high-resolution electron microscopy of glycoproteins… · high-resolution electron microscopy of...

jf. Cell Sci. 51, 295-321 (1981) 295Printed in Great Britain © Company of Biologists Limited 1981

HIGH-RESOLUTION ELECTRON MICROSCOPY

OF GLYCOPROTEINS: THE CRYSTALLINE

CELL WALL OF LOBOMONAS

K. ROBERTS, P. J. SHAW AND G. J. HILLSJohn limes Institute, Colney Lane, Norwich NR4 yUH, U.K.

SUMMARY

Lobomonas piriformis is a member of an order of green algae (Volvocales) that have crystallineglycoprotein cell walls. As part of a program of investigation of these glycoproteins and theirarchitecture we have studied the cell wall of Lobomonas by a variety of chemical, electron-microscopical and image-analysis techniques. Lobomonas and Vitreochlamys incisa show a verysimilar structure in their cell walls and represent 1 of the 4 classes into which all the structuresof the walls of these algae that we have so far examined fall. The 2 classes that we have previouslystudied in detail, represented by Chlamydomonas reinhardii and Chlorogonium elongatum, havea crystalline component of the wall that is a more or less smooth continuous surface overlyingan amorphous inner wall layer. Although Lobomonas also has this 2-layer structure, the crystal-line layer consists of distinct plate3, each of which is built around a single, very coherent crystallattice. The polar nature of the architecture of the cell wall is shown by sectioning and byexamination of the cell-wall surface by metal-shadowing of carbon replicas, both of intact cellsand of isolated cell-wall plates.

There are great similarities in chemical composition between the glycoproteins of the cellwall of C. reinhardii and those of Lobomonas. Both have a large content of hydroxyproline intheir amino acid composition and a sugar/hydroxyproline ratio of about 60, and both containsugar sulphates. Lobomonas however has a large gluco3e content, whereas Chlamydomonas hasalmost none. Electron micrographs of walls stained with methylamine tungstate and shadowedspecimens show that the Lobomonas crystal structure is entirely different from that of C.reinhardii, and that there is a distinctly different structure in the centre of the plates from thatat their edges, although the transition between the 2 areas occurs with no distortion of thecrystal lattice. Computer image analysis has been used to calculate reconstructed images of the2 areas, and by using minimal-dose techniques has yielded 2-dimensional maps of the negativelystained structure at a resolution of 1 -8 nm. The 2-sided plane group of both areas of the crystalis P2, and the centre area contains 2 distinct structural units, both centred on dyad axes,together with other more complex features. In the edge structure, one of the structural unitsappears unchanged, but the other unit has a considerably different appearance. The most likelyinterpretation of this is as a conformational or positional change in one of the subunits. However,because the underlying lattice is so accurately maintained across this transition, it seems prob-able that the basic structural arrangement that defines the lattice is common to the 2 areas.Some of the computational and mathematical techniques used in the image analysis have notbeen previously published and are described in detail and compared with published techniquesin an Appendix.

INTRODUCTION

The green algae of the order Volvocales have a cell wall characterized by a crystallineglycoprotein lattice. The glycoproteins involved show remarkable similarity to thehydroxyproline-containing glycoproteins characteristic of higher plant cell walls(Lamport, 1980) and provide a unique model system for studying their structure in

