The Three-dimensional Structure of The

7/28/2019 The Three-dimensional Structure of The

http://slidepdf.com/reader/full/the-three-dimensional-structure-of-the 1/14

J. Cell Sci. 68, 271-284 (1984) 2 71Printed in Great Britain © The Company of Biologists Limited 1984

THE THREE-DIMENSIONAL STRUCTURE OF THE

CELL WALL GLYCOPROTEIN OF CHLOROGONIUM

ELONGATUM

P. J. SHAW AND G. J . HI LL S

John Innes Institute, Colney Lane, Norwich NR4 7U H, U.K.

SUMMARYThe green alga Chlorogonium elongatum, a member of the Volvocales, possesses a crystalline cell

wall composed of hydroxyproline-rich glycoprotein similar to the primary cell wall glycoproteins ofhigher plants . Electron microscopy and co mpu ter image processing have been used to determine thecrystal structure of the Chlorogonium cell wall in three d imensions to a resolution of 2-0 nm . Th estructure is composed of heterologous dimers. Each subunit of the dimer comprises a long, thinspacer domain and a large globular domain, which is the site of the intra- and inter-dimer inter-actions. Th ere are also sites of intersu bunit interactions at the opposite ends of the rod dom ains. Wesuggest that the rods are composed predominantly of glycosylated polyproline helix, as has beensuggested for higher plant cell wall glycoproteins and has been shown for the cell wall glycoproteinof Chlamydomonas reinhardtii, which is closely related to Chlorogonium.

INTRODUCTION

Glycoproteins have been identified as important constituents of the extra-cellularmatrix of organisms ranging from the Archaebacteria to higher plants and animals.

Within the great range of glycoproteins that have been characterized, a distinction

may be made between those glycoproteins that contain hydroxyproline and those that

do not. It has been suggested (Lamport, 1977) that the acquisition of the enzymic

apparatus necessary to use molecular oxygen to hydroxylate proline residues

represents a distinct evolutionary step, and that this is reflected in the function and

distribution of the resulting glycoproteins. In the plant kingdom hydroxyproline

(Hyp)-containing glycoproteins have been shown to be impo rtant constituents of the

primary cell wall of higher plants, in /3-lectins (Allen, Desai, Neuberger & Geeth,

1978), and as the factors mediating sexual agglutination in certain algae

{Chlamydomonas reinhardtii (Cooper et al. 1983) and Chlamydomonas eugametos(Pijst, Zilver, M usgrave & Van den End e, 1983 )).

Th e latter organisms are members of a fairly large group of motile eucaryotic algae,

the Volvocales, which possess cell walls" composed solely of glycoproteins. These

glycoproteins contain a high proportion of hydroxyproline and, at least in the case of

C. reinhardtii, there is good evidence that the major cell wall glycoprotein is organized

into Hyp-rich domains, showing a polyproline II helix structure, and domains con-

taining little or no hydroxyproline (Homer & Roberts, 1979). It has been suggested

that the polyproline II helix, glycosylated by short oligosaccharides, is a common

structural feature of Hyp-containing plant glycoproteins, and evidence for the



272. P. J. Shaw and G. J. Hills

presence of this structure has also been presented for potato lectin (Allen et al. 1978)and the primary cell wall glycoprotein of higher plants (Lamport, 1977).

The cell wall glycoproteins of the Volvocalean algae are of particular interest to us

because, besides being Hyp-rich glycoproteins related to those found in higher plant

cell walls, they occur in vivo as crystalline lattices, which are thus amenable to detailed

structural study. Most of the glycoprotein of these cell walls forms the crystalline

outer layer and the remainder forms an amorphous and much more diffuse inner wall

layer. The inner layer is formed first during cell division and probably forms a tem-

plate on which the outer layer crystallizes (Roberts, 1974).

