed to isolate the active compounds in the complex tannin mixtures which potentiate either negative or positive staining. This is crucial since the ability to experimentally control the absolute contrast of crystal sections will greatly facilitate reliable interpretations. The problem of determining the absolute contrast of embedded specimen is not a simple one. Undoubtedly, errors have been made in the past in interpreting the embedded specimen due to a priori assumption of the incorrect contrast. This problem was circumvented in earlier studies of human yG1 ira-
munoglobulin crystals [24]. Although processed in a manner analogous to the catalase crystals studied by Longley [1], the sections were determined to be negatively stained by comparison with the projected structure of a 6 A X-ray structure [25]. Furthermore, crystal sections of the Bchl-protein from Chlorobiurn limicola prepared by conventional methods demonstrated both negatively and positively stained areas in the same sections [26]. The absolute contrast was again assigned using data from the known X-ray structure. It is possible that both negatively and positively contrasted specimens may contain complementary information about the structure. This appears to be the case with the (001) projection of trigonal catalase. The X-ray structure has shown that catalase is a flattened disk-shaped molecule of dimensions 105 × 60 × 105 A. An approximate molecular orientation of the catalase tetramer in the trigonal crystals may be deduced from the reconstruction in fig. 3c. The narrow ( - 6 0 ~,) protrusions from the large morphological units probably represent edge-on views of the disk-shaped molecule which have been resolved in the projection of the positively stained structure. Hence, the 105 × 105A surface of the tetramer may be closely aligned with the c-axis.
4. Conclusions
In this report, the crystal packing of a trigonal modification of catalase was re-investigated. Contrary to an earlier report [1], the crystal structure is composed of 6 catalase molecules packed tightly about a common triad screw axis when viewed along [001]. Information obtained from positively stained sections led to the conclusion that the tetramers are packed about the triad screw axis with their 105 × 105 ~, surfaces aligned approximately with the c-axis. This report has demonstrated the feasibility of using this crystal form as a model system for further development of methods for three-dimensional analysis of thin sections. It was also shown that resolutions of at least 20 can be obtained reproducibly either by careful selection of the tannic acid mixture used for fixation or by combining tannic acid fixation with low
c. 14I..Akey et al. / Trigonal catalase crystals
t e m p e r a t u r e embedding. Surprisingly, crystal sections prepared b y the latter m e t h o d were both well preserved a n d positively stained. The chemical basis for the contrast reversal requires further investigation, b u t the use of purified c o m p o n e n t s should eventually allow direct control of the absolute contrast of e m b e d d e d specimen. To date, a n u m ber of p r o t e i n arrays have been fixed with t a n n i c acid and e m b e d d e d b y c o n v e n t i o n a l methods [2,5,6,21,22]; in all cases, the specimen proved to be negatively contrasted based o n evidence from various sources. However, it is clear that the use of t a n n i c acid fixation does not guarantee a negatively stained specimen. The use of n o n - o p t i m a l batches of t a n n i c acid or the c o m b i n a t i o n of t a n n i c acid fixation with low temperature e m b e d d i n g can lead to structurally ordered, positively stained sections. Therefore, until purified c o m p o n e n t s of t a n n i c acid mixtures are available for fixation, a priori a s s u m p t i o n s c o n c e r n i n g the absolute contrast of crystalline specimen prepared b y the t a n n i c acid m e t h o d should n o t be made.
Acknowledgements W e wish to t h a n k T.A. Ceska for help with d e v e l o p m e n t of c o n d i t i o n s for low temperature e m b e d d i n g . We also would like to t h a n k R.H. C r e p e a u for helpful discussions d u r i n g the course of this work. The work was supported b y N a t i o n a l Science F o u n d a t i o n G r a n t PCM-7910462.
References [1] W. Longley, J. Mol. Biol. 30 (1967) 323 [2] C.W. Akey, J.K. Moffat, D.C. Wharton and S.J. Edelstein, J. Mol. Biol. 136 (1980) 19.