296 K. Roberts, P. J. Shaw and G. J. Hills

detail. The cell-wall architecture is species-dependent and at present 4 distinct types ofcell wall have been described on the basis of their crystal structures (Roberts, 1974).(In fact, 5 cell-wall groups were originally described but one of these, Chlamydomonasangulosa, has now been re-examined and found to be the same as class I, i.e. Chlamydo-monas reinhardii. This means that there are now only 4 classes represented by thefollowing species: class I, C. reinhardii; class II, Chlorogonium elongatum; class III,Chlamydomonas asymmetrica and class IV, Lobomonas piriformis.) It is proposed thatthese 4 basic cell-wall types have not only taxonomic but also evolutionary significance.For example, all the multicellular species examined (Volvox, Pandorina, Eudorina) havea cell-wall type (class I) identical to the single-celled C. reinhardii (Roberts, 1974;Roberts, Gurney-Smith & Hills, 1972; Hills, Gurney-Smith & Roberts, 1973), suggest-ing a common ancestral origin for these species. Most algae examined have a class IIcell-wall structure, which is postulated as being the original ancestral type of wall fromwhich the others have evolved (Roberts & Hills, 1976). The other 2 groups of algae(III and IV), which contain few representatives, have so far received only a briefdescriptive treatment (Roberts, 1974). This paper provides a more rigorous structuraland biochemical analysis of one of these remaining cell-wall classes (IV), using as thetype organism L. piriformis. A similar cell wall is found in Vitreochlamys incisa (formerlyChlamydomonas incisa) and a preliminary description of both has been published(Roberts, 1974). The Lobomonas cell wall consists of relatively rigid crystalline plates,with a high degree of long-range order, covering the outer surface of a thin amorphousinner cell-wall layer, and we concentrate here on a description of the glycoproteinarrangement within a 2-dimensional projection of the crystalline layer to a resolution ofi-8 nm. This has been determined by a combination of low-dose electron microscopyand digital image processing. The results are discussed in terms of the suggestion thatthe glycoproteins found in these algal cell walls are the ancient ancestors of thehydroxyproline-containing cell-wall glycoproteins and arabinogalactan proteins ofhigher plants (Lamport, 1980).

MATERIALS AND METHODS

Cultures of L. piriformis (Piingsheim) (strain 45/i) and V. incisa (Pringsheim) (strain 11/10,were obtained from the Culture Centre of Algae and Protozoa, 36 Storeys Way, Cambridge)U.K. Cells were grown in sterile conditions in liquid culture without aeration, using a Tris-buffered, yeast and peptone-supplemented medium (Catt, Hills & Roberts, 1978) undercontinuous illumination (80 /iEinsteins mol"1 s"1 in the waveband of 400-700 nm). Cell wallswere harvested from the medium (as cast-off mother cell walls) by differential centrifugation asdescribed by Hills, Phillips, Gay & Roberts (1975).

Negatively stained specimens were made on 400-mesh copper grids coated with thin evapo-rated carbon films stripped from freshly cleaved mica. Stains used were 5 % ammonium molyb-date, 5 % sodium tungstate (pH 6-8), or 2-5 % methylamine tungstate (Faberg6 & Oliver, 1974).Rotary-shadowed specimens were made using platinum/palladium at an angle of 50. For thinsections, walls and cells were fixed either by the method of Franke, Krien & Malcolm-Brown(1969) or using a modified tannic acid procedure (Catt et al. 1978). Subsequent processing isdescribed elsewhere (Roberts et al. 1972). Specimens were examined in either an AEI EM6B, aJEOL JEM 100B or a Siemens 102 electron microscope, all calibrated using catalase. Opticaldiffraction was carried out as described previously (Roberts et al. 1972). For routine examinationnormal doses of 5000-10000 electrons per square nm (e/nm1) were used. For high-resolution

Crystalline cell wall of Lobomonas 297

negative-stained pictures, minimal-dose conditions (500-1000 e/nm1) were used (see Hills,1981).

Images were selected for further processing by examining their optical diffraction patterns.The selected images had sharp unsplit spots extending to a resolution of about z nm (up to 9orders in b*), and focussing was such that the first zero of the contrast transfer function (Erickson,1973) occurred at a higher resolution than the highest resolution spots. The effect of the con-trast transfer function could therefore be neglected.

