JOURNAL OF ULTRASTRUCTURE RESEARCH
51,326-339 (1975)
An EM Study of Phosphorylcholine-Binding Fab' Immunoglobulin Fragment Crystals L. W. LABAW*, E. A. PADLANt, D. M. SEGAL~:, AND D. R. DAVIESt * Laboratory of Physical Biology and ~ Laboratory of Molecular Biology, National Institute of Arthritis, Metabolism, and Digestive Diseases, and $Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda. Maryland 20014 Received July 30, 1974, and in revised form December 17, 1974; accepted December 17, 1974 The Fab' fragment of McPC603 mouse myeloma imnmnoglobulin with phosphorylcholine-binding activity crystallizes into both a hexagonal and a cubic structure. Models of' these structures showing approximate molecular packing were deduced from an electron microscopic examination of stained crystal sections, a preliminary X-ray diffraction study, and the knowledge that both crystal structures grow simultaneously in the same crystallizing solution. When both of these models were modified slightly to agree with the more exact molecular packing in the hexagonal crystal from a 4.5 A resolution X-ray study, they were both still compatible with the EM data. INTRODUCTION
Th e crystallization and p r e l i m i n a r y X-ray investigation of the pepsin Fab' fragments from a mouse myeloma protein, IgA(K) McPC603, that binds phosphorylcholine has been reported (13) as part of a continuing study to elucidate the structure of the combining sites of antibody molecules. Crystallization in 43% saturated ammonium sulfate gave hexagonal prisms [see Fig. 3 (I3)] of space group P63 with unit cell dimensions a - b - 162.5, c - 60.8 J~ with one molecule per asymmetric unit. When these Fab' fragments were crystallized in. 1.7 molar potassium phosphate (K~HP04), both 1) regular rhombic dodecahedra, space group P213 or P23 with unit cell dimensions a = b = c = 148 A; and 2) the same hexagonal prisms obtained from the ammonium sulfate crystallization were formed, as shown in Fig. la. There was again only one molecule per asymmetric unit in these cubic crystals. The asymmetric unit being a single Fab' fragment with an approximate molecular weight of 50 000 gives the unit cell volume per dalton of protein (9) in the hexagonal crystal (where there are six asymmetric units per unit cell) of 4.6 A3/dalton, and in the cubic crystal (where there are 12 asymmetric
units per unit cell) of 5.4 A3/dalton, both very open structures. The hexagonal McPC603 Fab' crystals were stable enough to make a more detailed X-ray analysis practical. The structure of this Fab' fragment to 4.5 ~ resolution has recently been reported (I1). The present paper describes how an approximate molecular packing in both the hexagonal and the cubic crystals can be obtained by combining the preliminary X-ray data with data obtained from the electron microscopic examination of stained crystal sections.
326 Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
MATERIALS AND METHODS The Fab' crystals were fixed for several weeks in glutaraldehyde concentrations of 0.5%. The hexagonal crystals were transferred from the 43% saturated ammonium sulfate in which they were grown to saturated lithium sulfate to which glutaraldehyde was added to give 0.5%. This was done to avoid the reaction between the ammonium radical and glutaraldehyde. The cubic crystals were fixed in their mother liquor with glutaraldehyde in potassium phosphate added to give 0.5% glutaraldehyde. The use of a higher concentration of glutaraldehyde than the 0.03% used previously (4) was necessary to prevent the crystals dissolving during the dehydration procedure. This necessity for a higher concentration of cross-linking agent probably reflects the more open structure of these crystals. The crystals were then washed with 5 × l0 4 M phosphate buffer (pH 7.3), postfixed in
FAB' CRYSTALS 1% osmium tetroxide for 15 min, dehydrated in graded alcohols, and embedded in Maraglas. Sections of the hexagonal crystals were cut parallel to the (0001), (10i0), and (1120) planes; sections of the cubic crystals were cut parallel to the (100), (11i). and (110) planes. The proper orientations for these cuts could be made within one or two degrees since the hexagonal prism crystals had six well-developed (1120) type faces parallel to the c or long axis and the cubic crystals had 12 rhombic (110) type faces (see Fig. lal. The sections were stained with uranyl acetate for 60 rain followed by lead citrate for 1 min, then lightly coated with carbon for increased stability in the electron microscope. The average periodic pattern in the micrographs (first optical integration) was obtained by averaging over the periodicity in real space by repeated linear translation and superposition of the micrograph upon itself (4, 5, 6, 10). The signal-to-noise ratio was further improved and the contrast enhanced by performing a similar second averaging on the first (second optical integration), using a diflerent direction of translation. Micrographs were taken on a Philips 300 microscope that was calibrated using indanthrene olive T crystals coated with carbon for increased stability in the beam. These crystals acted as phase gratings with a 24.9 ~, periodicity as measured by X-ray powder diffraction patterns (3). This periodicity also was checked at about × 500 000 against the graphitized carbon spacing of 3.4 •. OBSERVATIONS AND DISCUSSION A t y p i c a l s t a i n e d section of the hexagonal F a b ' crystals c u t p e r p e n d i c u l a r to the c axis [parallel to the (0001) p l a n e s ] is s h o w n in Fig. lb. T h i s p r o j e c t i o n of the crystal along the c d i r e c t i o n has the a p p e a r a n c e of a h o n e y c o m b s t r u c t u r e . T h e d e n s i t y in the holes is similar to t h a t in the plastic o u t s i d e the c r y s t a l (not shown) a n d there is c o n s i d e r a b l e d e n s i t y v a r i a t i o n in the wall a r o u n d e a c h hole. First a n d s e c o n d o p t i c a l i n t e g r a t i o n s of this m i c r o g r a p h , shown in Figs. lc a n d d, respectively, d e m o n s t r a t e t h a t there is no c o n s i s t e n t p a t t e r n to this v a r i a t i o n as t h e r e was in b a c t e r i o c h l o r o phyll crystals of the s a m e space g r o u p (5). T h i s h o n e y c o m b p a t t e r n suggests t h a t there are large h e x a g o n a l l y a r r a n g e d c h a n nels parallel to t h e c axis filled w i t h e m b e d d i n g plastic a n d t h u s n o t stained, giving a very o p e n c r y s t a l s t r u c t u r e , as indic a t e d also b y t h e v o l u m e to p r o t e i n ratio of
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4.6 A3/dalton. B o t h c a t a l a s e (8) a n d b a c t e r i o c h l o r o p h y l l (5) h e x a g o n a l c r y s t a l s s h o w e d similar o p e n c h a n n e l s . T h e location of the s t a i n in this p r o j e c t i o n is also the location of the p r o j e c t e d protein. T h i s i n t e r p r e t a t i o n n e e d not m e a n t h a t the p r o t e i n itself stains. P r e v i o u s work (4) i n d i c a t e s t h a t the F a b ' m o l e c u l e does n o t stain very intensely. M o s t of the stain is p r o b a b l y , therefore, b e t w e e n the m o l e c u l e s where the e m b e d d i n g plastic could n o t p e n e t r a t e (see discussion of this p o i n t in (4 a n d 5). T w e n t y m i c r o g r a p h s , e.g., the one shown in Fig. lb, were m e a s u r e d to get an a v e r a g e value for a. In e a c h m i c r o g r a p h , the centert o - c e n t e r s e p a r a t i o n of the c h a n n e l s was m e a s u r e d along e a c h of' the three lattice directions. T h e a v e r a g e of these 60 m e a s u r e m e n t s gave a = 151.8 ± 7.9 A (stand a r d deviation), w h i c h is 6.6% below the X - r a y value. If only the highest m e a s u r e m e n t for e a c h m i c r o g r a p h was used, the average of these 20 gave a = 159.0 ± 5.3 A, or only 2.1% below the X - r a y value. T h e difference b e t w e e n these two averages reflects the d i s t o r t i o n in the sections resulting f r o m the c u t t i n g a n d m o u n t i n g procedures. T h e h e x a g o n a l s t r u c t u r e in Fig. lb, for e x a m p l e , is c o m p r e s s e d along the s h o r t page axis, which was also the c u t t i n g direction. T h e P63 space g r o u p s y m m e t r i e s (2) can be satisfied either b y h a v i n g the a s y m m e t ric u n i t s c l u s t e r e d a r o u n d the sixfold screw axes or a r o u n d the threefold r o t a t i o n axes. It is clear f r o m the m i c r o g r a p h in Fig. 1 t h a t the latter o b t a i n s for this crystal, with the centers of the o p e n c h a n n e l s being t h e sixfold screw axes. T h e three a s y m m e t r i c u n i t s t h a t m u s t lie a r o u n d one threefold r o t a t i o n axis would be, for this space group, at a level c/2 f r o m the three a s y m m e t r i c u n i t s s u r r o u n d i n g e a c h of t h e three a d j a c e n t threefold r o t a t i o n axes. It is ins t r u c t i v e to c o n s t r u c t a m o d e l of this crystal using two spheres joined t o g e t h e r as the a s y m m e t r i c unit, or m o l e c u l e in this case, where one sphere r e p r e s e n t s the v a r i a b l e
FIO. 1. (a) Optical micrograph of McPC603 Fab' crystals grown in potassium phosphate. (b) Electron micrograph of a stained hexagonal crystal section cut perpendicular to the c direction. (c) First optical integration of (b), translation for integration across page. (d) Second optical integration of (b), translation up to right. (e) Hexagonal crystal model viewed along the c direction, with one simulated molecule and three trimers beside it. (a) × 72; (b) x 400 000; (c) and (d) × 1 100 000.