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[3] E. Carlemalm, R.M. Garavito and W.J. Villiger,J. Microscopy 126 (1983) 123. [4] J.-C. J~sior, EMBO J. 1 (1982) 1423. [5] C.W. Akey and S.J. Edelstein, J. Mol. Biol. 163 (1983) 575. [6] C.W. Akey, V. Spitsberg and S.J. Edelstein, J. Biol. Chem. 258 (1983) 3222. [71 P.N.T. Unwin, Nature 269 (1977) 118. [8] L.A. Amos and T.S. Baker, Intern. J. Biol. Macromolecules 1 (1979) 146. [9] A. McPherson, Jr. and A. Rich, Arch. Biochem. Biophys. 157 (1973) 23. [10] P.N.T. Unwin, J. Mol. Biol. 98 (1975) 235. [11] M.R.N. Murthy, T.J. Reid III, A. Sicignano, N. Tanaka and M.G. Rossmann, J. Mol. Biol. 152 (1981) 465. [12] H. Tauber and E.L. Petit, J. Biol. Chem. 195 (1952) 703. [13] B.W. Low and F.M. Richards, J. Am. Chem. Soc. 24 (1951) 1660. [14] A.R. Spurr, J. Ultrastruct. Res. 26 (1969) 31. [15] H. Kim, L.F. Binder and J. L. Rosenbaum, J. Cell Biol. 80 (1979) 266. [16] E.S. Reynolds, J. Cell. Biol. 17 (1963) 208. [17] R.C. Williams and H.W. Fisher, J. Mol. Biol. 52 (1970) 121. [18] P.N.T. Unwin and R. Henderson, J. Mol. Biol. 94 (1975) 425. [19] R.H. Crepeau and E.K. Fram, Ultramicroscopy 6 (1981) 7. [20] B.F. McEwen, T.A. Ceska, R.H. Crepeau and S.J. Edelstein, J. Mol. Biol. 166 (1983) 119. [21] K. Fujiwara and L.G. Tilney, Ann. NY Acad. Sci. 253 (1975) 27. [22] R.L. Garrell, R.H. Crepeau and S.J. Edelstein, Proc. Natl. Acad. Sci. USA 76 (1979) 1140. [23] B.W. Matthews, in: The Proteins, 3rd ed., Eds. Neurath and Hill (Academic Press, New York, 1975) ch. 4, p. 468. [24] L.W. Labaw and D.R. Davies, J. Biol. Chem. 246 (1971) 3760. [25] V.R. Sarma, D.R. Davies, L.W. Labaw, E.R. Silverton and W.D. Tery, Cold Spring Harbor Symp. 36 (1971) 413. [26] B.W. Matthews, R.E. Fenna and S.J. Remington, J. Ultrastruct. Res. 58 (1977) 316.
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TRIGONAL CATALASE CRYSTALS: A NEW MOLECULAR PACKING ASSIGNMENT OBTAINED FROM SECTIONS PRESERVED WITH TANNIC ACID Christopher W. AKEY, Margot SZALAY and Stuart J. EDELSTEIN Section of Biochemistry, Molecular and Cell Biology, Division of Biological Sciences, Wing Hall, Cornell University, Ithaca, New York 14853, USA Received 1 November 1983; presented at EMSA Symposium August 1983
The molecular packing of a trigonal crystal form of catalase initially studied by Longley [l] has been re-evaluated. Sections of crystals fixed and preserved with tannic acid were obtained parallel to the (001) and (100) planes. Specimens prepared by either conventional or low temperature embedding maintained 20 ,~ resolution after sectioning. The space group of the crystals is either P3~21 or P3221 and the observed unit cell parameters for (001) and (100) are a = b = 174 A,, ~, = 119 ° and b = 189 ,~, c = 248 ,~ with a = 89.5 °. Computer-based reconstructions of two principal projections coupled with crystal density measurements allowed the deduction that there is one catalase tetramer per asymmetric unit. The crystal structure consists of 6 molecules packed closely about a c o m m o n triad screw axis. This interpretation differs from that proposed by Longley [J. Mol. Biol. 30 (1967) 323], because thin sections of embedded crystals were assumed a priori to be positively stained in the early work; in actuality the sections were negatively stained. We also demonstrate that tannic acid fixation can lead to well preserved, positively stained crystal sections under certain conditions.