Computer processing was carried out on a DEC PDP 11/60 mini-computer using one 28Mbyte disc (RK07), running the RSX 11M operating system. Programs were either writtenspecifically for this machine or modified to run on it. Most programs were written to take theirinput parameters and file specifications from the command line used to involve them. Thiswas so as to take advantage of the indirect command-file feature of the operating system; filescontaining sequences of image-processing operations could be set up and run as a single job or,alternatively, the programs could be invoked interactively as image-processing 'commands'.This approach offers a convenient and easily implemented compromise between image-processing systems consisting of' stand-alone' programs (Unwin & Henderson, 1975; Crowther& Klug, 1975) and elaborate fully integrated systems with specialized console monitor programssuch as SPIDER (Frank & Shimkin, 1978) and SEMPER (Saxton, Pitt & Homer, 1979). Allimages (scanned, intermediate or reconstructed) were stored in a common format, and programswere written so as to be able to operate on any image as far as possible. Some programs, such asscanning, trimming and interpolation, operate only on real images; others, such as spot latticerefinement, and amplitude and phase calculation, only on transform images. Other programswere written so as to operate on either type of image: grey-scale output; contour output;fast Fourier transformation (FFT). A final set of programs, for example, origin refinement,symmetry averaging and film-to-film scaling, operates on files comprising lists of indices,amplitudes and phases.

Images were scanned and stored on magnetic tape using, initially, a Scandig rotating-drumdensitometer (courtesy of W. O. Saxton) or, later, directly read onto disk using a flat-bedcomputer-controlled densitometer constructed in this laboratory (Shaw, Garner & Parker,1981). The scanning sample interval was matched as closely as possible to one third of theminimum spatial period required (i.e. about 0 7 nm, since the diffraction maxima extended to2 0 nm). Generally 512 by 512 points were scanned, although this was occasionally less whenonly a smaller area of the image was suitable for analysis. A mixed radix F F T program wasused: an implementation for this computer of the routines written by Ten Eyck (1973). Floatingof the images (De Rosier & Moore, 1970) was not generally necessary since the image filled thewhole of the Fourier transform interval and so there were no large edge effects. Occasionallythere were slight optical density gradients across the image, which gave rise to small originstreaks in the transform. In these cases a floating procedure was used in which a plane was fittedto the data by a least-squares calculation and then subtracted so as to give a mean opticaldensity of zero across the image. In practice this made very little difference to the final results.

The reciprocal lattices were determined and refined, and values for the amplitudes and phasesof the peaks were generally determined by a profile-fitting procedure. This differs from otherpublished methods and is compared with them in the Appendix. The best data sets were averagedtogether for the final maps of the negative-stained structure, and the 2-fold symmetry wasimposed exactly by setting the phases to the nearest theoretical phase for a 2-fold symmetricstructure (o° or 180°).

For scanning electron microscopy, cells were fixed, dehydrated, carbon dioxide critical-pointdried from amyl acetate (Anderson, 1951), coated with carbon and gold, and viewed in aPhillips type 501 scanning electron microscope. Light microscopy was carried out on a ZeissPhotomicroscope II.

Polyacrylamide gel electrophoresis on 5 % gels has been described (Catt, Hills & Roberts,1976) and carbohydrate assays were by the phenol/sulphuric acid method (Dubois et al. 1956).Hydroxyproline and other assays were as previously described (Roberts, 1979). Sugars wereestimated by gas chromatography of their trimethylsilylated derivatives (Bhatti, Chambers &Clamp, 197°) on a column of 3 % SE30 on GasChromQ. Amino acids were analysed as theirisobutyl iV(0)-heptafluorobutyl esters on the same column (MacKenzie & Tenaschuk, 1975).Sulphate content was determined using the barium chloride/gelatin technique (Dodgson &Price, 1962).