Many species within the Volvocales have now been examined by high-resolution

electron microscopy, and all possess one of only four types of crystal structures in their

cell walls (Roberts, Hills & Shaw, 1982). The use of the cell wall structure as aphylogenetic marker for the classification of the algae has been suggested (Roberts,

1974). We have previously determined the three-dimensional structure of a represen-

tative of one of these classes by electron microscopic analysis (Lobomonas piriformis;

Shaw & Hills, 1982) (class IV of Roberts et al. (1982)). We showed that the structure

is composed of two sets of dimers, each of which consists of two rod-shaped molecules

aligned in the plane of the crystal.

The subject of the present investigation, Chlorvgonium elongatum, displays by far

the most common cell wall structure found in the Volvocales - denoted as type II by

Roberts. A preliminary investigation of the wall has been reported by Roberts & Hills

(1976). As the structure was also apparently the simplest among the wall types, it was

suggested that it was closest to that of a hypothetical ancestral type, from which the

other wall types had diverged. Table 1 shows all the species currently categorized as

type II.

Table 1. Species of algae currently known to have type II cell walls

Species Collection no.

Brachiomonas submarina 7/I aCarteria crucifera 8/7aCarteria eugametos 8/3Carteria incisa 8/4Chlamydomonas applanata 11/2Chlamydomonas chlamydogama 11 /48aChlamydomonas dorsoventralis 11/4

Chlamydomonas dysosmos 11/31Chlamydomonas eugametos 1 l/ScChlamydomonas fimbriata 11 /6 9Chlamydomonas moetvusi 1 l/16fChlamydomonas pulsatilla 11 /4 4Chlamydomonas reginae 11 /78Chlamydomonas rosae 11 /6 6Chlamydomonas sphaerella 11 /2 7Chlorvgonium elongatum 12/1Chlorogonium euchlorum 12/3Haematococcus capensis 34/4bPolytoma uvella 62/2a



Three-dimensional structure of Chlorogonium cell wall 273

MATERIALS AND METHODS

Cultures of Ch. elongatum (Dangeard) (Strain 12/1) were obtained from the Culture Centre ofAlgae and Protozoa, 36 Storey's Way, Cambridge, U.K. and were grown in sterile conditions inliquid culture without aeration, using a Tris-buffered mediu m, supplem ented with yeast peptone(Catt, Hills & Roberts, 1978) under continuo us illumination (80 ̂ Einstein mol~

2s in the wave-

band 400- 700 n m) . Cells were disrupted with glass ballotini in a Mickle shaker, layered onto a 5 %to 120 % (w /v) glucose gradient and centrifuged in a swinging-bucket rotor for 5 min at 1500 # .The fastest sedimenting band consisted of almost pure cell walls.

Negatively stained specim ens were made on 400 mesh copper grids coated with thin carbon filmsmade by evaporation onto a freshly cleaved mica surface and stripped off by flotation onto water.The negative stains used were 2-5% (w/v) aqueous methylamine tungstate (MET) (Faberg6 &Oliver, 1974) or methylamine tungstate/tannic acid made by the addition of 0-2mg/ml tannic acid(Mallinckrodt, St Louis, U.S.A.) to 2-5% (w/v) methylamine tungstate solution (METT). The

latter solution must be made by the addition of solid tannic acid to the MET solution to avoidprecipitation, and the best results were obtained using the resulting solution within one day.

Specime ns were examined on a JE O L JEM 1200 EX electron microscope fitted with a eucentricsingle-axis tilt specimen holder or a tilt/rotatio n specimen ho lder. Micrograp hs were generally recor-ded at 80 kV, X 30000, u sing K odak SO 163 film. Since only a small proportion of specimen areas givediffraction spo ts to high (2 nm ) reso lution , the following strategy was used for the collection of tilteddata. M icrographs were recorded from 10-20 areas of a suitable grid and the coordinates of each areawere noted from the microscope's digital display. Th e micrograp hs were developed and examined inan optical diffractometer. T he best areas, which were rare, contained spo tsout to theA = 3row . I twa sthen p ossible to retur n to these areas and collect tilt series, generally abou t a single axis from +6 0 ° an d—60°, but occasionally ab out two p erpendicular axes. Additional zero tilt images recorded after theseries showed that no significant degradation of the image occurred during this procedure.