1. Introduction
Three methods of obtaining 20 ,~ resolution in sectioned protein crystals have recently been described: tannic acid fixation [2], low temperature embedding [3] and grid sectioning of negatively stained crystals [4]. Sectioning of protein crystals can provide a valuable adjunct to conventional methods of electron microscopy of thin crystals. The information obtained can be employed to determine the three-dimensional packing of a protein crystal [2,4-6] or to partially supplement the cone of missing data in a three-dimensional data set [7,8]. The ultimate goal in improving the structural preservation maintained in the embedded and sectioned specimen centers on the potential use of thin sectioned crystals for three-dimensional image reconstruction. To be useful for three-dimensional work, thin sections must possess suitable resolution, structural fidelity, and a known contrast. Tannic acid fixation appears to satisfy the above criteria based on studies of two-dimensional projections of crystal
sections of Pseudomonas cytochrome oxidase [2], orthorhombic beef liver catalase [5] and beef heart F1-ATPase [6]. However, a rigorous evaluation of a known structure in three dimensions is needed to define completely the fidelity of this method of sample preservation. Furthermore, a suitable model system would be beneficial in allowing an experimental evaluation of possible problems inherent in three-dimensional reconstruction of sections. Potential problems include non-integral numbers of unit cells, phase origin refinement in specimens with multiple unit cells along c, and limitations on specimen thickness as a result of multiple scattering. In order to develop methods of general applicability and to evaluate the potential usefulness of sections for three-dimensional analysis, we have focused on a trigonal crystal form of catalase initially characterized by Longley [1]. Catalase provides an excellent model system as 5-6 crystal forms are known [9] including at least 2 orthorhombic modifications suitable for conventional three-dimensional electron microscopy of nega-
0304-3991/84/$03.00 © Elsevier Science Publishers B.V. (North-Holland Physics Publishing Division)
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C 14". Akey et al. / Trigonal catalase cD,stals
tively stained specimen [4,5,10]. The space group parameters of the trigonal form employed in this study were obtained by X-ray diffraction [1], and crystals can be grown reproducibly to sizes suitable for direct manipulation and embedding. Furthermore, the X-ray structure has recently been solved at 2.5 A resolution [11], thereby allowing the determination of the fidelity of various microscopy derived structures by comparison at low resolution. In this report we verify that the space group of the crystals is P3121 (or P3221 ) and demonstrate that there are 6 molecules per unit cell packed tightly about a common triad screw axis. Based on crystal density measurements, there is one tetramer per asymmetric unit; therefore the 222 point group symmetry of the molecule is not expressed in the crystallographic packing. Hence, three-dimensional analysis will be aided by the presence of non-crystallographic symmetry. It is also demonstrated that crystal sections prepared by tannic acid can be positively stained, and under optimal conditions sections obtained by either conventional or low temperature embedding are structurally ordered to a resolution of at least 20 A.
2. Materials and methods
2.1. Crystals and density measurements Beef liver catalase was obtained as a twice-recrystallized suspension in 0.1% thymol from Sigma. The crystal form in this commercial preparation is predominantly trigonal. For thin section experiments, the trigonal prisms were recrystallized to obtain larger crystals free of inherent twinning found in the preparation. The trigonal crystals were grown by the procedure of Tauber and Petit [12], using 0.05 M potassium phosphate, pH 7.3, after the removal of thymol. Crystals grown by this procedure often exceeded 1 mm in length after 1-6 months and appeared as elongated, untwinned prisms with hexagonal cross-sections. Crystal buoyant density was measured using a linear gradient of water-saturated bromobenzene and xylene [13] with standardized CsC1 solutions for calibration.