r-r-1; , ^ ; ,- 5


RESULTS AND DISCUSSION

General morphology

L. piriformis is an approximately spherical biflagellate green alga of the familyChalmydomonacae and under the culture conditions used ranges in size from 10-20fim in diameter (Fig. 1). When the cells divide, 4 daughter protoplasts are usuallyformed within the mother cell wall, which splits and releases the progeny cells. Themother cell walls remain free in the growth medium and under the light microscopereveal a faceted surface (Fig. 2). Both in scanning electron micrographs (Fig. 3) and inthin sections it can be seen that these facets reflect the fact that the wall is made upfrom a series of abutting plates (Fig. 6). By differential centrifugation the cast-off cellwalls can be separated from the cells and provide the highly pure cell-wall preparations(Fig. 4) that were used for all the work described here. Thin sections of cell wallsfixed in the presence of tannic acid reveal further details of cell-wall construction(Figs. 8-11). Tannic acid was used as a cofixative to enhance the strong subsequentpositive staining of the negatively charged carbohydrate-rich wall. The plates, ofwhich there may by 50-400 to a cell, vary in size between 2 and 5 /<m across. Insection each plate shows a basic pattern of construction analogous to that found in 2of the other cell-wall classes, C. reinhardii (Roberts et al. 1972) and Chlorogoniumelongatum (Roberts & Hills, 1976), although these cases show no evidence of separateplates in the wall. The common element is an outer wall layer of constant thickness,which shows crystalline order, and an amorphous inner wall layer of variable thickness.The outer wall layer (Figs. 8-11) is about 26 nm in total thickness and consists of abilaminar structure with an electron-dense outer leaflet about 7 nm thick and an inner

Fig. 1. Phase-contrast optical micrograph of a cell of L. piriformis. x 1500.Fig. 2. Phase-contrast optical micrograph of a discarded cell wall of Lobomoiias showingthe characteristic faceted appearance, x 1500.Fig. 3. Scanning electron micrograph of a critical-point dried cell wall showingdetails of the facets, x 3000.Fig. 4. Optical micrograph of a Lobomonas cell wall preparation, x 500.Fig. 5. SDS/polyacrylamide gels stained with periodic acid-Schiff reaction, which isspecific for reducing sugars. A. C. reinhardii cell walls for comparison, B. L. piriformiscell walls minus /?-mercaptoethanol. c. L. piriformis cell walls incubated at 100 °C with/?-mercaptoethanol.Fig. 6. Section of a whole cell of Lobomonas showing the faceted appearance of the cellwall and its construction from separate plates, x 5000.Fig. 7. Basal body of the flagellum of Lobomonas showing the ribbed flagellar collar,x 75000.Figs. 8-11. Thin sections of tannic acid fixed cell walls in situ on the cell (8, 9, 11) andisolated from the medium (10). Figs. 8, 9 ( x 50000) clearly show the boundary of theindividual plate (arrows), the amorphous inner wall layer (1), and the bilaminarstructure of the outer wall layer (o). Periodicities can be seen in the outer leaflet of theouter wall layer and also (in Fig. 11, x 90000) in the central zone of the outer walllayer (3 arrows in Fig. 11). Fig. 10 ( x 110000) shows that the cast-off mother cell wallstend to lose much of the amorphous inner-wall layer leaving just the asymmetricbilaminar structure of the outer wall layer (0).

goo K. Roberts, P. J. Shaw and G. J. Hills


leaflet about 6-7 nm thick. These 2 leaflets are separated by a region of lower density.In both this gap region, and in the other leaflet, periodicities are clearly seen, theappearance and dimensions of which vary with the angle of section (Figs. 8-11). Eachplate has a clearly defined edge where it abuts neighbouring plates. Around the baseof each flagellum, and continuous with the cell wall, is a cylindrical collar in whichdistinct periodicities are visible (Fig. 7). The construction of this collar and the numberof striations contained corresponds closely to that described in C. reinhardii (Roberts,Phillips & Hills, 1975) and contrasts with the less-structured collar seen in the pre-sumably more primitive class II algae such as Chlorogonium elongation (Roberts &Hills, 1976).