FAB'CRYSTALS
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portion of the Fab' molecule and the other, pattern. The hexagonal crystal model is the constant portion. [References 4 (Fig. 3), seen looking along the [10i0] direction in 11, and 12 indicate that this is a reasonable Fig. 2d. T h e four lines of molecules parallel first approximation.) Such a model is to c showing shadows should correspond to shown, looking down the c direction, in Fig. the lines of m a x i m u m staining in Fig. 2c le. T h e angle t h a t the long axes of the since here you are looking more nearly simulated molecules make with a plane along molecular lengths (see Fig. le). (The perpendicular to c was made 28 ° (this crystal model shown in Fig. le was photochoice will be explained later) and the graphed in the direction of a 60 ° clockwise projections of these molecules on this plane rotation from the short page axis to obtain were taken to lie along lines joining adja- Fig. 2d; therefore, there are four lines with cent threefold rotation crystallographic shadows corresponding to m a x i m u m stain axes for convenience in making the model. lines in the micrograph.) If these lines with T h e separation of adjacent threefold rota- shadows in the model picture are e x a m i n e d tion axes in the model was a consequence of carefully, staggered prominences correclose-fitting a simulated molecule at one sponding the those in Fig. 2e are apparent. level along c with one from an adjacent While the second optical integration has threefold rotation center, which, as noted the general characteristics predicted by the above, was at a level c/2 away in the c model, the p a t t e r n is skewed and the direction. This can be seen in Fig. 2d. T h e measured value of c = 44.3 A is 27% ratio of a (the center-to-center distance smaller t h a n the X-ray value. This section between the channels in Fig. 1) to c in this was cut with the d i a m o n d knife moving in model is 2.55 compared to 2.67 from the the c direction and thus was considerably X-ray crystal m e a s u r e m e n t s , a difference compressed or folded in this direction; of only 4.5%. inadequacy of the subsequent t r e a t m e n t T h e micrograph in Fig. 2a is of a stained with chloroform vapor to unfold the section section cut parallel to the (1010) planes of on the water surface in the boat betbre it the hexagonal Fab' crystals. This projec- was picked up on the specimen grid m a y tion is equivalent to looking along the short account for this large discrepancy. T h e r e page axis at the crystal sections shown in also m a y have been some shrinkage in this Figs. lb, c, d, and e. T h e coarse periodicity direction during the embedding procedure. across Fig. 2a should thus be v/3a/2, which b u t this explanation is not as likely as the is 140.7 /~ for the X - r a y d e t e r m i n e d unit former. T h e most extreme measured value cell. M e a s u r e m e n t s on 46 micrographs of on the 46 micrographs leading to the aversections with this orientation gave an aver- age value of V~a/2 = 137.6 A cited above age value of 137.6 ± 8.7 }t, which is 2.2% was only 12.8% lower t h a n this average, b u t lower than the X - r a y value. It is difficult to all these sections were cut with the knife see any periodicity along the c direction, moving in the c direction, so this measurewhich is vertical in Fig. 2a, b u t a first m e n t was not affected by the section foldoptical integration of this in Fig. 2b, and a ing. second optical integration in Fig. 2c, demFigure 2e shows a typical stained section onstrate t h a t there is one. T h e direction of cut parallel to the (11-20) planes of the translation to obtain Fig. 2b from Fig. 2a hexagonal crystal. This projection is along was a p p r o x i m a t e l y at right angles to the c a direction 30 ° to t h a t for Fig. 2a and still direction, i.e., the lines in the micrograph, perpendicular to c. This projection down and to obtain Fig. 2c from Fig. 2b, along the [1120] direction is equivalent to looking the c direction. In the latter case, using a along the long page axis at the crystal translation interval along c twice the one sections in Fig. 1. Thus, any coarse perioshown here did not sensibly change the dicity should be a/2, with the darker lines
FIo. 2. (a) Stained hexagonal crystal section cut parallel to (10i0) planes. (b) First optical integration of (a), translation across page. (c) Second optical integration of' (a), translation along long page axis period. (d) Hexagonal crystal viewed along [1010] direction. (e) Stained hexagonal crystal section cut parallel to (llg0) planes. (t) Hexagonal crystal model viewed along [1120] direction. (a) and (el x 400 000; (b) × 600 000; (c) × 1 300 000. 330
FAB' CRYSTALS of stain parallel to c having less contrast than in Fig. 2a and corresponding to lines of molecules viewed end-on. The model photographed in the corresponding orientation in Fig. 2f shows no openings directly through the crystal in this direction. Therefore, one would not expect to observe any distinct periodicity in the staining along c. This was confirmed by not finding any consistent periodicity along c in any of the optical integrations performed on several micrographs of this projection. Measurements on 30 micrographs gave an average for a/2 of 81.0 ± 3.2 A, compared with the X-ray value of 81.3 ]~. Here again, sections were cut with the knife motion along c. It should be emphasized t ha t in making the hexagonal crystal model, the choice of the 28 ° angle the molecular axes make with a plane perpendicular to c was rather arbitrary, as was also the alignment of the molecules such that their axial projections on this plane are exactly along lines joining the adjacent threefold axes. Because the cell is so open, a large latitude is available in choosing the angle that the molecular axes make with the plane perpendicular to c. This can be anything from 0 ° to 60 ° without disagreeing with the electron micrographs. Likewise, the trimers at the threefold rotation centers could be rotated by up to 20 ° and still fit the EM data. Determination of the correct angles for these can only be made by further X-ray structure analysis. This will be discussed again after the data from the cubic crystal form have been presented. The fact that both the hexagonal and the cubic crystals grow side by side under the same conditions strongly suggests that the molecules are grouped together in both crystals in a similar fashion. This notion would be confirmed, fbr example, if the same trimer a r r a n g e m e n t of molecules around the threefold hexagonal crystals axes also obtained around the four threefold cubic crystal axes. It will now be shown that such an interpretation is compatible with the elec-