2.2. Tannic acid fixation Over a period of 5 years, the chemical characteristics of a batch of tannin from Merck changed as judged by the predominant contrast present in crystal sections under standard embedding conditions. Therefore, we recently have screened 5 batches of commercially available tannic acid from 4 suppliers, in an attempt to optimize our results with crystal sections. Optimal results were obtained with a purified tannic acid from Mallinckrodt (lot ZCN). Crystals are now routinely fixed in 8% tannic acid, 2% glutaraldehyde in 50 mM monobasic potassium phosphate, pH 7.3, overnight at room temperature. For this choice of buffer, the fixative solution must be brought to pH with N a O H to avoid precipitation. Following fixation, crystals were washed twice with 50 mM phosphate, 50 mM glycine for 5 min, then rinsed twice with 50 mM phosphate buffer without glycine. The crystals were pre-stained with 0.5% OsO 4 in 50 mM phosphate at pH 7.3 for 1 h at room temperature. This was followed by 3 rinses in phosphate buffer. The crystals were then processed for conventional embedding using a graded dehydration series of water-ethanol, followed by infiltration with a low viscosity resin [14]. After a final infiltration in 100% resin for 12 24 h, the crystals were aligned in flatbed molds and polymerized at 60°C. 2.3. Low temperature embedding The method of low temperature embedding [3] has been combined with tannic acid fixation. The Lowicryl kit was obtained from Balzers Union. Specimens were fixed and pre-stained as described above; however, the ethanol dehydration and subsequent infiltration steps were carried out at low temperatures, essentially as described in the Balzers Union instruction manual. Some steps were modified; the initial dehydration step was done at 4°C and all subsequent steps were carried out in a sample block thermally equilibrated with dry ice-ethanol. To achieve the requisite conditions of low temperature and dryness for successful embedding, all manipulations were carried out in a glovebag under N 2 atmosphere. The samples were
c.w. Akey et al. / Trigonal catalase crystals
kept in white plastic vials which were seated in an aluminum block which fit snugly into the top of a styrofoam box (20 × 10 x 20 cm). All manipulations of the crystals were monitored directly with a stereomicroscope which was fitted to the glovebag. The resin mixture was kept dry and consisted of K 4 M A 1 g, K4MB 6.5 g, initiator C 76 mg and 35 mg dithiothrettol. Crystals were aligned in flatbed molds and the samples were polymerized by UV irradiation in a sealed Thermos cooler box. The samples were kept cold by contact with a layer of dry ice. Samples prepared without D T T did not yield sufficiently hard blocks for sectioning. The conditions described herein must be considered approximate; refinements are needed to reproducibly obtain blocks with good thin sectioning quality. 2.4. Thin sections, electron microscopy and image processing
All blocks were sectioned with a diamond knife as described previously [2]. Sections (600-1200 ,~
105
thick) were picked up on either 400 mesh or Formvar-backed slot grids. Post-staining was carried out with either 2% uranyl acetate in 35% ethanol ( v / v ) and 12.5% methanol ( v / v ) [15], or methanolic 2% uranyl acetate. Sections were also post-stained with lead citrate [16]. Images were recorded on Kodak electron image film using a Philips EM 301 and minimum beam methods [17,18,2]. Micrographs were digitized at 30 micron steps on a Syntex AD-1 densitometer. The resultant arrays were processed on our laboratory computer system using Fourier [2] or a combination of real space-Fourier space analysis [19,20].
3. Results and discussion
A trigonal modification of catalase has been chosen for development of general methods for three-dimensional analysis of thin sections. This crystal form can be grown at p H 7.3 and was analyzed by Longley [1] using X-ray diffraction and thin section methods. The space group was
C
b Fig. 1. Micrographs of thin sections along (001) and (100). (a) Thin section ( - 600 ,~ thick) cut parallel to the (001) planes. The contrast is identical to that observed by Longley [1]. (b) Thin sections cut parallel to the (100) planes. The scale bar is 500 A.
C. I'lL Akey et al. / Trigonal catalase crystals
106
,
,,
(~
F
,j
b
~.
,
d
(001)
.,
---..5
c
(100)
Cl
Fig. 2. Reconstructions of projections of the (00l) and (100) planes of trigonal catalase. Heavy contours correspond to protein. (a) Unenforced reconstruction of a thin section of (001). The unit cell is indicated. The symmetry elements in this projection are shown in fig. 3a. The most prominent features of the m a p are the large morphological units connected by 6 crossbridges. (b) Symmetry-enforced reconstruction from the same area used for fig. 2a. The crossbridges are indicated (CB). Also note that each morphological unit is surrounded by 6 channels with 3-fold symmetry. (c) Symmetry-enforced reconstruction of the (100) planes from 3 areas. The true crystallographic unit cell is indicated. The major features of the m a p correspond to the morphological units designated A, B and C which are stacked along the c-axis, parallel to the triad screw axis.
determined to be either P3~21 or P3221 with a and b equal to 173 .~ and c = 237 A, 7 = 120°. However, the initial interpretation of the crystal packing was incorrect because the assumption was made that the thin sections were positively stained. In fact, we conclude that the sections were negatively stained for reasons discussed in more detail below.
