J. Mol. Biol. (1976)
101, 39-66
Surface Structure of in vitro Assembled Bacteriophage Lambda Polyheads M.
WURTZ.J.
KISTLER
AND T. HOHX
LJepartment of Microbiology, Biozentrum. 3056 Basel, Switzerhd
Klingelbergstr,
70
(Received 4 September 1975) Tubular aberrant phage h capsoids (polyheads) can be obtained by self-assembly of capsid protein. Three types of polyheads can be distinguished which correspond in their surface structure to petit A, enlarged petit h (D--head) and phage head. The latter type of polyhead consists of two different proteins (pE and pD) arranged in a composite hexagonal lattice, the other two of one type of protein (pE) arranged in hexagonal lattices. Each type has its own lattice constants. Polyheads were observed both after negative staining and freeze drying-shadowing and data were exploited by optical filtration methods. Models are proposed to elucidate the surface fine structure of polyheads and their spherical analogues, and the conformational changes occurring during maturation of the phage X head.
1. Introduction important biological processes such as regulation of enzyme activities (Monod et al., 1965), muscle contraction (Wakabayashi et al., 1975), membrane permeability (Singer, 1974) and assembly of complex subcellular structures (Wood, 1974; Kellenberger, 1972) are believed to be controlled by conformational changes of proteins. These shifts will be more easily detected if large arrays of regularly arranged identical subunits are involved. Thus, in morphopoietic transitions of viruses (Hohn et aZ., 1974,1976; Showe & Kellenberger, 1975), due to a “domino effect”, the shift of one subunit is transferred to all the other identical subunits and thus becomes eligible for electron microscopical observation. Still, the detailed interpretation of virus morphopoietic transitions by electron microscopy can be difficult because of: (I) superposition of the upper and lower surface of the particles; (2) curvature of the particle surfaces; (3) the different orientation of the individual particles on the grid and (4) the artifacts and distortions caused hy preparation techniques and the damage caused by the electron beam (Dubochet. 1975; Beer et al., 1975). In order to overcome these difficulties, techniques were developed to produce paracrystalline arrays of viruses (Horne & Pasquali-Ronchetti, 1974). Virus surfaces were studied on squashed particles (Mazza & Felluga, 1973 ; Williams & Richards, 1974; Hohn et al., 1974), where areas of subunits are oriented flat on the microscope grid, or elongated tubular viruses or aberrant tubular capsids (polyheads) were examined, where the multiple repetition of the basic structural elements along the long axis of the particle allows the application of optical filtration methods (De Rosier & Klug, 1972; Yanagida et al., 1972; Leonard & Kleinschmidt. 1972). Many
31.
40
\V1:HTZ
E?’ ..l I,
The bacteriophage X head is supposed to undergo several transitions during morphogenesis and DNA packaging (Hohn et al., 1974,1975,1976; Hendrix & Casjens, 1975). A prehead empty of DNA is formed first. It packages the DNA, swells thereby and adds the small capsid protein (pD) in amounts equimolar to the large capsid protein (pE). Accordingly, three types of head structures can be isolated from infected lysates: preheads, enlarged preheads (D--heads), both consisting of pE, and heads consisting of pE and pD. Conditions were sought and found to produce in vitro three types of h polyheads that are analogues in their protein composition and surface structure to these spherical particles. The lambda polyheads show a helical arrangement of surface clusters with a pitch angle of 30”. At this pitch angle the upper and the lower surface image cannot be separated by optical filtration of negatively stained specimens. To obtain one-sided images, a freeze drying-shadowing method was adapted from Nermut & Frank (1971).
2. Materials and Methods (a) Petit
h particles
and phage heads
Petit h particles and empty phage heads were isolated Their protein composition is listed in Table 1. (b) Dissociation
as described
by Hohn
et al. (1975).