Areas of the m icrographs were masked off for scanning u sing optical diffractometry. Areas wereused only if the defocus level was such th at the first zero of the contrast transfer function (Erick son,1973) occurred outside the highest-resolution spots. If possible, exactly the same area of each

member of a tilt series was used.Areas of the micrograph s were digitized using a microdensitometer cons tructed in this laboratory

(Shaw, Garn er & Parker, 1981) linked to a DE C P DP 11/6 0 m inicomp uter, w hich was used for allsubse quen t data proces sing. Th e scanning raster was set to 24 /im, which correspo nds to 0-8 nm atthe specimen, and either 512x512, or more usually 384x384 points were scanned. (The adequacyof the smaller scan size was checked by trimming down a 512x512 image and reprocessing thesmaller area. No significant difference was observed in the reconstructions.)

Processing of the images was carried out essentially as described previously (Shaw & Hills, 1982;Robe rts, Sh aw & Hills, 1 981; Shaw, 1981). The reciprocal lattice vectors were initially determinedby an interactive program, then refined by a search procedure that examines every significant spotin the transform and refines its position by a peak-profile analysis method. Amplitudes and phasesfor each spot in the transform were then determined by the profile analysis procedu re. Tw o furtherquantities were output for each spot: the ratio of peak to background amplitudes, and the ratio ofthe least-squares residual to average local background amplitude. This latter quantity is useful asa measure of how well the spots agree with the theoretical sine profile (Shaw, 1981). For some

images, amplitude and phase data around each spot were printed out and the spots were examinedmanually to check the validity of the automatic procedure.

Tilt angles and axes were determined by the calculation given by Shaw & Hills (1981). T herelative origins of the various data sets were determin ed using the phase search proced ure describedby Unw in & Hend erson (1975). Th e relative origins of four untilted images were determined to givea zero tilt starting set to which the tilted data sets were correlated in order of increasing tilt angle.Finally, each data set was refined in turn against all the others for three iterations of the entirelist, until no significant shifts in origins resulted. In refinement, only points on each z* linecloser than 0-025 nm"

1were used. A mplitudes were scaled using an unweighted least-squares scale

factor:

k = _ summ ed over all pointsLrt'



274 P. J. Shaw and C. J. Hills

closer than 0-025 nm~'. Various weighting schemes were tried for this calculation, but in all casesa slight drift of scale factors duri ng iteration of the refinemen t w as found. A similar problem has beendiscussed by Fox & Holmes (1966).

Regularly spaced values along the 2* lines were interpolated by a least-squares fit using sinefunctions (C rowth er, de Rosier & Klug , 1970). Th is method w orked well for all the phase curves,but occasionally the procedure, an unconstrained least-squares fit, yielded what was judged to bean unreasonably close fit to the data points for some of the weaker amplitude lines resulting in anoscillating curve. These lines were smoothed manually. The interpolated data were used as inputto a three-dimensional Fourier transform progTam, and the reconstructed three-dimensional mapwas contoured with equally spaced levels in sections of constant z.

RESULTS

Large, dislocation-free areas of the cell wall of Ch. elongatum can easily be ob-tained. This is a consequence of the shape of the cells and the way in which the two-

dimensional lattice is fitted around the closed surface of the cell. The cell wall is an

elongated ellipsoid with pointed ends, where large numbers of lattice faults occur.

The central portion of the ellipsoid is an approximate cylinder, however, and a nearly

Table 2. Statistics relating to the two-dimensional structure determination

A. Pairwise phase comparison of individual zero-tilt data sets

2012G

2032G358G317G

2012G

35-6

29-927-4

31-9

In the lower left-hand triangle

2032G

14-5

23-0'25-426-4

one film of each DE

3S8G

9-9

10-734-3

26'1

lir has been rotated thro

317G

12-7

10-711-033-7

ueh 180°. 2012Gand2032G stained with M E T T ; 358G and 317G stained with M ET alone.Phase residuals/?^ in degrees defines as : R$ = 1/2/V [£(4>u* —<p2**)]*, where <J>IA* are the phases forthe first im age; 4>2A* are th e phas es for the seco nd image with the origin shifted to give the best phasecorrelation, sum med over all spots whose peak-to-background ratio is greater than 2 0 .