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tron microscopic data. The choice of the 28 ° angle the molecular axes make with a plane perpendicular to the threefold rotation axes in the hexagonal crystal model was dictated by the fact that this same angle between the molecular axes and planes perpendicular to the four threefold cubic crystal axes greatly facilitates making an extended cubic crystal model that agrees with the EM data and gives a structure sufficiently open to make this agreement clear. Regular rhombic dodecahedra have two types of vertices (I): (1) those at the intersection of three faces, and (2) those at the intersection of four faces. If all of the 12 rhombic faces are equal, the crystal structure should have threefold rot at i onal symmetry around lines joining opposite three-face vertices (which are also the diagonals of this truncated cube) and have twofold screw or rotational symmetry around lines joining opposite four-face vertices (these lines are parallel to the truncated cube edges) in accord with the preliminary X-ray finding (13) that these crystals belong to either the P213 or P23 space groups. Fortunately, the structure of this cubic Fab' crystal is sufficiently open, and the molecules are aligned well enough in the directions of the cube diagonals and cube edges, so that stained sections cut perpendicular to these directions show cross-periodicities. The nature and orientation of these periodicities are sufficient to 1) eliminate P23 as a possible structure, and 2) determine the approximate molecular packing in the P2~3 structure. The periodicities in stained sections of this cubic Fab' crystal indicate that there are planes of density parallel to each face of the rhombic dodecahedron, spaced just over 100 • from one another in a direction perpendicular to that face. [Lange's Bgranules (7) had a similar appearance but different spacing.] Thus, in sections cut perpendicular to the cube diagonals, or [111] direction, three equal periodicities 60 ° or 120 ° apart in angle give the hexago-
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nal pattern shown in Fig. 3a. These three intersecting sets of lines in this projection are parallel to the sides of the hexagon that outline the crystal when it is viewed down the threefold axis (see lower right crystal in Fig. la). The average line separation from 30 measurements on 10 micrographs was 101.7 i 4.3 A. Sections cut perpendicular to the twofold axes (lines joining opposite four-face vertices) or unit cell edges showed two intersecting sets of lines at right angles to each other, as shown in Fig. 4a. These lines are parallel to the sides of the square t h a t outline a rhombic dodecahedron viewed down one of these twofold axes and are therefore parallel to the projections of the cube diagonals in this direction. Thirty measurements on 15 micrographs gave the average line separation in this projection of 103.3 ~: 4.6 A. On sections cut parallel to a face of the rhombic dodecahedron crystals (projection in the [110] direction), the lines of density, as seen for example in Fig. 5a, were parallel to the short axis of the face. The average line separation on 20 micrographs showing this projection was 104.0 ± 4.2 •. The X-ray data leading to the space group assignments showed a very prominent spacing equal to 1/2 the diagonal of a cube face or 1/2 (V~a) = 104..7 A. The spacings measured on the electron microbraphs, as indicated above, are essentially identical. The average stain pattern looking in the direction of the cube axes, as shown in the first and second optical integrations in Figs. 4b and 4d respectively, implies that there are rectangularly arranged holes through the crystal in these three directions. The first and second optical integrations of Fig. 3a in Figs. 3b and 3d show an average stain pattern in which each lightly stained center is surrounded by three trigonally arranged holes and three more heavily stained regions, also trigonally arranged. Thus, along each of the four cube diagonal directions there are empty channels through the crystal arranged hexago-
nally. This very open structure agrees with the large volume of 5.4/~3/dalton of protein mentioned earlier. Since the stain density in the projection down the threefold rotation axes, as seen in Fig. 3d, contains the clue to the molecular packing in the crystal, first and second optical integrations were performed on several other micrographs of sections similarly cut. All of the second integrations had density patterns that could be characterized as having hexagonally arranged weakly stained regions, each trigonally surrounded by three regions of no stain and three densely stained regions. Two of these second optical integrations are shown in Figs. 3c and 3e. With the measured average line separation on micrographs of this projection being 101.7/~, the separation of the darker stained areas (center-to-center), or the lightly stained areas, or the holes, becomes 117.4 A or about 120 A, which is equal to the projection of the X-ray unit cell edge onto this plane, both in magnitude (120.8 A) and direction. Thus, any of these features of the repeating pattern could be at the threefold rotation center of the unit cell. The preliminary X-ray data identified the space group for this crystal as either P23 or P213. Both of these (2) have 12 asymmetric units in the unit cell, with threefold rotation axes. This means that at each of four positions in either unit cell there are three asymmetric units arranged with rotational symmetry. In the P23 space group, these four positions are at the corners of a tetrahedron, and these centers can only be somewhere along the diagonals of the unit cell. Now, if the lightly stained areas in this projection correspond to one of these centers, the three darkly stained areas trigonally arranged around them should correspond to the other three trimer centers, which for the P23 space group should each be directly between two lightly stained areas, since for this space group they must be along the cube diagonals. The fact that they are off to one side of this line
Fro. 3. (a) Stained cubic crystal section cut perpendicular to [111] direction. (b) First optical integration of' (a), translation across page. (d) Second optical integration of (a). translation up to right. (c) and (e) Second optical integrations of two other micrographs similar to (a). (f) Cubic crystal model viewed along [111] direction. (a) × 300 000; (b) and (d) × 1 400 000; (c) and (e) × 1 300 000. 333
FIG. 4. (a) Stained cubic crystal section cut perpendicular to [100] direction. (b) First optical integration of (a), translation diagonally up to letl. (c) Cubic crystal model viewed along [100] direction. (d) Second optical integration of (a), translation across page. (e) Unit cell of the cubic crystal model with a trimer along side (stereoscopic pair). One of the twofold screw axes in each of the three unit cell edge directions and the threefold rotation axis along the unit cell diagonal are indicated by sticks. (a) × 300 000; (b) and (d) × 1 300 000.
FIG. 5. (a) Stained cubic crystal section cut parallel to the (110) planes or rhombic crystal face. (b) Second optical integration of (a), translation along long page axis. (c) First optical integration of (a), translation skewed from across page. (d) Cubic crystal model viewed along [110] direction. The two sticks indicate two of the cube diagonal directions. (e) Cubic unit cell model of Fig. 4e viewed along the threefold rotation cube diagonal with the single trimer uppermost. (i) Cubic unit cell model of Fig. 4e viewed along the same diagnoal from the opposite direction. (a) and (c) × 300 000; (b) × 600 000.