and rewsembly
In a typical experiment, 1 ml petit h or emptied h heads (40 mg/ml in TMA buffer (Hohn et al., 1975)) was mixed with 1.4 g guanidinium*HCl and 15 ~1 dithiothreitol (0.1 M) and was kept for 1 h at 37°C. Then 9 ml 0.1 M-ammonium carbonate buffer (pH 8) (or another buffer, if indicated) were added and the solution dialysed overnight at room temperature against 100 ml of the same buffer. Precipitates that formed were removed by centrifugation and the solution was concentrated IO-fold by pressure dialysis through an Amicon PM10 filter. The concentrate contained polyheads and other assemblies. (c) Procedure
for polyhead
enrichment
from
crude lysates
Crude solutions of polyheads were a side product of the isolation of petit X particles. After centrifugation through a sucrose gradient in the zonal rotor, where petit h move to containing polythe middle of the gradient (see Hohn et al., 1975), the cushion fraction, heads and other phage-related materials and debris was used. It was diluted to 1 1 with TMA buffer and centrifuged for 5 min at 5000 revs/min in the GS3 rotor of the Sorvall centrifuge. The supernatant was centrifuged again for 10 min at 8000 revs/min and the pellet was resuspended in 100 ml TMA buffer. After two further cycles of centrifugation at 5000 and 8000 revs/min, the pellet was resuspended in 5 ml TMA buffer and used for electron microscopy of polyheads. (d) Preparation (i) Negative
for electron
microscopy
staining
Negative staining was either performed with 2% uranyl acetate (pH 4.2), or with sodium phosphotungstate (pH 7.2). All electron micrographs presented are stained the latter method. (ii)
Freeze drying
2% by
and shadowing
Carbon-coated collodion grids were glow discharged and then placed on a drop of particle suspension for adsorption, The grids were washed with several drops of distilled water and put upsidedown on a wet filter paper; thus a very thin layer of water is formed. After a few seconds the grids were removed and immediately frozen in liquid nitrogen. Freeze drying was done with a Balzers apparatus BA-511 M equipped with an electron gun evaporation source and a quartz film-thickness monitor. Sublimation was at -30°C
LAMBDA
POLYHEADS,
IN
41
I’ITRO
within a vacuum of better than 5 x low7 Torr. The dried particles were shadowed with tungsten at an angle of 30’ with a mass thickness of about 5 d. For stabilization, carbon was evaporated at an angle of 90” onto the specimen. The vacuum during evaporation was about 4 x 1O-6 Torr. (e) Electron
microscopy
and image processiny
The specimens were observed in a Philips 300 electron microscope. Optical diffraction and filtration work were done according to the prescriptions of Aebi et al. (1973). The shape of the window was chosen so that its diffraction does not, interfere with the diffraction pattern of the polyheads.
3. Results (a) In vitro aggregation of h capsid protein Petit X particles and empty X heads isolated from certain mutants of phage and host and dissolved in guanidinium *HCI constitute different types of “instant” protein
mixtures
for self-assembly
experiments
(Table
1). Thus for instance
Nu3
defective petit h protein consists of main capsid protein pE ; or head protein consists of small capsid protein pD and a set of minor proteins in addition to pE. After restoring favourable ionic conditions to the protein mixtures by dilution with the assembly buffer and concentration by pressure dialysis, the protein aggregates. About 30 to 50% of the input pE precipitates and is removed. The soluble fraction contains various types of assemblies, i.e. roundish particles of the size of petit X, smaller round&h particles (petit, petit X), a few larger roundish particles, various kinds of abnormally formed particles (monsters, spirals, sheets, long tubular structures (polyheads)) and amorphous aggregates of peanut shape (Fig. 1). All types of protein mixtures shown in Table 1 yielded these general types of assemblies in similar relative amounts, except for pNu3-containing mixtures, where the proportion of normal-sized petit h particles seemed to be higher, and for the pD-containing mixtures, where fine structures of the assembly products were different. The proportions of particles did vary, however, with ionic strength and pH of the assembly TABLE 1 Protein
composition of petit h and empty heads used as a source of protein for in vitro self-assembly experiments
Particle as source of proteins
Protein PLNU3 (16H
composition PI) (1W
Minor
proteins
Polyheads obtained
*Vu3-
petit
X
None
A> (B)f
groE-
petit
h
Unprocessed
A> (B)
Processed
:I, (B)
Processad
(1
I)
p&t
Empty
-I-
A (prehead) head
-
/-
t Molecular weight x 10 -3. 1 Minor fraction in mixture. Data from Hohn et al. (1976). The last column shows the types specific mixtures. More rarely occurring type in parentheses.
of polyheads
obtained
with
the
Figs 1 and 2. Particles obtained after self-assembly of phage h capsid protein. Electron micrographs from negatively stained preparations with 2% sodium phosphotungstate (pH 7.2). Particles are named spirals (e.g. in l(c)), petit petit X (in l(e) and 2(b)), petit A (e.g. in 2(a)),
LAMBDA
pD.pE
POLYHEADS.