• The factor of i is to give values comparable to those obtained with multiple film scaling in twoand three dimensions, where the discrepancy calculated is of individual films from an average orinterpolated value.

B. Overall statistics for two-dimensional scaling4 data sets included2-dimensional data included for 43 spots

fl<f>#= 14-3°

The summations are over all common points.




perfect crystal lattice can be laid down in this region. The purification procedure

produces mainly entire or nearly entire envelopes, and back-to-back double layers arethus usually observed in the electron microscope. However, with careful searching

areas of single layers can be found. Unfortunately these often show poor crystal orde r,

which we believe is due to inadequate preservation by the negative stain. With simple

methylamine tungstate as a stain we have only ever obtained three or four areas with

diffraction spots extending to 2-0nm (this included one tilt series). T he ad dition of

Fig. 1. Electron micrograph of portions of the cell wall of Chlorogonium stained withM ET T. Bar, 100 nm.

Fig. 2. Optical diffraction pattern of negatively stained Chlorogonium cell wall showingspots in addition to the basic lattice spots.

Fig . 3. Optical diffraction p atter n of an area of the Chlorogonium wall that does not display

any extra spots.






500

180-

-180-

*

/+

+ /

i j, .hi r4*^

1 1 1 1

(-5,0)

— 1 1——1 1 —-0 -4 - 0 -2 n m - i 0-2 0-4

3000-

-0 -4 -0 -2 0-4

0-4 -0 -2 - i 0-2 -0 -4

-0 -4

Fig. 5. Selection of lattice lines obtained during the three-dimensional reconstruction.

the wall structure (see below), but since their total intensity is small we ignored them

in our initial structura l analysis. Althoug h the indexing system we have used is not the

most obvious one, it is the only one that gives integral h indices for these extra spots.

The unit cell dimensions for this lattice are:

a = 28-6nm = 7-2nm y= 128-5°.



278 P. J. Shaw and G. J. Hills

Table 3. Statistics relating to the three-dimensional structure determination

4 untilted images were included37 tilted images were included30 2* line were finally included (only those lines with more tha n 20 data poin ts)

summed over all N data points.

- 0 -5

-0 -6

Fig. 6. Reciprocal space resolution plot of the 2* lines used in the three-dimensionalreconstruction.




Table 2 shows statistics on the agreement of four individual two-dimensional images.

A two-dimensional reconstruction produced by averaging data from the four imagesis shown in Fig. 4. The basic structure is of rows of subunits packed closely together

along the b axis, but rather widely separated by less-dense fibrillar interconnections

in the other dimension. Although the structu re appears to have approximate twofold

symm etry, there are very distinct and quite consistent departures from this sym metry,

Table 2 shows this in a quantitative fashion, by pairwise comparison of images with

and without relative twofold rotations. The structure must therefore be assigned to

the two-sided plane group PI (Holser, 1958).

Some statistics of the three-dimensional structure determination are shown in

Fig. 7. Stereo pair of the stack of contoured sections of the three-dimensional re construc-tion. A an d B, large and small subunits.

Fig. 8. Wooden model of the three-dimensional reconstruction showing the envelope

included within the lowest positive contour in Fig. 7.

Fig. 9. Th e wooden model taken apart to show the subun it struc ture.




to

so

u

$

z(nm)

Fig. 10. Graph of the maximum contrast (i.e. the difference between the maximum andminimum density) plotted against z.