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indicates that the cubic Fab' crystal does not belong to this space group, and ~hus it must belong to P213 instead. Several models were built to test this, and none with P23 symmetry was compatible with this projection in the [111] direction. For P213 symmetry, the four trimer positions in the unit cell lie along the diagonals of four of the eight small cubes, with edges a/2 that fit into the unit cell cube, such that there is a twofold screw axis at positions (a/2, a/4) and (a/2, 3a/4) in each projection along a cube axis. The [111] projection of an extended Fab' cubic crystal model built with this P213 symmetry is shown in Fig. 3f. The trimer is the same as that used to build the hexagonal Fab' crystal model shown in Figs. le, 2d, and 2f, and each of the trimers was placed I/4 the way along a small cube diagonal in accordance with the three pairs of nonintersecting twofold screw axes for this space group. This is seen more clearly in Fig. 4e, which is a stereoscopic pair of photographs of a model of this cubic unit cell viewed approximately down one of the cube axes. One of the trimers is to the right of the unit cell model. The unit cell model in Fig. 4e has a twofold screw axis indicated by a stick at the center of an open channel in each of the three cube axial directions together with a stick representing the threefold rotation axis along a cube diagonal. Even without stereoscopic viewing, the different thicknesses of the stick images give a clue to their relative positions. The trimer at the upper left in the unit cube, which is also the one closest to the viewer, is centered on the cube diagonal about 1/s the way along this diagonal from the upper left cube corner, but it is not in contact with the other three trimers in the unit cell. These other three trimers are in contact through one molecule or asymmetric unit in each. The angle that the molecular axes in the trimer make with a plane perpendicular to the threefold rotation axis of the trimer was taken as 28o8 ' so that these three contacting molecules would lie in a plane
and be in close contact with each other, as shown in Fig. 5f. Here the unit cube is viewed along the threefold rotation axis, looking directly at the triangle formed by the contacting molecules of the three trimers. In Fig. 5e, the view is down the opposite direction of this same threefold rotation axis, looking directly at the isolated trimer that was seen at the upper left in Fig. 4e. The connection between this isolated trimer and the three trimers in contact in each unit cell, which is necessary to give a self-supporting structure, is made through the molecules of adjacent unit cells. Just as the P213 space group allows any angle between the molecular axes of a trimer and the plane perpendicular to the rotation axis, the angle around the c u b e diagonal or threefold rotation crystal axis at which the first threefold symmetrically arranged trimer is placed in the unit cell can have any value. Once this first trimer is placed, of course, the positions of the other three are specified by the symmetries. The cubic model was constructed so that the projections of the three arms of this particular trimer Were, in the [111] projection, each approximately perpendicular to one of three sides of the hexagon outlining the unit cell (see Fig. 5e). This rotational position of' the first trimer and its location along the diagonal from a cube corner of about 1/8 the diagonal length were determined by changing these two until the best correspondence was found between the model and the [111] projection data as seen in the second optical integrations in Fig. 3. For checking this correspondence, the centers of the lightly stained areas in the second optical integration were taken as the projection of the cube diagonal about which there is threefold rotational symmetry. The lighter staining here reflects the relative lack of protein at these positions since, along this direction, the first trimer is separated from the plane of the connecting arms of the other three trimers by half a cube diagonal. The darker
FAB' CRYSTALS stained regions trigonally arranged around each of these would then approximately correspond to the centers of the three connecting unit cell trimers, at which projection positions you are looking along the molecules that join the first trimer with the plane of the connecting molecules of' the other three trimers, half the cube diagonal away. The three unstained areas trigonally arranged around each lightly stained area in this projection would correspond to the open channels. The choice of the linear position along the diagonal of the first trimer and its rotational position are both critical for the correspondence between the model and the [111] optical integrations. The 28 ° angle the molecular axes make with a plane perpendicular to the trimer rotation axis is not critical. It will be shown Iater that this angle can have other values and still give a model in agreement with the EM data. Choosing the 28 ° angle permitted the extended model to be made ac¢urately with ease and gave a model sufficiently open to allow the connection between the molecules to be traced in the model photographs. Having chosen the arbitrary parameters for this cubic crystal model to satisfy the EM data on the [111] projection, will this model also predict the EM data in the other two projections? A favorable comparison between this cubic model photographed in Fig. 4c, looking down one of the three cube axes, and the EM [100] projection in Fig. 4d, gives an affirmative answer for the unit cell edge directions. The stained crystal section in Fig. 5a, which was cut parallel to a set of (110) planes, or rhombic face of the crystal, has striations running in the direction of the short rhombic face axis (bottom to top of page) separated by about 102 A. The first optical integration of this in Fig. 5c, where the direction of travel for the averaging was nearly at right angles to the striae (shown by the streaking direction), shows periodicity along the striations. This periodicity of about 127 A is brought out more clearly in a
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second optical integration in Fig. 5b, where the direction of travel for averaging was along the striations. This 127 ]t periodicity is 14% lower than the comparable 148 A (unit cell length) determined by X-ray diffraction, but here again the knife travel in cutting these sections was along the striations, so this difference could be caused by the uncorrected section compression in this direction. A first glance at the model photographed in the [110] direction in Fig. 5d gives the impression that the period along the striations should be about 0.7 times their separation, but a more careful examination shows that the true repeat is about twice this or 1.4 times their separation. The adjacent protrusions along the striae being on opposite sides, as seen in Fig. 5b, is also understandable from the model photographs. Thus, in this [110] direction also, the model agrees with the EM data. Having demonstrated that the same trimer configuration can be used to construct a hexagonal and a cubic Fab' crystal model that agrees with the EM data, let us examine these models more critically to see if they are compatible with the more recent X-ray study of the hexagonal crystal (1I). If the hexagonal crystal model shown in Figs. 1 and 2 is correct, the X-ray determined unit cell length of a = 162.5 ~ would be satisfied by an Fab' molecular length of 63.9 A (each of the two spheres representing the molecule would be 31.9 ~ in diameter). If the cubic crystal model shown in Figs. 3, 4, and 5 is correct, the X-ray determined unit cell length of a = 148 would be satisfied by a molecular length of' 60.4 A. These molecular lengths predicted by the two models are reasonably close to each other but quite different from the molecular length of about 80 A found in 11. However, this same X-ray study on the Fab' hexagonal crystal showed that the correct angle between the molecular axes and a plane perpendicular to the c direction is about 60 ° , rather than the 28 ° used in making the models in Figs. 1-5. Using
FIG. 6. (a) Modified (see text) hexagonal (a) viewed along [10i0] direction. (c) Same crystal model viewed along [111] direction. model as in (d) viewed along [110] direction. (d), (e) and (f).
crystal model viewed along [0001] direction. (b) Same model as in model as in (a) viewed along [1120] direction. (d) Modified cubic (e) Same model as in (d) viewed along [100] direction. (f') Same The directions of the four cube diagonals are indicated by sticks in
FAB'CRYSTALS this 60 ° angle to make new models, as shown in Fig. 6, the molecular lengths predicted from these new hexagonal and cubic crystal models are 72.8 X and 71.1 A, respectively. The ratio a / c is equal to 2.58 in this new hexagonal crystal model. Here again, the molecular length predicted by these modified Fab' crystal models are very close together but still lower than the a p p r o x i m a t e length given in I 1 . However, the molecule is now so situated in the new cubic model (Figs. 6d, 6e, 6f) t h a t it can be longer without sensibly changing the unit cell dimensions. The increased length would just protrude farther into the open channel. T h e same thing would be true for the new hexagonal model if the trimers were rotated slightly around their threefold axes, such t h a t the projections of the molecular axes onto a plane perpendicular to c no longer coincided with lines joining adjacent threefold rotational crystal axes. This is, in fact, the way the molecules are placed in the real hexagonal crystal as d e t e r m i n e d in I I . These modified crystal models in Fig. 6 still fit the E M data. If the hexagonal model shown here were modified still further by rotating the trimers slightly, the comparison would still be favorable. This would u n d o u b t e d l y also be true if the more exact molecular shape, as determined by the X - r a y s t u d y (11) were used in the model, since the E M data from stained sections of d e h y d r a t e d crystals e m b e d d e d in plastic are very coarse or of low resolu-
339
tion c o m p a r e d to the X - r a y data. The models in Figs. 1-5 were retained because their more open structure makes the density variations along the striae t h a t one sees in Figs. 2c and 5d easier to see in the model photographs. These same variations along the striations obtain in the model p h o t o g r a p h s in Figs. 6b and 6f, b u t they are not as apparent. We are grateful to Mr. C. H. Hanna for cutting the sections. REFERENCES 1. Dana's Textbook of Minerology, John Wiley and Sons, Inc. New York, 1955. 2. International Tables for X-ray Crystallography, Voh I, Kynock Press, Birmingham, England 1952. 3. LABAW,L. W., J. Ultrastruct, Res. 5, 409 (1961). 4. LABAW,L. W., ANDDAVIES,D. R., J. Ultrastruct. Res. 40, 349 (1972). 5. LABAW,L. W., ANDOLSON,R. A., J. Ultrastruct. Res. 31,456 (1970). 6. LABAW,L. W., ANDROSSMANN,M. G., J."Ultrastruct. Res. 27, 105 (1969). 7. LANGE,R. H., J. Ultrastruct. Res. 46, 301 (1974). 8. LONGLEY,W., J. Mol. Biol. 30, 323 (1967). 9. MATTHEWS, B. W., J. Mol. Biol. 33, 491 (1968). 10. MCLACHLAN,D., Proc. Nat. Acad. Sci. U.S.A. 44, 948 (1958). 11. PADLAN,E. A., SEGAL, D. M., SPANDE, T. F., DAVIES, D. R., RUDIKOFF,S,, ANDPOTTER, M., Nature New Biol. 245, 165 (1973). 12. POLJAK,R. J., AMZEL,L. M., AvEY,H. P., BECKA, L. N., ANDNISONAFF,A., Nature New Biol. 235, 137 (1972). 13. RUDIKOFF,S., POTTER,M., SEGAL~D. M., PADLAN, E. A.. AND DAVIES, D. R., Proc. Natl. Acad. Sci. U.S.A. 69, 3689 (1972).