IS
T’ZTRO
43
sheets (l(a)), pE sheets (l(b)), polyheads of type -4 (2(a)), type B (2(b)) and type C (2(c)), peanut-shaped particles (P(c)). Bar length represents 100 nm. Kote that the dimensions of polyheads and spherical particles cannot be directly compared, since the former ones are oriented completely flattened on the grid, while t,he latter ones are not, much collapsed.
RI. WURTZ
44
ET
.4L.
TABLE 2 Main products obtained after self-assembly of petit h protein in dependence on pH and molarity of the ammonium carbonate buffer used
6
Petit petit A, sheets
Petit petit A, amorphous aggregates, spirals
Roundish particle of various size, sheets, spirals
8
Petit petit A, spirals, sheets
Pctit A, spirals, sheets, polyheads
Spherical particles various size
Spherical particles of various size, including petit A, sheets
Yolyheads and roundish particles often in state of degradation Free capsomers?
Roundish particles, often in state of degradation Free capsomers?
10
of
The radii of self-assembled petit h particles are similar to those of preheads produced in viwo, i.e. at 2413 nm. The radii of most of the petit petit h are at 16&2 nm (for instance, Fig. 2(b)). The smallest ones observed (smaller ones in Fig. l(e)) at IO&l nm. Theoretical values for T = 7, T = 4, T = 3 and 7’ = 1 petit A-like particles are 23.7 nm (standard), 17.9 nm, 1.55 nm and 9 nm. The radius of phage heads, for comparison, is 28.6 run.
buffer employed (Table 2). Optimal conditions for polyhead formation so far were found at ionic strength I = 0.1 and pH 8, where up to a few per cent of the input protein assemblies are polyheads. These polyheads are the ones used for the most part in this study. (b) Polyheads produced in vivo Lambda polyheads have been reported to be produced in cells infected by certain phage head mutants (Kemp et al.? 1968; Kemp, 1971; Murialdo & Siminovitch, 1972). If properly enriched (see Materials and Methods), some polyheads can be detected in wild-type infected lysates too, as well as in lysates of all types of h head amber mutants, except those in gene E. Polyheads produced in vivo and polyheads produced in, vitro resemble each other in all details. (c) Three types of polyheads Three structurally different types of polyheads (type A, type B and type C) can be distinguished. They are depicted in Figures 2, 3 and 4 and their properties are listed in Table 3. Protein mixtures derived from petit h yield mainly type A polyheads (Fig. 2(a)), which are narrow tubes with a rough surface appearance built from one type of cluster. Since Nu3 defective petit h, which consists of pE only (Table I), is sufficient to provide the building blocks, it can be concluded that pE is the only protein needed to form these polyheads. In viva, type A polyheads so far have been found in Ddefective lysates only. Type B polyheads (Fig. 2(b)) are obtained in minor amounts (1 to 59/o) in the same preparations that yielded type A. They are wider and they appear smoother; clustering is not so easily visible. Their proportion in polyhead mixtures increases after prolonged storage or after treatment with 4 M-Urea (Hohn et al., manuscript in preparation) or with EDTA (Kemp, 1971).
LAMBDA
POLYHEADS,
IN
VITRO
45
and filtering of negatively stained polyheads. A, B, C: type A, type B. FIG. 3. Optical diffraction type C polyheads, respectively. (1) Electron microgrephs of samples stained as described in the legend to Fig. 2. Bar length represents 40 nm. (2) Optical diffraction patterns of (1). (3) Optical filtered images. Bar length represents 20 nm.
Jr. KURT2
ET
.4 I,
E’IG. 4. Polyheads of’ type A, B anti C as swt~ in t,he electron microscope after freeze drying untl shadowing with tungsten. One of the polyheads (AB) is half t,ype A and half t,ypo R. Bar length represents 100 nm.
F
r\’
F
s
F
A
l-3
B
c
C 22
20
10
21
27
24
No. of particles measured
3
I 1.7
56.O+
54.7&
57.3_
1.1
1.4
1.7
53.8+-‘7.”
45.5
41.8,2
Width (nm) of flattened particle (UJ)
4
94
7.8
10.4
(4
Thickness (nm) of particle
5
13+~0.2
lY.Of
12.9&0.2
13.0&0.4
0.2
10.7~1-0.2
10.8*0.14
(nm) (1)
Lattice constant,
7
59.3 & 1.4 60.5&3.3 59.0+ 1.4 60.0 i 1.3 58.5+ 1.3 59.7i2.4 59.5* 1 59.4+ 2 58.3= 1.8 58.73~ 1.3 58.2+0.6 58.0+0.6
-Wle (Y) (“) between lattice vect~ors
h polyheads
Xeasurements 6
of bacteriophage
3
10
10
10
10
10
10
(n)
8 No. of longitudinal rows around circumference the
Calculations 10
17.8
17.4
18.2
17.1
14.5
13.3
18.3
18.2
18.1
18.2
16.0
15.1
Itadius of tube (nm) calculated from columns 4, 6 and 8
9
(For
the symbols
see tho column
headings).