Table 3, a selection of z* lines in Fig. 5, and a projection plot showing the distribution of

data measured in reciprocal space in Fig. 6. Th e lack of symm etry in most of the #* lines,

particularly in am plitude, confirms that the symm etry group is only P I . A stereo-pair

picture of the stacked contour plot is shown in Fig. 7, and a wooden model of the en-

velope enclosed by the lowest positive contour in Figs 8 and 9. Th e crystalline layer has

a total thickn ess of 6—7 nm; Fig. 10 shows a graph of image contrast as a function of z.

Delineation of the su bunits from which the structure is composed is quite straight-

forward and there is little or no ambiguity. There are two types of subunit, which,

however, are very similar. The subunits comprise two domains: a large globular

domain of approximate dimensions 4nmX6nmX6nm and a very thin long, rod-like

domain about 7—8nm in length, which bends and thickens at the end. The chief

difference between the two types of subunit lies in the size of the large globular

domains; the A subunits in Fig. 7 containing significantly more stain-excluding

material. The subunits are associated as heterologous dimers with the dimer interfacebeing formed by the large domains, and the dimers in turn packing to form lines of

the large doma ins. T he rod-like arms project ou t of this line on either side in the plane

of the crystal and interlock at their ends with those from the neighbouring rows in a

second, m uch less dense line of stru ctu re. T he effect is to give on one side of the crystal

rows of raised units that project about 3-0—4-0 nm from the surrounding structure,

and on the other side a rather flat network. The two types of pore through the

structure, although crystallographically different, are nevertheless almost identical in

size and shape, being approximately circular and 4-0—5-0 nm in diameter. The struc-

ture is illustrated diagrammatically in Fig. 11.




Fig. 11. Diagrammatic representation of the subunit structure of the Chlorogonium cellwall.

For the three-dimensional analysis we ignored the extra spots that are frequently

observed. They are very variable in intensity and often entirely absent from the

transform, and at their strongest are of rather small intensity compared to the simple

lattice spots. With the definition of b* we have used, these spots may be indexed by

simple fractional k indices. We have observed image transforms with extra spots

corresponding to k = 2n/5, k — 2w/6 and k = 2n/7, as well as transforms where the

spots are 'smeared' in the direction parallel to b*. The results from analysis of an

image with particularly strong and coherent super-lattice spots are shown in Figs

12—14. The two-dimensional reconstruction using only the basic lattice spots is

shown in Fig. 12. A reconstruction including the extra spots is shown in Fig. 13. The

changes resulting from the extra spots are quite small, and are centred mainly around

the subsidiary lines of stru cture, where the subunit 'tails' interdigitate. Fig . 14 shows

reconstruction using data only from the extra peaks, i.e. it represents the difference

between Figs 12 and 13. This shows very clearly rows of associated positive and

negative peaks. We interpret this as being due to a periodic displacement of the ends

of the tails, which repeat in this case every three, unit cells (or every six tails). We

presume that the spots that we have observed on other images at k = 2n/5, k = 2n/l

arise from a similar replacement repeating every five and every seven tails, respect-

ively. Two explanations of this phenomenon have occurred to us. The first is that

this is a genuine super-lattice structure arising from longer-range interactions than

nearest neighbour, and is displayed by the wall structure in vivo in the cell. Thesecond is that it is produced by flattening an originally curved surface onto the

specimen grid. This would introduce strains into the sheet, which for certain

geometries might be relieved by periodic distortions along what is likely to be the

weakest part of the structure. We favour the first explanation, chiefly because there

does not appear to be any correlation between the presence of the super-lattice and

position on the wall; an area near the 'equator' where curvature is minimal is just

as likely to display the super-lattice as an area near the end where curvature is

greater.

CEL68



282 P.J. Shaw and G.J. Hills

Figs 12—14. Two-dimensional reconstructions of an area of the Chlorogomum cell wallshowing strong super-lattice spots.

Fig. 12. Without the additional spots.

Fig. 13. With the additional spots.

Fig. 14. With only the additional spots included. The contour level is approximatelyhalf of that in Figs 12 and 13.