Micrographs of negatively stained cell walls show that each cell wall plate has acrystalline structure (Fig. 12) that is quite different from that of the other cell wallclasses. Of the negative stains tried, methylamine tungstate gave consistently goodimages as judged by optical diffraction and was chosen as the negative stain for allsubsequent work. Optical diffraction analysis also showed that the centre of manycell-wall plates has a different structure from the edge. Figs. 13 and 14 show enlargedareas of the edge and centre, respectively. Although the appearance of the 2 areas isdifferent in detail, the unit cell parameters are the same for the 2 areas (a = 24 nm,b = 16 nm, y = 109°) and the crystal lattice is continuous. The unit cell, which has avolume of approximately 7000 nm3, is larger than those of the other algae we haveexamined in detail. The large number of unit cells per cell-wall plate (10000-50000),together with the fact that optical diffraction analysis showed the long-range orderwithin each plate to be exceptionally high, makes high-resolution analysis possible.

Directional and rotary shadowing of carbon replicas of the cell walls with platinum/palladium also clearly reveals the 2 crystalline domains within most plates (Figs. 15—17). Replicas of whole cells show the presence of non-crystalline material at the bound-ary where 2 or more plates abut (Fig. 18). Since we can be certain we are looking at theouter face with such replicas, we can deduce the absolute configuration of the 2-sidedlattice. All the plates that we have examined so far have the same absolute configura-tion. All the images and reconstructions we present are as if viewed from outside thecell. The 2 sides of the bilaminar outer wall layer can be seen in Fig. 22, which shows afolded piece of cell wall; the optical diffraction patterns of the edge region of bothsides (a and b) are shown in Figs. 23 and 24. We assume that the side that shows littleevidence of crystalline structure arises from the amorphous material of the innerleaflet of the outer wall layer as the plate dries down obscuring its crystalline structure.The alternative explanation, that the structure is just very smooth, contradicts theevidence from thin sections. All the images of replicas analysed further are from theouter face of the plate.

Fig. 12. Negatively stained cell-wall plate taken under normal-dose conditions(5000-10000 e/nm1). Differences in appearance are evident betweenthe central andedge areas. Enlargements of the boxed areas are shown in Figs. 13, 14. x 50000.

Fig. 13. Enlargement of the marked area of the edge, x 180000.

Fig. 14. Enlargement of the marked area of the centre, x 180000.


V

• ; • • • * '

: C

• *

c

. - < - . • . * • •


Chemical composition

Sodium dodecyl sulphate/polyacrylamide gel electrophoresis of the Lobomonaswalls shows a more complex pattern than C. reinhardii. Staining for carbohydrate andprotein in paired gels reveals 4 clear bands for C. reinhardii, but for Lobomonas 1major glycoprotein together with about a dozen lesser bands, which do not appear to bein stoichiometric amounts (Fig. 5 A, B). When the Lobomonas wall samples wereincubated with /5-mercaptoethanol the position of several of the minor bands, but notthe major band, changed, indicating the presence of disulphide bridges linking someof the sub units (Fig. 5 c). Extensive studies of the C. reinhardii cell-wall glycoproteinhave shown that it is characterized by short oligosaccharide side-chains and portions ofpolypeptide chain rich in hydroxyproline (Roberts, 1979; Homer & Roberts, 1979;Catt et al. 1976, 1978). Table 1 presents a comparison of the amino acid compositions ofC. reinhardii and Lobomonas. The overall pattern of composition is very similar and,notably, the hydroxyproline content of Lobomonas is also very large. The sugar/hydroxyproline ratio for Lobomonas is 6-o, suggesting again short oligosaccharideside-chains; however, the sugar composition is quite different from that in C. rein-hardii (see Table 2 of O'Neill & Roberts, 1981). As in Chlamydomonas, however, asimilar low percentage ( I - I %) of sulphate is found (Roberts, Gray & Hills, 1980).

Crystal structure

The computer analysis of the images is discussed in detail in the Appendix. Briefly,values for the amplitudes and phases of the spots were extracted from the Fouriertransforms by a peak-profile analysis procedure. Spots were regarded as significantif the peak to least-squares residual ratio (see Appendix) was at least 2-0 and only thesespots were used in reconstruction or further processing.