Other
(column
explanations
r = t
are given
9) or r =
in the text.
n .I . cos a 2rr (column
lo),
ci -
30”
Particle thickness (column 5) as moasurrd from the length of the shadow according to the evaporation angle and the orientation of the particle. Lattice crxlstant (column 6) : values mediated from the three dire&ions of t,he lattice are given. (column 7) Two of the angles, namely the ones between the lattice vectors and the long axis of the particle, are given. The radius of the polyheads was e&her calculated according to
N
(‘9
Negative staining (N)/freeze dryingshadowing
2
A
Sample
1
Propertkr
TABLE
48
11. WUH’I’Z
F2’ I
.1I 2.
Type C polyheads (Fig. 2(c)) are obtained whenever pD is present in addition to pE (Table 1) and, if the amount of pD is sufficient, they are the only species observed. They are as wide as type B and t’heir rough surface appearance is made up of two types of clusters. In viuo bhey are found in wild-type lysates and in all types of headdefective lysates, except t’hose mutant in genes D or E.
(d) Fine structure of polyheads A survey to determine structural details of the three types of polyheads involved observation of at least 20 well-preserved species of each kind. One series was studied by negative staining and another one by freeze drying and shadowing. After optical diffraction, a minimum of five selected micrographs was used for image reconstruction. Structural details are already discernible on some of the untreated electron micrographs (Fig. 2). Images, however. can be improved by optical filtering (Figs 3 and 5). Usually the polyheads were found to rest as flat double ribbons on the grid (Fig. 4). Assuming that the polyheads in solution are cylindrical, their mean radii (Table 3, column 9) can be calculated from the width (Table 3, column 4) of the ribbon. Wall thickness can be determined from the freeze dried specimens. Type A polyheads have the thickest walls and type B polyheads the thinnest ones. Optical diffraction patterns of all three types of polyheads indicate hexagonal lattices (Fig. 3 A(2), B(2) and C(2)). Lattice constants are 10.8 nm for type A and 13.0 nm for type B and C, and pitch angles are 30” & 2” in each case. In the diffraction patterns of type B polyheads the intensity of low-order spots is very low (Fig. 3 B(2)), indicating a relatively fine structure on t’he polyhead surface. Negatively stained micrographs of type A polyheads show rings with approximately 65 nm diameter between the centres of mass concentration (Fig. 3A(l)). On the other hand, relatively compact, protuberances. which seem to be connected by bridges, are revealed in shadowed preparations (Fig. 5A( 1)). The bridges are of little mass thickness and lie deeply within the surface structure, since they can hardly be seen in negative staining and are only weakly shadowed. In type B polyheads, ringlike structures can be discerned also. However, they have a smaller diameter (4.5 nm) and can be seen both in negatively stained and freeze dried preparations (Figs 3B( 1) and 5B( 1)). The surface is rather smooth and the ring structures protrude only slightly from the surrounding matter. Type C polyheads clearly show two types of clusters, confirming the results of Williams & Richards (1974). Rings of approximately the same size as in type B polyheads are surrounded by six clusters of a different type, each lying symmetrically between three of the ringlike clusters. They appear very bright in negative stain (Fig. 3C(l)) and are strongly shadowed in freeze dried preparations (Fig. 5C(l)). The arrangement of the clusters on the polyheads can be described in terms of a helix. Ten of the clusters in type A and type B polyheads and ten of the ringlike clusters in t*ype C polyheads are needed for one t’urn of the helix with a pitch angle of 30”. Helices of this type show longitudinal rows. which indeed are easily discernible on all types of X polyheads. The lattice constant and the number of clusters per turn allow another way of calculation of the radius of the tube (Table 3, column 10). The values obtained are in agreement with the ones described above, which were obtained from ribbon width (Table 3, column 9).