DISCUSSION

It is a somewhat unexpected feature of our structural results that the dimer should

be heterologous. However, this finding is consistent with results obtained by SDS

(sodium dodecyl sulphate)/polyacrylamide gel electrophoresis of the cell wall

glycoprotein (R oberts, 1974). For Chlorogonium two bands were obtained, both w ith

approximate relative molecular mass of 150-200( X103). Th is figure is consistent with

the volume enclosed by the lowest positive contour in our reconstruction. Further

support for this correlation of the SD S/polyacrylam ide gel patterns with the structu re

comes from another species showing the same class of cell wall structure,

Chlamydomonas dysosmos. This wall gives only one band on SDS/polyacrylamide

gel electrophoresis, with a mobility close to that of the larger of the two Chlorogonium

bands (Roberts, 1974). Our preliminary analysis of micrographs of this cell wall has

shown that although the overall structure is very similar to that of Chlorogonium, th e

C. dysosmos structur e shows good twofold symm etry. We therefore surmise that this

structure is composed of symmetric dimers. We suggest that a species such as C.

dysosmos appears to have a single cell wall glycoprotein subunit that is closest to a

hypothetical ancestral type. One might then propose a simple evolutionary step from

a glycoprotein subu nit that dimerizes (i.e. self—self recognition) to two closely similar

glycoproteins, which recognize each other and thus form heterologous dimers.

The rod-like arms of the subunits are comparable in width to the resolution of the

reconstru ction (2 nm) an d would be entirely consistent with the model for Hyp-rich

glycoproteins proposed by Lamport (1980). In this model regions very high in

hydroxyproline residues form a polyproline II helix, and the oligosaccharidesattached to the hydroxyl groups of the proline residues pack around the backbone

helix to give stable and strong rods, which would have a diameter of approximately

2 nm. There is strong evidence for the presence of a large domain of this structure in

the cell wall glycoprotein of the closely related species C. reinhardtii, both from

circular dichroism measurements of the intact glycoprotein and large proteolytic

fragments (Homer & Roberts, 1979), and from chemical studies of amino acid com-

position and pa ttern s of glycosylation (R ober ts, 1979). T he m odel we suggest for each

of the subunits of the Chlorogonium cell wall glycoprotein, therefore, is of a long rod

composed of a glycosylated polyhydroxyproline helix, which acts as a structural

spacer, together with a large globular domain, which forms strong dimer associations

and inter-dimer contacts. There is then a region at the opposite end of the rods thatforms a second pair of intersubunit associations. The Chlorogonium wall glycoprotein

appears to display in a particularly simple manner structural features that may well

be shared by other hydroxyproline-containing plant glycoproteins; namely, long rod-

like spaced dom ains together w ith globular domains that form interaction and associa-

tion sites. We might expect a similar general structure for such glycoproteins as the

flagellar sexual agglutination factors in Chlamydomonas (Cooper et al. 1983; Pijst et

al. 1983), and the class of plant cell wall glycoproteins tha t has been term ed extensin

(Lamport, 1977). There is already some evidence for this type of architecture in

potato lectin (Allen et al. 1978).




REFERENCES

AKEY, C. W. & EDELSTEIN, S. J. (1983). Equivalence of the projected structure of thin catalasecrystals preserved for electron microscopy by negative stain, glucose embedding or embedding

in the presence of tannic acid. J . molec. Biol. 163, 575-612.ALLEN, A. K., DESAI, N. N., NEUBERGEB, A. & G E E T H , J. M. (1978). Properties of potato lectin

an d the nature of its glycoprotein linkages. Biochem.J. 171, 665-674.CATT, J. W., HILLS, G. J. & ROBERTS, K. (1978). Cell wall glycoproteins from Chlamydomonas

reinhardii, and their self-assembly. Planta 138, 91-98 .COOPER, J. B., A D A IR, W. S., M E CH A M , R. P., H E U S E R, J. E. & G O O D E N O U G H , U. W. (1983).