51,326-339 (1975)
An EM Study of Phosphorylcholine-Binding Fab' Immunoglobulin Fragment Crystals L. W. LABAW*, E. A. PADLANt, D. M. SEGAL~:, AND D. R. DAVIESt * Laboratory of Physical Biology and ~ Laboratory of Molecular Biology, National Institute of Arthritis, Metabolism, and Digestive Diseases, and $Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda. Maryland 20014 Received July 30, 1974, and in revised form December 17, 1974; accepted December 17, 1974 The Fab' fragment of McPC603 mouse myeloma imnmnoglobulin with phosphorylcholine-binding activity crystallizes into both a hexagonal and a cubic structure. Models of' these structures showing approximate molecular packing were deduced from an electron microscopic examination of stained crystal sections, a preliminary X-ray diffraction study, and the knowledge that both crystal structures grow simultaneously in the same crystallizing solution. When both of these models were modified slightly to agree with the more exact molecular packing in the hexagonal crystal from a 4.5 A resolution X-ray study, they were both still compatible with the EM data. INTRODUCTION
Th e crystallization and p r e l i m i n a r y X-ray investigation of the pepsin Fab' fragments from a mouse myeloma protein, IgA(K) McPC603, that binds phosphorylcholine has been reported (13) as part of a continuing study to elucidate the structure of the combining sites of antibody molecules. Crystallization in 43% saturated ammonium sulfate gave hexagonal prisms [see Fig. 3 (I3)] of space group P63 with unit cell dimensions a - b - 162.5, c - 60.8 J~ with one molecule per asymmetric unit. When these Fab' fragments were crystallized in. 1.7 molar potassium phosphate (K~HP04), both 1) regular rhombic dodecahedra, space group P213 or P23 with unit cell dimensions a = b = c = 148 A; and 2) the same hexagonal prisms obtained from the ammonium sulfate crystallization were formed, as shown in Fig. la. There was again only one molecule per asymmetric unit in these cubic crystals. The asymmetric unit being a single Fab' fragment with an approximate molecular weight of 50 000 gives the unit cell volume per dalton of protein (9) in the hexagonal crystal (where there are six asymmetric units per unit cell) of 4.6 A3/dalton, and in the cubic crystal (where there are 12 asymmetric
units per unit cell) of 5.4 A3/dalton, both very open structures. The hexagonal McPC603 Fab' crystals were stable enough to make a more detailed X-ray analysis practical. The structure of this Fab' fragment to 4.5 ~ resolution has recently been reported (I1). The present paper describes how an approximate molecular packing in both the hexagonal and the cubic crystals can be obtained by combining the preliminary X-ray data with data obtained from the electron microscopic examination of stained crystal sections.
326 Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
MATERIALS AND METHODS The Fab' crystals were fixed for several weeks in glutaraldehyde concentrations of 0.5%. The hexagonal crystals were transferred from the 43% saturated ammonium sulfate in which they were grown to saturated lithium sulfate to which glutaraldehyde was added to give 0.5%. This was done to avoid the reaction between the ammonium radical and glutaraldehyde. The cubic crystals were fixed in their mother liquor with glutaraldehyde in potassium phosphate added to give 0.5% glutaraldehyde. The use of a higher concentration of glutaraldehyde than the 0.03% used previously (4) was necessary to prevent the crystals dissolving during the dehydration procedure. This necessity for a higher concentration of cross-linking agent probably reflects the more open structure of these crystals. The crystals were then washed with 5 × l0 4 M phosphate buffer (pH 7.3), postfixed in
FAB' CRYSTALS 1% osmium tetroxide for 15 min, dehydrated in graded alcohols, and embedded in Maraglas. Sections of the hexagonal crystals were cut parallel to the (0001), (10i0), and (1120) planes; sections of the cubic crystals were cut parallel to the (100), (11i). and (110) planes. The proper orientations for these cuts could be made within one or two degrees since the hexagonal prism crystals had six well-developed (1120) type faces parallel to the c or long axis and the cubic crystals had 12 rhombic (110) type faces (see Fig. lal. The sections were stained with uranyl acetate for 60 rain followed by lead citrate for 1 min, then lightly coated with carbon for increased stability in the electron microscope. The average periodic pattern in the micrographs (first optical integration) was obtained by averaging over the periodicity in real space by repeated linear translation and superposition of the micrograph upon itself (4, 5, 6, 10). The signal-to-noise ratio was further improved and the contrast enhanced by performing a similar second averaging on the first (second optical integration), using a diflerent direction of translation. Micrographs were taken on a Philips 300 microscope that was calibrated using indanthrene olive T crystals coated with carbon for increased stability in the beam. These crystals acted as phase gratings with a 24.9 ~, periodicity as measured by X-ray powder diffraction patterns (3). This periodicity also was checked at about × 500 000 against the graphitized carbon spacing of 3.4 •. OBSERVATIONS AND DISCUSSION A t y p i c a l s t a i n e d section of the hexagonal F a b ' crystals c u t p e r p e n d i c u l a r to the c axis [parallel to the (0001) p l a n e s ] is s h o w n in Fig. lb. T h i s p r o j e c t i o n of the crystal along the c d i r e c t i o n has the a p p e a r a n c e of a h o n e y c o m b s t r u c t u r e . T h e d e n s i t y in the holes is similar to t h a t in the plastic o u t s i d e the c r y s t a l (not shown) a n d there is c o n s i d e r a b l e d e n s i t y v a r i a t i o n in the wall a r o u n d e a c h hole. First a n d s e c o n d o p t i c a l i n t e g r a t i o n s of this m i c r o g r a p h , shown in Figs. lc a n d d, respectively, d e m o n s t r a t e t h a t there is no c o n s i s t e n t p a t t e r n to this v a r i a t i o n as t h e r e was in b a c t e r i o c h l o r o phyll crystals of the s a m e space g r o u p (5). T h i s h o n e y c o m b p a t t e r n suggests t h a t there are large h e x a g o n a l l y a r r a n g e d c h a n nels parallel to t h e c axis filled w i t h e m b e d d i n g plastic a n d t h u s n o t stained, giving a very o p e n c r y s t a l s t r u c t u r e , as indic a t e d also b y t h e v o l u m e to p r o t e i n ratio of
327
4.6 A3/dalton. B o t h c a t a l a s e (8) a n d b a c t e r i o c h l o r o p h y l l (5) h e x a g o n a l c r y s t a l s s h o w e d similar o p e n c h a n n e l s . T h e location of the s t a i n in this p r o j e c t i o n is also the location of the p r o j e c t e d protein. T h i s i n t e r p r e t a t i o n n e e d not m e a n t h a t the p r o t e i n itself stains. P r e v i o u s work (4) i n d i c a t e s t h a t the F a b ' m o l e c u l e does n o t stain very intensely. M o s t of the stain is p r o b a b l y , therefore, b e t w e e n the m o l e c u l e s where the e m b e d d i n g plastic could n o t p e n e t r a t e (see discussion of this p o i n t in (4 a n d 5). T w e n t y m i c r o g r a p h s , e.g., the one shown in Fig. lb, were m e a s u r e d to get an a v e r a g e value for a. In e a c h m i c r o g r a p h , the centert o - c e n t e r s e p a r a t i o n of the c h a n n e l s was m e a s u r e d along e a c h of' the three lattice directions. T h e a v e r a g e of these 60 m e a s u r e m e n t s gave a = 151.8 ± 7.9 A (stand a r d deviation), w h i c h is 6.6% below the X - r a y value. If only the highest m e a s u r e m e n t for e a c h m i c r o g r a p h was used, the average of these 20 gave a = 159.0 ± 5.3 A, or only 2.1% below the X - r a y value. T h e difference b e t w e e n these two averages reflects the d i s t o r t i o n in the sections resulting f r o m the c u t t i n g a n d m o u n t i n g procedures. T h e h e x a g o n a l s t r u c t u r e in Fig. lb, for e x a m p l e , is c o m p r e s s e d along the s h o r t page axis, which was also the c u t t i n g direction. T h e P63 space g r o u p s y m m e t r i e s (2) can be satisfied either b y h a v i n g the a s y m m e t ric u n i t s c l u s t e r e d a r o u n d the sixfold screw axes or a r o u n d the threefold r o t a t i o n axes. It is clear f r o m the m i c r o g r a p h in Fig. 1 t h a t the latter o b t a i n s for this crystal, with the centers of the o p e n c h a n n e l s being t h e sixfold screw axes. T h e three a s y m m e t r i c u n i t s t h a t m u s t lie a r o u n d one threefold r o t a t i o n axis would be, for this space group, at a level c/2 f r o m the three a s y m m e t r i c u n i t s s u r r o u n d i n g e a c h of t h e three a d j a c e n t threefold r o t a t i o n axes. It is ins t r u c t i v e to c o n s t r u c t a m o d e l of this crystal using two spheres joined t o g e t h e r as the a s y m m e t r i c unit, or m o l e c u l e in this case, where one sphere r e p r e s e n t s the v a r i a b l e
FIO. 1. (a) Optical micrograph of McPC603 Fab' crystals grown in potassium phosphate. (b) Electron micrograph of a stained hexagonal crystal section cut perpendicular to the c direction. (c) First optical integration of (b), translation for integration across page. (d) Second optical integration of (b), translation up to right. (e) Hexagonal crystal model viewed along the c direction, with one simulated molecule and three trimers beside it. (a) × 72; (b) x 400 000; (c) and (d) × 1 100 000.