Our initial analyses of images of negatively stained cell walls showed that consider-ably higher resolution was obtainable when minimal electron-dose techniques (Williams& Fisher, 1970) were used. With normal doses (estimated 5000—10000 e/nm*)approximately 4 orders in b* were seen in diffraction patterns, whereas with minimaldoses (500-1000 e/nm2) 9 orders could be obtained with optimal focussing. Figs. 26and 27 show reconstructed images from normal and minimal-dose micrographs. Forthe purpose of comparison, in the minimal-dose reconstruction only data from thosespots also present in the normal-dose transform have been used, so that the resolutionis the same in both cases. As both pictures have been normalized to the same (low)resolution they should look the same if irradiation had no effect (cf. values for agree-ment between different films in Table 3), but it is clear that the higher electron dose

Fig. 15. A carbon replica of a cell-wall plate rotary-shadowed with platinum/palladium.The differences between the 2 areas are more marked than with negative staining.Enlargements of the boxed areas are shown in Figs. 16, 17. x 40000.Fig. 16. Enlargement of the marked area of the edge, x 130000.Fig. 17. Enlargement of the marked area of the centre, x 130000.Fig. 18. Part of a rotary-shadowed replica of the whole cell showing where 3 cell-wallplates abut. The centre structure of each plate is labelled c.

3°4 K. Roberts, P. J. Shaw and G. J. Hills

•22


has caused a significant change in the stain distribution as well as degrading theresolution. The peaks (regions of stain exclusion) have become somewhat higher andmore rounded in character and the general impression is of a less-continuous structure.These observations would be consistent with a general contraction of the stain uponirradiation, although some stain migration is also probable. Images taken with stilllower electron doses (50-100 e/nm2) (Fig. 25), which are sufficiently low to preservethe protein structure (Unwin & Henderson, 1975), show the same structure as theminimal-dose pictures. Therefore, we may conclude that these minimal-dose imagesrepresent the undamaged negative-stain picture.

For the final negative-stain images shown in Fig. 29 the 3 best minimal-dose centreand edge images of the many analysed have been averaged. Statistics on the averagingare presented in Table 3. The mean phase-error between the different films is com-parable to the mean discrepancy of the average phases from o° or 1800, confirming thepresence of a 2-fold axis, at least to the resolution we have so far obtained. When thisinformation is added to that obtained from sections and replicas of folded walls wededuce that the 2-sided plane group (Holser, 1958) is P2. For all the reconstructionimages shown, therefore, we have imposed the 2-fold symmetry by setting the phasesto the ideal phases (0° or 1800) nearest those experimentally determined. ComputedFourier transforms, displayed as intensity plots, of the centre and edge structures, asseen with both negative stain and with rotary shadowing of carbon replicas, are shownin Fig. 28. The diffraction maxima for the negative-stained centre images extend toabout i-8 nm; the resolution in the edge images is somewhat less, reflecting, pre-sumably, a slightly less-ordered structure. We cannot say whether such a lowerordering is inherent or is an artefact of the specimen preparation. Distortions in thelattice might be expected to be greatest at the edges of the wall plates and splitting issometimes visible at the edge of isolated plates. In general, however, the plates areremarkably coherent single crystals, often with no visible dislocations and imperfections.The transition from centre to edge apparently occurs with no disruption of the crystallattice (Fig. 19).

In interpreting the negative-stained reconstructed maps it must be remembered thatthese pictures are projections of the 3-dimensional structure onto a plane. In inter-preting such a complicated structure some caution is necessary. The central area

Fig. 19. Rotary-shadowed replica showing lines of centre structure interdigitating withthe edge structure, x 200000.Fig. 20. Computer reconstruction of the rotary-shadowed centre structure (cf. Fig.17). Contours have been drawn at equal, arbitrary intervals, and regions of high metaldeposition have been indicated by dark shading.Fig. 21. Computer reconstruction of the rotary-shadowed edge structure (cf. Fig. 16).Fig. 22. Rotary-shadowed replica of a folded cell-wall plate showing both sides. Theareas marked a and b have been used for the optical diffraction patterns in Figs. 23, 24.x 40000.

Fig. 23. Optical diffraction pattern of area a that corresponds to the external face ofthe wall.Fig. 24. Optical diffraction pattern of area b that corresponds to the internal face ofthe wall.