3.1. The (001) projection The (001) projection is of interest since it gives a view along the unique c-axis. Sections from crystals prepared by either the method described by Longley [1] or with tannic acid-fixation have identical contrast; however, the resolution is improved at least two-fold in the latter. A representative micrograph of the (001) projection is presented in fig. la. This crystal form has a c-axis repeat of 248 A, therefore sections 600-1200 ,~ thick are about 2.5-5 unit cells thick. Variability was encountered in reconstructions from 8 sections ( - 6 0 0 ,~ thick), undoubtedly caused by non-integral numbers of unit cells. A reconstruc-
tion from a section judged to be either 2 or 3 unit cells thick, based on apparent section thickness and symmetry preservation, is presented in fig. 2a. The symmetry-enforced reconstruction is shown in fig. 2b. The unit cell consists of a large triangular unit with interconnecting crossbridges (CB) which link the morphological units together. The thick contours have been identified as protein, based on the following considerations. First, tannic acid fixation has consistently yielded negatively stained sections of a number of macromolecular arrays including fibers of tubulin [12] and hemoglobin S [22] and crystals of Pseudomonas cytochrome oxidase [2], orthorhombic catalase [6] and beef heart F1-ATPase [6]. Second, a series of embeddings were carried out comparing both trigonal and thin orthorhombic catalase crystals under identical fixation conditions. Sections of the trigonal form demonstrated the same contrast as in fig. l a while sections of the orthorhombic form were negatively stained as deduced elsewhere [5]. The two crystal forms of catalase are expected to stain similarly, hence the trigonal crystals must be negatively stained. Further evidence is presented later,
C.w. Akey et al. / Trigonal catalase crystals in the section on positively stained sections. T h e unit cell p a r a m e t e r s f r o m 8 m i c r o g r a p h s were obt a i n e d a n d are a = b = 184 ,~ with ~ = 119 °.
107
excluded. T h e m e a s u r e d crystal d e n s i t y was 1.078 + 0.012 g / c m 3 for crosslinked crystals in water (n = 8). This c o r r e s p o n d s to a c a l c u l a t e d molecular weight of 223 k d + 34 kd, b y the m e t h o d of M a t t h e w s [23]. T h e t e t r a m e r m o l e c u l a r weight is 228 k d [11], therefore the correct value is 1 molecule p e r a s y m m e t r i c unit with 6 molecules p e r unit cell. This crystal form has a Vm of 5.3 (,~3/dalton) a n d a solvent c o n t e n t of 77%. A p a c k i n g m o d e l for the trigonal crystals with 6 molecules p e r unit cell in space g r o u p P3121 is shown schematically in fig. 3a. T h e catalase t e t r a m e r s are d r a w n as spheres for ease of representation, a n d are p a c k e d tightly a b o u t a triad axis to generate the large m o r p h o l o g i c a l unit o b s e r v e d in the (001) projection. The molecules are labelled b y height along c ( 1 - 6 ) a n d the 2 sets of 31-screw axis related molecules are 1, 3, 5 a n d 2, 4, 6. In space group P3z21, details of the p a c k i n g w o u l d differ b e c a u s e the s y m m e t r y axes l o c a t e d at 1 / 6 (see fig. 3a) w o u l d be l o c a t e d at 1 / 3 , while those shown at 1 / 3 in the d i a g r a m w o u l d be at 1 / 6 . N o t e that the (100) a n d (001) p r o j e c t i o n s in space g r o u p P3t21 are indistinguishable. T h e o n l y difference concerns the p o s i t i o n o f the 21-screw a n d 2-fold axes which
3.2. The (100) projection A m i c r o g r a p h of a section parallel to (100) is p r e s e n t e d in fig. l b . A s y m m e t r y - e n f o r c e d reconstruction f r o m 3 areas is p r e s e n t e d in fig. 2c. The true unit cell b-vector is twice the o b s e r v e d r e p e a t in this p r o j e c t i o n due to the s y m m e t r y of this space group which has a 21-screw axis a n d a 2-fold axis o v e r l a p p i n g in a direction parallel to b. The m e a s u r e d unit cell c o n s t a n t s are b = 179 ,~, c = 248 with a = 89.5 °. T h e r e are 3 m a j o r m o r p h o l o g i cal units labelled A, B a n d C stacked along the c-axis in this projection.
3.3. Packing model T h e possible n u m b e r of catalase t e t r a m e r s p e r unit cell in the trigonal space g r o u p is either 3, 6 or 12. However, b a s e d on c o n s i d e r a t i o n of the possible values of the crystal p r o t e i n specific v o l u m e [23], the value of 3 molecules p e r unit cell can be
tt
11
v
.
.
.
.
,. . . .
.
. . . . . . . .
,. . . .