FAB'CRYSTALS
329
portion of the Fab' molecule and the other, pattern. The hexagonal crystal model is the constant portion. [References 4 (Fig. 3), seen looking along the [10i0] direction in 11, and 12 indicate that this is a reasonable Fig. 2d. T h e four lines of molecules parallel first approximation.) Such a model is to c showing shadows should correspond to shown, looking down the c direction, in Fig. the lines of m a x i m u m staining in Fig. 2c le. T h e angle t h a t the long axes of the since here you are looking more nearly simulated molecules make with a plane along molecular lengths (see Fig. le). (The perpendicular to c was made 28 ° (this crystal model shown in Fig. le was photochoice will be explained later) and the graphed in the direction of a 60 ° clockwise projections of these molecules on this plane rotation from the short page axis to obtain were taken to lie along lines joining adja- Fig. 2d; therefore, there are four lines with cent threefold rotation crystallographic shadows corresponding to m a x i m u m stain axes for convenience in making the model. lines in the micrograph.) If these lines with T h e separation of adjacent threefold rota- shadows in the model picture are e x a m i n e d tion axes in the model was a consequence of carefully, staggered prominences correclose-fitting a simulated molecule at one sponding the those in Fig. 2e are apparent. level along c with one from an adjacent While the second optical integration has threefold rotation center, which, as noted the general characteristics predicted by the above, was at a level c/2 away in the c model, the p a t t e r n is skewed and the direction. This can be seen in Fig. 2d. T h e measured value of c = 44.3 A is 27% ratio of a (the center-to-center distance smaller t h a n the X-ray value. This section between the channels in Fig. 1) to c in this was cut with the d i a m o n d knife moving in model is 2.55 compared to 2.67 from the the c direction and thus was considerably X-ray crystal m e a s u r e m e n t s , a difference compressed or folded in this direction; of only 4.5%. inadequacy of the subsequent t r e a t m e n t T h e micrograph in Fig. 2a is of a stained with chloroform vapor to unfold the section section cut parallel to the (1010) planes of on the water surface in the boat betbre it the hexagonal Fab' crystals. This projec- was picked up on the specimen grid m a y tion is equivalent to looking along the short account for this large discrepancy. T h e r e page axis at the crystal sections shown in also m a y have been some shrinkage in this Figs. lb, c, d, and e. T h e coarse periodicity direction during the embedding procedure. across Fig. 2a should thus be v/3a/2, which b u t this explanation is not as likely as the is 140.7 /~ for the X - r a y d e t e r m i n e d unit former. T h e most extreme measured value cell. M e a s u r e m e n t s on 46 micrographs of on the 46 micrographs leading to the aversections with this orientation gave an aver- age value of V~a/2 = 137.6 A cited above age value of 137.6 ± 8.7 }t, which is 2.2% was only 12.8% lower t h a n this average, b u t lower than the X - r a y value. It is difficult to all these sections were cut with the knife see any periodicity along the c direction, moving in the c direction, so this measurewhich is vertical in Fig. 2a, b u t a first m e n t was not affected by the section foldoptical integration of this in Fig. 2b, and a ing. second optical integration in Fig. 2c, demFigure 2e shows a typical stained section onstrate t h a t there is one. T h e direction of cut parallel to the (11-20) planes of the translation to obtain Fig. 2b from Fig. 2a hexagonal crystal. This projection is along was a p p r o x i m a t e l y at right angles to the c a direction 30 ° to t h a t for Fig. 2a and still direction, i.e., the lines in the micrograph, perpendicular to c. This projection down and to obtain Fig. 2c from Fig. 2b, along the [1120] direction is equivalent to looking the c direction. In the latter case, using a along the long page axis at the crystal translation interval along c twice the one sections in Fig. 1. Thus, any coarse perioshown here did not sensibly change the dicity should be a/2, with the darker lines
FIo. 2. (a) Stained hexagonal crystal section cut parallel to (10i0) planes. (b) First optical integration of (a), translation across page. (c) Second optical integration of' (a), translation along long page axis period. (d) Hexagonal crystal viewed along [1010] direction. (e) Stained hexagonal crystal section cut parallel to (llg0) planes. (t) Hexagonal crystal model viewed along [1120] direction. (a) and (el x 400 000; (b) × 600 000; (c) × 1 300 000. 330
FAB' CRYSTALS of stain parallel to c having less contrast than in Fig. 2a and corresponding to lines of molecules viewed end-on. The model photographed in the corresponding orientation in Fig. 2f shows no openings directly through the crystal in this direction. Therefore, one would not expect to observe any distinct periodicity in the staining along c. This was confirmed by not finding any consistent periodicity along c in any of the optical integrations performed on several micrographs of this projection. Measurements on 30 micrographs gave an average for a/2 of 81.0 ± 3.2 A, compared with the X-ray value of 81.3 ]~. Here again, sections were cut with the knife motion along c. It should be emphasized t ha t in making the hexagonal crystal model, the choice of the 28 ° angle the molecular axes make with a plane perpendicular to c was rather arbitrary, as was also the alignment of the molecules such that their axial projections on this plane are exactly along lines joining the adjacent threefold axes. Because the cell is so open, a large latitude is available in choosing the angle that the molecular axes make with the plane perpendicular to c. This can be anything from 0 ° to 60 ° without disagreeing with the electron micrographs. Likewise, the trimers at the threefold rotation centers could be rotated by up to 20 ° and still fit the EM data. Determination of the correct angles for these can only be made by further X-ray structure analysis. This will be discussed again after the data from the cubic crystal form have been presented. The fact that both the hexagonal and the cubic crystals grow side by side under the same conditions strongly suggests that the molecules are grouped together in both crystals in a similar fashion. This notion would be confirmed, fbr example, if the same trimer a r r a n g e m e n t of molecules around the threefold hexagonal crystals axes also obtained around the four threefold cubic crystal axes. It will now be shown that such an interpretation is compatible with the elec-
331
tron microscopic data. The choice of the 28 ° angle the molecular axes make with a plane perpendicular to the threefold rotation axes in the hexagonal crystal model was dictated by the fact that this same angle between the molecular axes and planes perpendicular to the four threefold cubic crystal axes greatly facilitates making an extended cubic crystal model that agrees with the EM data and gives a structure sufficiently open to make this agreement clear. Regular rhombic dodecahedra have two types of vertices (I): (1) those at the intersection of three faces, and (2) those at the intersection of four faces. If all of the 12 rhombic faces are equal, the crystal structure should have threefold rot at i onal symmetry around lines joining opposite three-face vertices (which are also the diagonals of this truncated cube) and have twofold screw or rotational symmetry around lines joining opposite four-face vertices (these lines are parallel to the truncated cube edges) in accord with the preliminary X-ray finding (13) that these crystals belong to either the P213 or P23 space groups. Fortunately, the structure of this cubic Fab' crystal is sufficiently open, and the molecules are aligned well enough in the directions of the cube diagonals and cube edges, so that stained sections cut perpendicular to these directions show cross-periodicities. The nature and orientation of these periodicities are sufficient to 1) eliminate P23 as a possible structure, and 2) determine the approximate molecular packing in the P2~3 structure. The periodicities in stained sections of this cubic Fab' crystal indicate that there are planes of density parallel to each face of the rhombic dodecahedron, spaced just over 100 • from one another in a direction perpendicular to that face. [Lange's Bgranules (7) had a similar appearance but different spacing.] Thus, in sections cut perpendicular to the cube diagonals, or [111] direction, three equal periodicities 60 ° or 120 ° apart in angle give the hexago-
332
LABAW E T AL.