Table i. Amino acid composition of the cell walls of C. reinhardii and of L. piriformis

Total amino acids (mole %)

ino acid

AlaGlyValThrSerLeuHeProHypAspPheGluLysTyrArgHis

Chlamydomonas

9 0

1 2 76-56 06-664368 18-77-73-7

io-o4 9I S3"7o-8

Lobomonai

7 9IO-26 0

7 98-96-S3-86 3

io-610-94-47 83 32-62-20 9

Table 2. Sugar composition of the cell walls of C. reinhardii and of L. piriformis

Sugar

XyloseRhamnoseArabinoseGalactoseMannoseGlucose

Sugar (mole

Chlamydomonas

2 9—

38-S2 9 82 3 16-6

%)

Lobomonas

3-76-s7-7

10-927-343-8

reconstruction (Fig. 29A) shows 2 main structural units centred on 2-fold axes (sub-units labelled A and B). They are both dimers and the subunits of both dimers arerather elongated. The first set of units (A) have a characteristic ' Z' shape composed of4 distinct lobes. Preliminary tilting experiments have shown this structure even athigh tilt angles, so we are fairly confident that it is a real structure rather than a super-position of subunits at different heights. These units form lines packing head-to-tailin adjacent unit cells. The other dimers (B) are rather more compact. The putativesubunits are roughly elliptical in shape and show some substructure. Adjacent units ofthis type are quite widely separated. The remaining material forms rather intricatelines of structure between the 2 basic units and appears to provide the connexionsbetween the 2 types of unit. However, it would be hazardous to interpret these lines asa continuous structure; they may be produced by superposition of subunits at differentheights, and their appearance changes quite substantially in the edge structure(Fig. 29 B). The C units in the edge structure correspond to the A in the centre structure

Crystalline cell wall of Lobomonas 3°7

Fig. 25. Low-dose (50 e/nm1) micrograph of negatively stained centre area of thecell-wall plate, xnoooo.Fig. 26. Computer-reconstructed image of centre area of a normal dose (5000-10000e/nm1) micrograph of negatively stained cell wall.Fig. 27. Computer reconstructed image of centre area of a minimal dose (500-1000e/nm1) micrograph of negatively stained cell wall. The resolution has been reducedto be equivalent to that in Fig. 26.

(although the lower resolution discussed above is noticeable). The other major unitsare completely different, however. We have outlined a region of high density (D) in theedge structure, which may correspond with the B units in the centre, but this ismainly for the purpose of describing the structure. We feel it is not possible tointerpret it unambiguously in projection. A possible explanation is that the unitlabelled B in the centre has changed its conformation and/or orientation in such a way


Table 3. Statistics on the phase and amplitude agreement between the images averagedfor the centre and edge negative-stained reconstructions

Centre images (3 images)

Weighted Unweighted

R$* before averaging (°)

RA"$o after averaging (°)

(130 independent spots were included in the final reconstruction)

Edge images (3 images)Weighted Unweighted

RQ before averaging (°) 13-6 25-6RF 0-115 0133R$ (°) 9 0 13 2Rtj>o after aveiaging (°) io-o 18-0

(58 independent spots were included in the final reconstruction)

9-90-1066 47 2

2O-I

0-148IO-I

I I - I

> r> / • u^ j \

• Rj (weighted) =

R^g (unweighted) = ^

where ^»i is the measured phase of spot (h, k) shifted to the best phase origin, and 0OJU is the2-fold symmetric phase (0° or 1800) closest to this value.

fRP (weighted) =

RP (unweighted) =

where F u is the average amplitude for spot {h, k) taken over all the images, and F(At is theamplitude for spot (h, k) in the image i.