LAMBDA
POLYHEADS,
IAX VI!l’lZO
FL h in infected cells (Hohn ef al.. 1975,1976). (1) Similar clusters. i.e. hexagonally packed rings are seen on “squashed” preheads and on type A polyheads (compare Figs 6(a) and (b) and 2(a)). The same is true for squashed heads and type C polyheads (compare Figs 6(c) and (d) and 2(c)). (2) Type A polyheads are 20 9 0 narrower than types B and C. A similar size difference is observed between preheads on one side: and enlarged preheads and heads on the other side (Hohn et al.. 1947.1976) (Figs 2, 3, 4: 5 and 6). (3) Treatment with urea (Hohn et al.. 1976 and manuscript in preparation) transforms both the prehead and the type A polyhead int,o their enlarged forms, i.e. enlarged prehead and type B polyhead. The enlarged forms can be transformed to heads and type C polyheads. respectively. by adding protein PD. (4) Assuming similar lattice constants for polyheads and their icosahedral analogues, one can calculate the radius of the approximated spheres according to the relation
+ 4rr P = 20 TT12. where r is the radius, T is the triangulation ~nmber (= 7) and 1 is the lattice constant. Radii thus obtained are 23.7 nm for the prehead and 28.6 nm for enlarged prehead and head. These values are in excellent agreement with those obt,ained by light scattering (P. Kiinzler, this Institute, unpublished measurements), by low-angle X-ray scattering (W. Earnshaw, personal communication) and by negative staining (Eiserling et al.. 1970 and own measurements). There is however one interesting deviation: the circumference of the polyheads corresponds to the circumference of an icosahedron of the triangulation number (Caspar & Klug, 1962) T =- 3. Jn contrast’ the T numbers of the h prehead and head are T = 7 (Williams & Richards, 1974; Hohn ef al., 1974). Thus the polyheads are not simply elongated versions of their icosahedral analogues. After either negative sta,ining or freeze drying. the polyheads are completely flattened and appear as double ribbons. In the first case, t’he flattening can be explained by surface tension (Anderson. 1962) cxcrtcd cvhen sucking off t’lre fluid. In the second case, it is probably an effect’ of t~hrrnmal oscillat,ion of the polyhcad walls, which start’s when the ice has sublimed and t hta particle lies free on the grid. As a consequence, the tubular form collapses and rests as a ribbon because of the stickiness of proteins in vacuum (thermal collapse) (Anderson, 1954). All three types of X polyheads (t’ypes A. B and C) show a helical arrangement in a hexagonal lattice. which is defined by the lat,tice constant, (I) and thri pit’ch angle (a). In addition an orientation angle (6) of tlrcl capsorner in respect to the lat’ticc, line
FIG. 6. Subunit and (d)). Clusters Xopntire staining
clustering correspond as in Fig.
on squashed petit h ((a) ant1 (b)) and on squashed to those in polyheads type A4 and t,ypr C. Compare ‘7. Bar length reprcsrnt,s 100 urn.
phage heads ((c) with Fig. 2 or 3.
52
RI.
~VUlZTZ
El’
_-lL .
has to be taken into account (Pig. 7). In all three cases the pitch angle was det,ermined to be 30”. At this angle the two sets of diffraction spots are superimposed. which indicates some unknown synthesis of both the motifs of the upper and lower polyhead wall. As a consequence, the orientation angle of the capsomers cannot be determined from negatively stained specimens. The superposition of the diffraction spots does not necessarily mean a superposition of the lattice points (i.e. the capsomers) of the upper and lower wall. Figure S(a),(b) shows two extreme models of orientation of the polyhead walls. In case (a) t,he capsomers of the upper and lower wall coincide. In this case, along the polyhead four rows of capsomer pairs are visible, flanked by two slender rows of single capSomers, either broken or bended. These flanking capsomers alternate in position right or left. This is the appearance of nearly all of the negatively stained type A polyheads (Fig. 2(a)) and some of the freeze dried ones. In case (b) the capsomers of the upper and lower wall are displaced laterally and each capsomer of the upper wall is located between t,wo of the lower wa,ll. In this case five unflanked rows are visible and for each capsomer on the left side there is a corresponding one on the right side. This is the appearance of most of the freeze dried type A polyheads (see the type A polyhead part of Fig. 4 AB). Figure 8(c) shows a model for coincidence for a composite lattice as it corresponds to the appearance of most of the type C polyheads. In bacteriophage T4 polyheads the pitch angle differs from 30” and accordingly in diffraction diagrams of negatively stained preparations two sets of spots are observed corresponding to the upper and the lower wall. In this case the images of the upper and the lower wall can easily be separated by optical filtration and the orientation angle determined (De Rosier & Klug, 1972; Yanagida et al., 1972; Steven et al., manuscript in preparation). In order to overcome the difficulties resulting from the superimposed diffraction spots in h polyheads one can follow three approaches: (i) selection of one-sided stained preparations, (ii) screening for rare polyheads with a pitch angle different from 0” and 30”, and (iii) applying freeze drying-shadowing techniques. where only the upper layer will contribute information to the image. So far, we have taken the third approach; micrographs