nal pattern shown in Fig. 3a. These three intersecting sets of lines in this projection are parallel to the sides of the hexagon that outline the crystal when it is viewed down the threefold axis (see lower right crystal in Fig. la). The average line separation from 30 measurements on 10 micrographs was 101.7 i 4.3 A. Sections cut perpendicular to the twofold axes (lines joining opposite four-face vertices) or unit cell edges showed two intersecting sets of lines at right angles to each other, as shown in Fig. 4a. These lines are parallel to the sides of the square t h a t outline a rhombic dodecahedron viewed down one of these twofold axes and are therefore parallel to the projections of the cube diagonals in this direction. Thirty measurements on 15 micrographs gave the average line separation in this projection of 103.3 ~: 4.6 A. On sections cut parallel to a face of the rhombic dodecahedron crystals (projection in the [110] direction), the lines of density, as seen for example in Fig. 5a, were parallel to the short axis of the face. The average line separation on 20 micrographs showing this projection was 104.0 ± 4.2 •. The X-ray data leading to the space group assignments showed a very prominent spacing equal to 1/2 the diagonal of a cube face or 1/2 (V~a) = 104..7 A. The spacings measured on the electron microbraphs, as indicated above, are essentially identical. The average stain pattern looking in the direction of the cube axes, as shown in the first and second optical integrations in Figs. 4b and 4d respectively, implies that there are rectangularly arranged holes through the crystal in these three directions. The first and second optical integrations of Fig. 3a in Figs. 3b and 3d show an average stain pattern in which each lightly stained center is surrounded by three trigonally arranged holes and three more heavily stained regions, also trigonally arranged. Thus, along each of the four cube diagonal directions there are empty channels through the crystal arranged hexago-
nally. This very open structure agrees with the large volume of 5.4/~3/dalton of protein mentioned earlier. Since the stain density in the projection down the threefold rotation axes, as seen in Fig. 3d, contains the clue to the molecular packing in the crystal, first and second optical integrations were performed on several other micrographs of sections similarly cut. All of the second integrations had density patterns that could be characterized as having hexagonally arranged weakly stained regions, each trigonally surrounded by three regions of no stain and three densely stained regions. Two of these second optical integrations are shown in Figs. 3c and 3e. With the measured average line separation on micrographs of this projection being 101.7/~, the separation of the darker stained areas (center-to-center), or the lightly stained areas, or the holes, becomes 117.4 A or about 120 A, which is equal to the projection of the X-ray unit cell edge onto this plane, both in magnitude (120.8 A) and direction. Thus, any of these features of the repeating pattern could be at the threefold rotation center of the unit cell. The preliminary X-ray data identified the space group for this crystal as either P23 or P213. Both of these (2) have 12 asymmetric units in the unit cell, with threefold rotation axes. This means that at each of four positions in either unit cell there are three asymmetric units arranged with rotational symmetry. In the P23 space group, these four positions are at the corners of a tetrahedron, and these centers can only be somewhere along the diagonals of the unit cell. Now, if the lightly stained areas in this projection correspond to one of these centers, the three darkly stained areas trigonally arranged around them should correspond to the other three trimer centers, which for the P23 space group should each be directly between two lightly stained areas, since for this space group they must be along the cube diagonals. The fact that they are off to one side of this line
Fro. 3. (a) Stained cubic crystal section cut perpendicular to [111] direction. (b) First optical integration of' (a), translation across page. (d) Second optical integration of (a). translation up to right. (c) and (e) Second optical integrations of two other micrographs similar to (a). (f) Cubic crystal model viewed along [111] direction. (a) × 300 000; (b) and (d) × 1 400 000; (c) and (e) × 1 300 000. 333
FIG. 4. (a) Stained cubic crystal section cut perpendicular to [100] direction. (b) First optical integration of (a), translation diagonally up to letl. (c) Cubic crystal model viewed along [100] direction. (d) Second optical integration of (a), translation across page. (e) Unit cell of the cubic crystal model with a trimer along side (stereoscopic pair). One of the twofold screw axes in each of the three unit cell edge directions and the threefold rotation axis along the unit cell diagonal are indicated by sticks. (a) × 300 000; (b) and (d) × 1 300 000.
FIG. 5. (a) Stained cubic crystal section cut parallel to the (110) planes or rhombic crystal face. (b) Second optical integration of (a), translation along long page axis. (c) First optical integration of (a), translation skewed from across page. (d) Cubic crystal model viewed along [110] direction. The two sticks indicate two of the cube diagonal directions. (e) Cubic unit cell model of Fig. 4e viewed along the threefold rotation cube diagonal with the single trimer uppermost. (i) Cubic unit cell model of Fig. 4e viewed along the same diagnoal from the opposite direction. (a) and (c) × 300 000; (b) × 600 000.
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LABAW ET AL.