~
~
,
~
;
~
Fig. 3. Packing diagram for trigonal crystals viewed along [001] and reconstructions of (001) from 1200 A thick sections. (a) Schematic diagram of crystal packing in trigonal modification of catalase, space group P3121. Tetramers are represented by spheres. Triad-screw-axis-related molecules are shaded in a similar manner and are 1, 3, 5 and 2, 4, 6. The molecules are numbered 1-6 in order of increasing height along c. (b) Reconstruction of (001) from section in fig. 4a. The expected 32 symmetry is strongly preserved in the unenforced reconstruction. The section was approximately 4-5 unit cells thick. (c) Reconstruction of (001) from section in fig. 4b. The 32 symmetry is again strongly preserved in the unenforced reconstruction. The molecular edges (M) of the disk-shaped catalase tetramer appear to be resolved near the periphery of the morphological units. Note that there are now 12 channels of 15 ,~, diameter surrounding a given morphological unit. The horn-like (H) features which occur on alternate molecules around the periphery of the morphological unit may be an artifact caused by multiple scattering or may represent a real feature of this projection,
108
C. 144 Akev et al. / Trigonal catalase crystals
overlap in projection. In the (100) projection this overlap occurs at c = + 1 / 3 and + 5 / 6 (see fig. 2c). The three morphological units in the (100) projection probably arise from superposition of two catalase tetramers, viewed in projection. A complete three-dimensional analysis should reveal the correct space group and the structure of the catalase tetramer preserved by tannic acid fixation. 3.4. Improved resolution in sections
Two approaches were investigated in an attempt to improve the structural order maintained in crystal sections. The two methods consisted of determining the best source of tannic acid for fixation and combining tannic acid fixation with low temperature embedding. Initially, tannic acid
8
Fig. 4. Micrographs of 1200 ~, thick sections of trigonal catalase. (a) Section cut parallel to (001). Crystal was fixed with tannic acid and embedded by conventional methods. The section is negatively contrasted and appears similar to those obtained by Longley [1]. (b) Section cut parallel to (001), Crystal was fixed with tannic acid and embedded using low temperature methods. The section is positively stained. The clarity of the structural preservation is good enough to count the 12 "'channels" which encircle a morphological unit. The mean diameter of the channels is 15 ,~ and is a measure of the resolution. Scale bar is 1000 ~,.
obtained from Merck was used in thin section studies of Pseudomonas cytochrome oxidase [2!, and gave negatively stained sections with 20 A resolution. The same batch, when used with trigonal catalase crystals, rarely preserved the structural order !o better than 35 A; hence~ this system was chosen for further studies aimed at improving resolution in sections. As described earlier, various tannin mixtures were screened and a batch which consistently gave 20 A resolution with the trigonal crystals was obtained. The quality of optical transforms of sections was also improved by employing a 100 micron objective aperture during imaging to enhance image contrast. In earlier studies no objective aperture was used (see ref (18]). An optical transform of the section ( - 1200 A thick) in fig. 4a is presented in fig. 5a and demonstrates reflections to 1//20 A resolution. Protein crystals become disordered during preparation of thin sections as a result of three major factors; dehydration damage, thermal denaturation and damage arising from the sectioning process. Diffraction studies with orthorhombic crystals [5] have demonstrated that tannic acid can protect the protein structure from dehydration, in a manner analogous to glucose embedding [18]. It was therefore reasoned that tannic acid might be partially protecting the crystal structure from dehydration during embedding. By combining tannic acid fixation with low temperature embedding, the dual effects of dehydration and thermal denaturation during embedding should be ameliorated. A section of a crystal fixed with tannic acid and embedded using low temperature methods is presented in fig. 4b. The structural order is preserved to better than 1/20 A as judged from optical transforms (see fig. 5b). However, the absolute contrast of the section is reversed when compared with fig. 4a. Sections with positively stained areas were also obtained with Spurr-embedded crystals when certain older batches of tannin were used. In these cases, the sections generally contained both negatively and positively stained areas as well as are was with intermediate characteristics. In these instances, the positive staining was optimized by employing methanolic 2% uranyl acetate as a post-stain.
C.W. ,4key et al. / Trigonal catalase crystals
II
.......
/
¢
b
109
/
¢
hi0
Fig. 5. Optical transforms from 1200 ,~ sections fixed with tannic acid. (a) Optical transform from the section shown in fig. 4a. The resolution extends to 7 diffraction lines in some directions. The expected 3 m m symmetry of the pattern is only approximate due to an optical artifact associated with the high contrast in the original image• (b) Optical transform from the section shown in fig. 4b. The resolution extends to 8 diffraction lines in some directions. The optical artifact is more marked in this transform, which displays a strong 3-fold distribution of intensities.
Optical transforms from micrographs of 1200 A thick sections imaged with an objective aperture demonstrated an unusual 3-fold intensity distribution as a result of breakdown in Friedel symmetry. The optical artifact was caused by high image contrast and the resultant variable emulsion thickness associated with extremely dense silver grain development. This results in the micrograph becoming a complex object for visible light scattering. Computed transforms were free of the artifact but maintained resolutions equivalent to those observed in optical transforms. Thicker sections were used at this point because earlier studies suggested that sections 4-5 unit cells thick were virtually free of problems caused by non-integral numbers of unit cells generated during the sectioning process [2,5,6]. Because of the large c-axis repeat of the trigonal crystals, 1000-1200 .~ thick sections were used.