of shadowed preparations (Fig. 5) provide a three-dimensional aspect’ of the polyhead surface and render interpretations of the structural transitions much easier. Nevertheless, thermal collapse effects during freeze drying and the limited resolution of shadowing material do not allow the protomers (i.e. monomeric subunits) to kcome visible, which would be vital for determining the orientation angle (Fig. 7). Thus on one hand the technical procedure still has to be improved. On the other hand, imaging of the inner polyhead surface could probably provide the necessary information. Freeze dried preparations of structurally related sheets, like the one shown in Figure 1 would help to overcome this problem and give further insight into the surface relief at the same time. In spite of these technical difficulties and with the caution necessary in interpreting negatively stained electron micrographs, but in relation to the biological data (Hohn et al., 1975,1976), one arrives at a model for the polyheads and the morphological transitions between them (Figs 7(b) and 9). In type A polyheads pE clusters emerge from the surface and seem to be connected by bridges at the base. Comparison of shadowed and negatively stained pictures indicate that these clusters are hollow on the inner side. Enlarged polyheads (type B) still consist of pE only. They appear much smoother and might be derived from type B by flattening the
LAMBI).%
POLYHEADS,
IN
VITRO
(b)
Pro. 7. Hypothetical lattices of preheads and type A polyheads (top line), expanded preheads and type B polyheads (middle line), and heads and type C polyheads (lower line). The white areas on the subunits represent the protruding parts of the molecule, whereas the thin lines represent, the molecule contours and thus the intermolecular contacts, Pitch angle of polyheads (a), subunit orientation angle (p), lattice vector angle (y) and lattice constant (1) are drawn in the left upper lattice for illustration of these parameters. The orientation angle (j3) is arbitrarily chosen, the pitch angle, lat,tioe angle and relative lattice constants correspond to the measured values.
FIG. 8. (a), (b) Ten TOWVIof plastic caps arranged on a flattened tube folded from a hexagonal net, with a pitch angl? of 30”. The flattening of the tube i* done in two ways. (a) One row of subunits on each side of the ribbon is tilted 90’ with respect. t,o those in the plane. In t.hiy case the caps on the upper aud lower ribbon coincide and the caps on the left. and right flanking rows alternate. A similar situation (nut shown) would OPCIII’ if the flanking POWPof caps were cut in half with uric half remaining with the upper and the other with the lower ribbon. (b) The tube is folded between the rows. In this case caps of the upper and the lower rihbon do not coincitle, and for each cap of the left rim thwr is a corrwponding one for the right rim. The two models correspond to the appearance of type A polyheads on the electron microscope grid. (c) A model as in (a) but built on a composite lattice with caps and triangles as used in Fig. 7(b). This model corresponds to the appca~~~~rcc of tFpt% (’ polyheads on the electron microscope grid.
LAMBDA
POLYHEADS,
I
/
IN
VITRO
55
pE monomer I
TYPE
FIG. 9. Hypothetical sections through type A, type B and type C polyheads along the lattice line (i.e. at 60” to the long axis of the polyhead lattices, as in Fig. 7, right). Half-hexamers of pE are shown that flatten during transition of type A to type B polyheads. This makes binding sites for pD trimers accessible. Shaded parts are located back in the plane. The drawing does not account for the structural shifts of the capsid subunit which is likely to cause the reorientation. capsomers and pushing them apart. That type A and type B polyheads are related is also shown by the existence of polyheads that are partially type A and partially type B (Fig. 4 AB). Type C polyheads are derived from type B by adding protein pD trimers around the centre of the pE hexamers. The pD clusters emerge far from the polyhead surface and are thus nearly covering the pE central rings from shadowing. Negatively stained pictures show these surrounding structures to be very bright, indicating a high mass concentration due Do the addition of the masses of the pD molecules to the masses of the pE bases. This interpretation stands in contradiction to the alternative model (Williams & Richards, 1974; Hohn et al., 1974). according to which, trimers of pE protein are surrounded by hexamers of D protein (Fig. 7(a)). From the structural point of view this model cannot be excluded. It is unlikely, however, since the transition of type B to type C lattices for this case would involve considerable rearrangements of the main capsid protein. Further biochemical studies by chemical crosslinking of protein subunits, and electron microscopical ones by imaging the inner polyhead side (or by imaging one-layer “sheets”. as shown in Figure 1) will help to clarify this question. The expert technical assistance of Hcndrikje Flick is gratefully acknowledged. We thank Elisabeth Leutlrardt for typing that manuscript and Regina Oetterli for the photographic work. We thank Barbara Hohn and Edward Kellenherger for many discussions throughout tlie work and A. Steven for critical reading of the manuscript. This work was funded hg a grant (30650.73) from the Swiss National Foundation for scient,ific research.