indicates that the cubic Fab' crystal does not belong to this space group, and ~hus it must belong to P213 instead. Several models were built to test this, and none with P23 symmetry was compatible with this projection in the [111] direction. For P213 symmetry, the four trimer positions in the unit cell lie along the diagonals of four of the eight small cubes, with edges a/2 that fit into the unit cell cube, such that there is a twofold screw axis at positions (a/2, a/4) and (a/2, 3a/4) in each projection along a cube axis. The [111] projection of an extended Fab' cubic crystal model built with this P213 symmetry is shown in Fig. 3f. The trimer is the same as that used to build the hexagonal Fab' crystal model shown in Figs. le, 2d, and 2f, and each of the trimers was placed I/4 the way along a small cube diagonal in accordance with the three pairs of nonintersecting twofold screw axes for this space group. This is seen more clearly in Fig. 4e, which is a stereoscopic pair of photographs of a model of this cubic unit cell viewed approximately down one of the cube axes. One of the trimers is to the right of the unit cell model. The unit cell model in Fig. 4e has a twofold screw axis indicated by a stick at the center of an open channel in each of the three cube axial directions together with a stick representing the threefold rotation axis along a cube diagonal. Even without stereoscopic viewing, the different thicknesses of the stick images give a clue to their relative positions. The trimer at the upper left in the unit cube, which is also the one closest to the viewer, is centered on the cube diagonal about 1/s the way along this diagonal from the upper left cube corner, but it is not in contact with the other three trimers in the unit cell. These other three trimers are in contact through one molecule or asymmetric unit in each. The angle that the molecular axes in the trimer make with a plane perpendicular to the threefold rotation axis of the trimer was taken as 28o8 ' so that these three contacting molecules would lie in a plane
and be in close contact with each other, as shown in Fig. 5f. Here the unit cube is viewed along the threefold rotation axis, looking directly at the triangle formed by the contacting molecules of the three trimers. In Fig. 5e, the view is down the opposite direction of this same threefold rotation axis, looking directly at the isolated trimer that was seen at the upper left in Fig. 4e. The connection between this isolated trimer and the three trimers in contact in each unit cell, which is necessary to give a self-supporting structure, is made through the molecules of adjacent unit cells. Just as the P213 space group allows any angle between the molecular axes of a trimer and the plane perpendicular to the rotation axis, the angle around the c u b e diagonal or threefold rotation crystal axis at which the first threefold symmetrically arranged trimer is placed in the unit cell can have any value. Once this first trimer is placed, of course, the positions of the other three are specified by the symmetries. The cubic model was constructed so that the projections of the three arms of this particular trimer Were, in the [111] projection, each approximately perpendicular to one of three sides of the hexagon outlining the unit cell (see Fig. 5e). This rotational position of' the first trimer and its location along the diagonal from a cube corner of about 1/8 the diagonal length were determined by changing these two until the best correspondence was found between the model and the [111] projection data as seen in the second optical integrations in Fig. 3. For checking this correspondence, the centers of the lightly stained areas in the second optical integration were taken as the projection of the cube diagonal about which there is threefold rotational symmetry. The lighter staining here reflects the relative lack of protein at these positions since, along this direction, the first trimer is separated from the plane of the connecting arms of the other three trimers by half a cube diagonal. The darker
FAB' CRYSTALS stained regions trigonally arranged around each of these would then approximately correspond to the centers of the three connecting unit cell trimers, at which projection positions you are looking along the molecules that join the first trimer with the plane of the connecting molecules of' the other three trimers, half the cube diagonal away. The three unstained areas trigonally arranged around each lightly stained area in this projection would correspond to the open channels. The choice of the linear position along the diagonal of the first trimer and its rotational position are both critical for the correspondence between the model and the [111] optical integrations. The 28 ° angle the molecular axes make with a plane perpendicular to the trimer rotation axis is not critical. It will be shown Iater that this angle can have other values and still give a model in agreement with the EM data. Choosing the 28 ° angle permitted the extended model to be made ac¢urately with ease and gave a model sufficiently open to allow the connection between the molecules to be traced in the model photographs. Having chosen the arbitrary parameters for this cubic crystal model to satisfy the EM data on the [111] projection, will this model also predict the EM data in the other two projections? A favorable comparison between this cubic model photographed in Fig. 4c, looking down one of the three cube axes, and the EM [100] projection in Fig. 4d, gives an affirmative answer for the unit cell edge directions. The stained crystal section in Fig. 5a, which was cut parallel to a set of (110) planes, or rhombic face of the crystal, has striations running in the direction of the short rhombic face axis (bottom to top of page) separated by about 102 A. The first optical integration of this in Fig. 5c, where the direction of travel for the averaging was nearly at right angles to the striae (shown by the streaking direction), shows periodicity along the striations. This periodicity of about 127 A is brought out more clearly in a
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second optical integration in Fig. 5b, where the direction of travel for averaging was along the striations. This 127 ]t periodicity is 14% lower than the comparable 148 A (unit cell length) determined by X-ray diffraction, but here again the knife travel in cutting these sections was along the striations, so this difference could be caused by the uncorrected section compression in this direction. A first glance at the model photographed in the [110] direction in Fig. 5d gives the impression that the period along the striations should be about 0.7 times their separation, but a more careful examination shows that the true repeat is about twice this or 1.4 times their separation. The adjacent protrusions along the striae being on opposite sides, as seen in Fig. 5b, is also understandable from the model photographs. Thus, in this [110] direction also, the model agrees with the EM data. Having demonstrated that the same trimer configuration can be used to construct a hexagonal and a cubic Fab' crystal model that agrees with the EM data, let us examine these models more critically to see if they are compatible with the more recent X-ray study of the hexagonal crystal (1I). If the hexagonal crystal model shown in Figs. 1 and 2 is correct, the X-ray determined unit cell length of a = 162.5 ~ would be satisfied by an Fab' molecular length of 63.9 A (each of the two spheres representing the molecule would be 31.9 ~ in diameter). If the cubic crystal model shown in Figs. 3, 4, and 5 is correct, the X-ray determined unit cell length of a = 148 would be satisfied by a molecular length of' 60.4 A. These molecular lengths predicted by the two models are reasonably close to each other but quite different from the molecular length of about 80 A found in 11. However, this same X-ray study on the Fab' hexagonal crystal showed that the correct angle between the molecular axes and a plane perpendicular to the c direction is about 60 ° , rather than the 28 ° used in making the models in Figs. 1-5. Using
FIG. 6. (a) Modified (see text) hexagonal (a) viewed along [10i0] direction. (c) Same crystal model viewed along [111] direction. model as in (d) viewed along [110] direction. (d), (e) and (f).
crystal model viewed along [0001] direction. (b) Same model as in model as in (a) viewed along [1120] direction. (d) Modified cubic (e) Same model as in (d) viewed along [100] direction. (f') Same The directions of the four cube diagonals are indicated by sticks in
FAB'CRYSTALS this 60 ° angle to make new models, as shown in Fig. 6, the molecular lengths predicted from these new hexagonal and cubic crystal models are 72.8 X and 71.1 A, respectively. The ratio a / c is equal to 2.58 in this new hexagonal crystal model. Here again, the molecular length predicted by these modified Fab' crystal models are very close together but still lower than the a p p r o x i m a t e length given in I 1 . However, the molecule is now so situated in the new cubic model (Figs. 6d, 6e, 6f) t h a t it can be longer without sensibly changing the unit cell dimensions. The increased length would just protrude farther into the open channel. T h e same thing would be true for the new hexagonal model if the trimers were rotated slightly around their threefold axes, such t h a t the projections of the molecular axes onto a plane perpendicular to c no longer coincided with lines joining adjacent threefold rotational crystal axes. This is, in fact, the way the molecules are placed in the real hexagonal crystal as d e t e r m i n e d in I I . These modified crystal models in Fig. 6 still fit the E M data. If the hexagonal model shown here were modified still further by rotating the trimers slightly, the comparison would still be favorable. This would u n d o u b t e d l y also be true if the more exact molecular shape, as determined by the X - r a y s t u d y (11) were used in the model, since the E M data from stained sections of d e h y d r a t e d crystals e m b e d d e d in plastic are very coarse or of low resolu-
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tion c o m p a r e d to the X - r a y data. The models in Figs. 1-5 were retained because their more open structure makes the density variations along the striae t h a t one sees in Figs. 2c and 5d easier to see in the model photographs. These same variations along the striations obtain in the model p h o t o g r a p h s in Figs. 6b and 6f, b u t they are not as apparent. We are grateful to Mr. C. H. Hanna for cutting the sections. REFERENCES 1. Dana's Textbook of Minerology, John Wiley and Sons, Inc. New York, 1955. 2. International Tables for X-ray Crystallography, Voh I, Kynock Press, Birmingham, England 1952. 3. LABAW,L. W., J. Ultrastruct, Res. 5, 409 (1961). 4. LABAW,L. W., ANDDAVIES,D. R., J. Ultrastruct. Res. 40, 349 (1972). 5. LABAW,L. W., ANDOLSON,R. A., J. Ultrastruct. Res. 31,456 (1970). 6. LABAW,L. W., ANDROSSMANN,M. G., J."Ultrastruct. Res. 27, 105 (1969). 7. LANGE,R. H., J. Ultrastruct. Res. 46, 301 (1974). 8. LONGLEY,W., J. Mol. Biol. 30, 323 (1967). 9. MATTHEWS, B. W., J. Mol. Biol. 33, 491 (1968). 10. MCLACHLAN,D., Proc. Nat. Acad. Sci. U.S.A. 44, 948 (1958). 11. PADLAN,E. A., SEGAL, D. M., SPANDE, T. F., DAVIES, D. R., RUDIKOFF,S,, ANDPOTTER, M., Nature New Biol. 245, 165 (1973). 12. POLJAK,R. J., AMZEL,L. M., AvEY,H. P., BECKA, L. N., ANDNISONAFF,A., Nature New Biol. 235, 137 (1972). 13. RUDIKOFF,S., POTTER,M., SEGAL~D. M., PADLAN, E. A.. AND DAVIES, D. R., Proc. Natl. Acad. Sci. U.S.A. 69, 3689 (1972).