Reconstructions of the (001) plane from relatively thick sections of both conventional and low temperature embedded crystals are presented in figs. 3b and 3c. The reconstructions were obtained using correlation methods and have not been symmetry-enforced. The contrast in fig. 3c was reversed to allow direct comparison with the maps of negatively stained sections. Fig. 3b is similar to the reconstructions in figs. 2a and 2b with large morphological units interconnected by crossbridges. In addition, the expected 32 symmetry is strongly preserved in the thicker sections, with alternate crossbridges about the periphery of the triangular unit demonstrating a weaker connectivity, similar to the enforced reconstruction in fig. 2b. Comparison of the reconstruction of a positively stained section in fig. 3c with the reconstruction in fig. 3b demonstrates both similarities and
110
C. 14d Akev et al. / Trigonal catalase crystals
differences. First, the large morphological units are packed in a similar manner. However, the units are not interconnected by crossbridges. Second, the substructure of the morphological units in fig. 3c is more strongly defined. Third, the 6 putative solvent channels centered on triad screw axes in the reconstruction in fig. 3b have been reduced in size along with the appearance of 6 additional channels occupying positions formerly filled by the apparent crossbridges in fig. 3c. These differences are reproducible and must be a consequence of the different types of contrast in the two sections. It is apparent at this resolution that the positively contrasted specimen is not merely a contrast-reversed image of the negatively stained structure. Further analysis and comparison of both negatively and positively contrasted specimens in three dimensions will be needed to fully evaluate differences in the two reconstructions. There are a number of possible explanations for the observed differences in the two projections. The first possibility is that both structures are faithful copies of the absolute structure in three dimensions, but that the projections differ intrinsically because of the contrast mechanisms at low resolution. In fig. 3b, the contrast is provided by stain versus protein; in fig. 3c, the contrast is provided by stain (protein) versus either plasticfilled or empty solvent channels in the crystal. The second alternative is that both structures are truly different due to intrinsic differences in the staining mechanisms. While the mechanism of negative staining in tannic-acid-fixed sections is reasonably well understood [5], the mechanism of positive staining has not been elucidated. Along these lines, further work is needed to isolate the active compounds in the complex tannin mixtures which potentiate either negative or positive staining. This is crucial since the ability to experimentally control the absolute contrast of crystal sections will greatly facilitate reliable interpretations. The problem of determining the absolute contrast of embedded specimen is not a simple one. Undoubtedly, errors have been made in the past in interpreting the embedded specimen due to a priori assumption of the incorrect contrast. This problem was circumvented in earlier studies of human yG1 ira-
munoglobulin crystals [24]. Although processed in a manner analogous to the catalase crystals studied by Longley [1], the sections were determined to be negatively stained by comparison with the projected structure of a 6 A X-ray structure [25]. Furthermore, crystal sections of the Bchl-protein from Chlorobiurn limicola prepared by conventional methods demonstrated both negatively and positively stained areas in the same sections [26]. The absolute contrast was again assigned using data from the known X-ray structure. It is possible that both negatively and positively contrasted specimens may contain complementary information about the structure. This appears to be the case with the (001) projection of trigonal catalase. The X-ray structure has shown that catalase is a flattened disk-shaped molecule of dimensions 105 × 60 × 105 A. An approximate molecular orientation of the catalase tetramer in the trigonal crystals may be deduced from the reconstruction in fig. 3c. The narrow ( - 6 0 ~,) protrusions from the large morphological units probably represent edge-on views of the disk-shaped molecule which have been resolved in the projection of the positively stained structure. Hence, the 105 × 105A surface of the tetramer may be closely aligned with the c-axis.