56
M. WURTZ
El’
AL.
REFERENCES Aebi,
U., Smith, P. R., Dubochet, J., H enry, C. & Kellenberger, E. (1973). .I. Supramol. Struct. 1, 498-522. Anderson, T. 11’. (1952). Amer. Naturalist, 86, 91-100. Anderson, T. ZI’. (1954). Trans. N. Y. Acad. Sci. 16, 242-249. Beer, RI., Frank, J., Hanszen, K. F., Kellrnberger. E. & Williams, R. (‘. (1975). Quart. Rev. Biophys. 7, 211-238. Caspar, D. L. II. & Klug, A. (1962). Cold Spring Harbor Symp. f&ant. Bio/. 27, 1 24. De Rosier, D. J. & Klug, A. (1972). J. Mol. BioZ. 65, 469--488. Dubochet, J. (1975). ,J. Cltrastruct. Res. 52, 276288. Eiserling, F. A., Geiduchek, E. P., Epstein, R. H. & Metter, E. -7. (1970). J. I’irol. 6, 865-876. Fujisawa, H. & Matsuo, H. (1973). Virology, 54, 313-317. Hendrix, R. & Casjens, S. (1975). J. Mol. BioZ. 91, 187.-199. Hohn, B., Wurtz, M.. Klein, B., Lustig, A. & Hohn, T. (1974). J. Supramol. Struct. 2, 302-317. Hohn, T., Flick, H. & Hohn, B. (1975). J. Mol. Biol. 98, 1077120. Hohn, T., Wurtz, M. & Hohn, B. (1976). Phil. Trans. Roy. Sot. London, in the press. Horne, R. W. & Pasquali-Ronchetti, I. P. (1974). J. Ultrastruct. Res. 47, 361-383. Kellenberger, E. (1972). In Polymerisation in Biological Systems. Ciba Symp. no. 7, pp. 189-203, Assoc. Scientific Publ., Amsterdam. Kemp, C. L. (1971). 1.irology, 44, 569-575. Kemp, C. L., Howatson, A. F. & Siminovitch, L. (1968). Virology, 36, 490-502. Leonard, K. R., Kleinschmidt, A. K. (1972). J. Mol. BioZ. 71, 201-216. Mazza, A. & Felluga, B. (1973). .J. Ultrastruct. Res. 45, 259-278. Monod, J., Wyman, J. & Changeux, J. P. (1965). J. Mol. BioZ. 12, 88-118. Murieldo, H. & Siminovitch, L. (1972). Virology, 48, 824-835. Nermut, M. 1’. & Frank, H. (1971). J. Gen. V&+oZ. 10, 37-51. Showe, M. & Kellenberger, E. (1975). In Control processes in virus muZtipZplication (Burka & Russel, eds), pp. 407-438, Cambridge University Press, Cambridge. Singer, S. J. (1974). Anna Rev. Biochem. 43, 805833. Wakabayashi, T., Huxley, H. E., Amos, L. A. & Klug, A. (1975). J. Mol. BioZ. 93,477 497. Williams, R. C. & Richards, K. E. (1974). J. Mol. BioZ. 88, 5477550. Wood, W. B. (1974). J. Suprarnol. Struct. 2, 512-514. Yanagida, M., De Rosier, D. .J. & Klug, A. (1972). J. Mol. BioZ. 65, 4599499. Note added in proof: While this contribution was in press, a study by Howatson and Kemp (1975), Virology 67, 80-84, appeared, describing phage /\ polyheads obtained in vieo. Type I and Type II tubules in that publication correspond to our type A and type C polyheads, respectively. Note that the interpretations of type C (type II) polyheads are different in the two publications.
101, 39-66
Surface Structure of in vitro Assembled Bacteriophage Lambda Polyheads M.
WURTZ.J.