X R4 (weighted) = X F > t | ^ - ^ m |

R4 (unweighted) = E — 10At - <j>m\,

where (pu, is the average phase for spot (h, k) taken over all the images, and 0<»i is the phase forspot (h, k) in image i.

that most of its bulk is placed further away from the 2-fold axes. There appears to bemore contact between adjacent tentative D units in the edge. We frequently observethe structural transition occurring in lines (see Fig. 19) and this might be consistentwith a conformational change propagated along contacts between the D units. How-ever, as the crystal lattice is maintained with great accuracy through the transitionbetween the 2 areas it seems likely that much of the basic structural arrangement of thesubunits is unchanged; the structural differences would also be consistent with arepositioning of a smaller morphological unit than the entire unit labelled D, but


28A

C D

Fig. 28. Computer Fourier transforms displayed as intensity plots. The directions ofthe reciprocal lattice vectors are labelled a* and b*. A. Minimal dose negative-stainedcentre pattern; B, minimal dose negative-stained edge pattern; c, normal doseshadowed replica: centre pattern; D, normal dose shadowed replica: edge pattern.

which is not resolved individually in 2-dimensional projection. The presence ofdistinct edge and centre structures is almost certainly indicative of the mechanismsby which the cell wall grows. Each plate must grow by addition of subunits at its edgesince there are no disclinations in the crystal lattice (as there are in Chlamydomonasor in Chlorogonium) to provide additional sites. This implies that the edge structure isthe first structure assembled and that a transition then occurs to the centre structure.The centre structure could than be regarded as the stable final wall structure. Newplates would have to arise at the interstices of existing plates (Fig. 18) and theirgenesis is possibly related to the amorphous material that is found there (Figs. 18, 15).

K. RoberU, P.J. Shaw and G.J. HiUs


The reconstructions of shadowed replicas confirm the negative-stain picture, albeitat much lower resolution. By following the difference between the centre and edge, wededuce that the units that are virtually unchanged in the 2 pictures (E and F in Figs.20, 21) are the same as subunits A and C in negative stain. The changes between the2 areas are almost wholly confined to the other units (G and H in Figs. 20 and 21,respectively). Again these changes would be consistent with a large conformationalchange, although in this case we are looking at prominent parts of the surface relief,rather than the distribution of stain-excluding material.

It is difficult to reconcile the fact that gel electrophoresis shows only 1 majorglycoprotein species to be present with a structure that appears to contain severaldistinct morphological units. Of the explanations that we can think of, the most un-likely is that the minor species seen on gels are in fact present in equimolar pro-portions and thus are contained in each repeating cell, but show atypical staining.This seems improbable as the minor gel bands show a certain variability between cellwall batches and such atypical staining has never been encountered before. Theseminor bands are more easily explained in other ways; for example, they may arise fromadsorbed degradation products of the cast-off walls or from molecules mediating theattachment between the plates and the inner wall layer. A second explanation is thateach asymmetric unit of the structure we see does in fact represent a single foldedglycosylated polypeptide. We consider that this is also unlikely as normal packingdensities for the estimated unit cell volume would give an impossibly large molecularweight for the major gel band. Even taking a much smaller value for the packingdensity in view of the thin-section appearance of the wall, this volume would stillrequire a single-subunit molecular weight of over hah0 a million. A third explanationis that the 2 main units in the centre structure are different orientations or conforma-tions of the same glycoprotein. If this is the case we might expect to confirm it byusing the 3-dimensional map we are currently calculating by combining images ofnegatively stained tilted specimens. Still further information could be obtained byobservation of unstained wall plates. Although glucose-embedding (Unwin& Hender-son, 1975) has not so far produced any useful information for this specimen, theinherent order and size of the crystals should be sufficient for unstained low-dosework. Therefore, we are investigating the possibility of examining frozen hydratedspecimens.

Fig. 29. Computer reconstruction of negative-stained structure. A. Centre structure( x 2200000); B, edge structure ( x 2200000). In each case data from 3 minimal-doseimages have been averaged. One unit cell is indicated. Two morphological units havebeen tentatively outlined for descriptive purposes and labelled A and B in A, andC and D in B.

3 i 2 K. Roberts, P. J. Shaw and G. J. Hills

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(Received 11 February 1981)

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