4. Conclusions
In this report, the crystal packing of a trigonal modification of catalase was re-investigated. Contrary to an earlier report [1], the crystal structure is composed of 6 catalase molecules packed tightly about a common triad screw axis when viewed along [001]. Information obtained from positively stained sections led to the conclusion that the tetramers are packed about the triad screw axis with their 105 × 105 ~, surfaces aligned approximately with the c-axis. This report has demonstrated the feasibility of using this crystal form as a model system for further development of methods for three-dimensional analysis of thin sections. It was also shown that resolutions of at least 20 can be obtained reproducibly either by careful selection of the tannic acid mixture used for fixation or by combining tannic acid fixation with low
c. 14I..Akey et al. / Trigonal catalase crystals
t e m p e r a t u r e embedding. Surprisingly, crystal sections prepared b y the latter m e t h o d were both well preserved a n d positively stained. The chemical basis for the contrast reversal requires further investigation, b u t the use of purified c o m p o n e n t s should eventually allow direct control of the absolute contrast of e m b e d d e d specimen. To date, a n u m ber of p r o t e i n arrays have been fixed with t a n n i c acid and e m b e d d e d b y c o n v e n t i o n a l methods [2,5,6,21,22]; in all cases, the specimen proved to be negatively contrasted based o n evidence from various sources. However, it is clear that the use of t a n n i c acid fixation does not guarantee a negatively stained specimen. The use of n o n - o p t i m a l batches of t a n n i c acid or the c o m b i n a t i o n of t a n n i c acid fixation with low temperature e m b e d d i n g can lead to structurally ordered, positively stained sections. Therefore, until purified c o m p o n e n t s of t a n n i c acid mixtures are available for fixation, a priori a s s u m p t i o n s c o n c e r n i n g the absolute contrast of crystalline specimen prepared b y the t a n n i c acid m e t h o d should n o t be made.
Acknowledgements W e wish to t h a n k T.A. Ceska for help with d e v e l o p m e n t of c o n d i t i o n s for low temperature e m b e d d i n g . We also would like to t h a n k R.H. C r e p e a u for helpful discussions d u r i n g the course of this work. The work was supported b y N a t i o n a l Science F o u n d a t i o n G r a n t PCM-7910462.
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[3] E. Carlemalm, R.M. Garavito and W.J. Villiger,J. Microscopy 126 (1983) 123. [4] J.-C. J~sior, EMBO J. 1 (1982) 1423. [5] C.W. Akey and S.J. Edelstein, J. Mol. Biol. 163 (1983) 575. [6] C.W. Akey, V. Spitsberg and S.J. Edelstein, J. Biol. Chem. 258 (1983) 3222. [71 P.N.T. Unwin, Nature 269 (1977) 118. [8] L.A. Amos and T.S. Baker, Intern. J. Biol. Macromolecules 1 (1979) 146. [9] A. McPherson, Jr. and A. Rich, Arch. Biochem. Biophys. 157 (1973) 23. [10] P.N.T. Unwin, J. Mol. Biol. 98 (1975) 235. [11] M.R.N. Murthy, T.J. Reid III, A. Sicignano, N. Tanaka and M.G. Rossmann, J. Mol. Biol. 152 (1981) 465. [12] H. Tauber and E.L. Petit, J. Biol. Chem. 195 (1952) 703. [13] B.W. Low and F.M. Richards, J. Am. Chem. Soc. 24 (1951) 1660. [14] A.R. Spurr, J. Ultrastruct. Res. 26 (1969) 31. [15] H. Kim, L.F. Binder and J. L. Rosenbaum, J. Cell Biol. 80 (1979) 266. [16] E.S. Reynolds, J. Cell. Biol. 17 (1963) 208. [17] R.C. Williams and H.W. Fisher, J. Mol. Biol. 52 (1970) 121. [18] P.N.T. Unwin and R. Henderson, J. Mol. Biol. 94 (1975) 425. [19] R.H. Crepeau and E.K. Fram, Ultramicroscopy 6 (1981) 7. [20] B.F. McEwen, T.A. Ceska, R.H. Crepeau and S.J. Edelstein, J. Mol. Biol. 166 (1983) 119. [21] K. Fujiwara and L.G. Tilney, Ann. NY Acad. Sci. 253 (1975) 27. [22] R.L. Garrell, R.H. Crepeau and S.J. Edelstein, Proc. Natl. Acad. Sci. USA 76 (1979) 1140. [23] B.W. Matthews, in: The Proteins, 3rd ed., Eds. Neurath and Hill (Academic Press, New York, 1975) ch. 4, p. 468. [24] L.W. Labaw and D.R. Davies, J. Biol. Chem. 246 (1971) 3760. [25] V.R. Sarma, D.R. Davies, L.W. Labaw, E.R. Silverton and W.D. Tery, Cold Spring Harbor Symp. 36 (1971) 413. [26] B.W. Matthews, R.E. Fenna and S.J. Remington, J. Ultrastruct. Res. 58 (1977) 316.