Chlamydomonas agglutinin is a hydroxyproline-rich protein. Proc. natn. Acad. Set. U.SA. 80,

5898-5901.CROWTHER, R. A., D E ROSIER, D. J. & K L U G , A. (1970). The reconstruction of a 3-dimensional

structure from projections and its application to electron microscopy. Proc. R. Soc. Land. A 317,

319-340.ERICKSON, H. P. (1973). Th e Fourier transform of an electron micrograph - first order and second

order theory of image formation. Adv. opt. Elect. Microsc. 5, 163-199.FABERGE, C. & OLIVER, R. M. (1974). Methylamine tung9tate, a new negative stain. J . Microsc.

20, 241-246.Fox , G. C. & HOLMES, K. C. (1966). An alternative method of solving the layer scaling equation s

of Hamilton, Rollett & Sparks. Ada crystallogr. 20, 886- 891 .HOLSER, W. T. (1958). Point groups and plane groups in a two-sided plane and their subgroups.

Z. Kristallogr. Kristallgeom. 110, 266- 281 .HOMER, R. B. & ROBERTS, K. (1979). Glycoprotein conformation in plant cell walls. Planta 146,

217-222.LAMPORT, D. T. A. (1977). Structure, biosynthesis and significance of cell wall glycoproteins. In

Recent Advances in Phytochemistry , vol. II (ed. Loewus & Runeckles). New York: Plenum.LAMPORT, D. T. A. (1980). Structure and function of plant glycoproteins. In The Biochemistry of

Plants, vol. 3 (ed. P. K. Stumpf & E. E. Conn) , pp . 501- 541 . New York, London: AcademicPress.

MITZUHIRA, V. & FUTAESAKU, Y. (1972). New fixation for biological mem branes using tannic acid.Acta Histochem. Cytochem. 5, 233-236.

PI J ST , H. L. A., ZILVER, R. J., MUSGRAVE, A. & VAN DEN ENDE, H. (1983). Agglutination factor

in the cell body of Chlamydomonas eugametos. Planta 158, 403—409.ROBERTS, K. (1974). Crystalline glycoprotein cell walls of algae: their structure composition and

assembly. Phil. Trans. R. Soc. Land. B 268, 129-146.ROBERTS, K. (1979). Hydroxyproline: its asymmetric distribution in a cell wall glycoprotein.

Planta 146, 275-279.ROBERTS, K. & HILLS, G. J. (1976). Th e crystalline glycoprotein cell wall of the green alga

Chlorogonium elongatum: a structural study. J. Cell Sci. 21, 5 9 - 7 1 .ROBERTS, K. , HILLS, G. J. & SHAW, P. J. (1982). The structure of algal cell walls. In Electron

Microscopy of Proteins, vol. 3 (ed. Harr is) . New York, London: Academic Press.ROBERTS, K. , SHAW, P. J. & HILLS, G. J. (1981). High-resolution electron microscopy of

glycoproteins: the crystalline cell wall of Lobomonas. J. Cell Sci. 51, 295-313 .SHAW, P. J. (1981). A peak profile analysis procedure for extracting unit cell transform data from

the Fourier transforms of periodic arrays. J. Cell Sci. 51, 314—321.SHAW, P. J., GARNER, R. T. & PARKER, E. A. (1981). A flat bed scanning microdensitometer for

computer processing of electron micrographs. Micron 12, 103-114.SHAW, P. J. & HILLS, G. J. (1981 ). Tilted specimen s in the electron microsco pe: a simple specimen

holder and the calculation of tilt angles for crystalline specimens. Micron 12, 279—282.SHAW, P. J. & HILLS, G. J. (1982). Three-dimensional structure of a plant cell wall glycoprotein.

J. molec. Biol. 162, 459- 471 .

UNWIN, P. N. T. & HENDERSON, R. (1975). Molecular structure determination by electron micro-scopy of unstained crystalline specimens..?, molec. Biol. 94, 425—440.

{Received 16 January 1984-Accepted 2 February 1984)

Documents

The Three-dimensional Structure of The