KISTLER
AND T. HOHX
LJepartment of Microbiology, Biozentrum. 3056 Basel, Switzerhd
Klingelbergstr,
70
(Received 4 September 1975) Tubular aberrant phage h capsoids (polyheads) can be obtained by self-assembly of capsid protein. Three types of polyheads can be distinguished which correspond in their surface structure to petit A, enlarged petit h (D--head) and phage head. The latter type of polyhead consists of two different proteins (pE and pD) arranged in a composite hexagonal lattice, the other two of one type of protein (pE) arranged in hexagonal lattices. Each type has its own lattice constants. Polyheads were observed both after negative staining and freeze drying-shadowing and data were exploited by optical filtration methods. Models are proposed to elucidate the surface fine structure of polyheads and their spherical analogues, and the conformational changes occurring during maturation of the phage X head.
1. Introduction important biological processes such as regulation of enzyme activities (Monod et al., 1965), muscle contraction (Wakabayashi et al., 1975), membrane permeability (Singer, 1974) and assembly of complex subcellular structures (Wood, 1974; Kellenberger, 1972) are believed to be controlled by conformational changes of proteins. These shifts will be more easily detected if large arrays of regularly arranged identical subunits are involved. Thus, in morphopoietic transitions of viruses (Hohn et aZ., 1974,1976; Showe & Kellenberger, 1975), due to a “domino effect”, the shift of one subunit is transferred to all the other identical subunits and thus becomes eligible for electron microscopical observation. Still, the detailed interpretation of virus morphopoietic transitions by electron microscopy can be difficult because of: (I) superposition of the upper and lower surface of the particles; (2) curvature of the particle surfaces; (3) the different orientation of the individual particles on the grid and (4) the artifacts and distortions caused hy preparation techniques and the damage caused by the electron beam (Dubochet. 1975; Beer et al., 1975). In order to overcome these difficulties, techniques were developed to produce paracrystalline arrays of viruses (Horne & Pasquali-Ronchetti, 1974). Virus surfaces were studied on squashed particles (Mazza & Felluga, 1973 ; Williams & Richards, 1974; Hohn et al., 1974), where areas of subunits are oriented flat on the microscope grid, or elongated tubular viruses or aberrant tubular capsids (polyheads) were examined, where the multiple repetition of the basic structural elements along the long axis of the particle allows the application of optical filtration methods (De Rosier & Klug, 1972; Yanagida et al., 1972; Leonard & Kleinschmidt. 1972). Many
31.
40
\V1:HTZ
E?’ ..l I,
The bacteriophage X head is supposed to undergo several transitions during morphogenesis and DNA packaging (Hohn et al., 1974,1975,1976; Hendrix & Casjens, 1975). A prehead empty of DNA is formed first. It packages the DNA, swells thereby and adds the small capsid protein (pD) in amounts equimolar to the large capsid protein (pE). Accordingly, three types of head structures can be isolated from infected lysates: preheads, enlarged preheads (D--heads), both consisting of pE, and heads consisting of pE and pD. Conditions were sought and found to produce in vitro three types of h polyheads that are analogues in their protein composition and surface structure to these spherical particles. The lambda polyheads show a helical arrangement of surface clusters with a pitch angle of 30”. At this pitch angle the upper and the lower surface image cannot be separated by optical filtration of negatively stained specimens. To obtain one-sided images, a freeze drying-shadowing method was adapted from Nermut & Frank (1971).
2. Materials and Methods (a) Petit
h particles
and phage heads
Petit h particles and empty phage heads were isolated Their protein composition is listed in Table 1. (b) Dissociation
as described
by Hohn
et al. (1975).
and rewsembly
In a typical experiment, 1 ml petit h or emptied h heads (40 mg/ml in TMA buffer (Hohn et al., 1975)) was mixed with 1.4 g guanidinium*HCl and 15 ~1 dithiothreitol (0.1 M) and was kept for 1 h at 37°C. Then 9 ml 0.1 M-ammonium carbonate buffer (pH 8) (or another buffer, if indicated) were added and the solution dialysed overnight at room temperature against 100 ml of the same buffer. Precipitates that formed were removed by centrifugation and the solution was concentrated IO-fold by pressure dialysis through an Amicon PM10 filter. The concentrate contained polyheads and other assemblies. (c) Procedure
for polyhead
enrichment
from
crude lysates
Crude solutions of polyheads were a side product of the isolation of petit X particles. After centrifugation through a sucrose gradient in the zonal rotor, where petit h move to containing polythe middle of the gradient (see Hohn et al., 1975), the cushion fraction, heads and other phage-related materials and debris was used. It was diluted to 1 1 with TMA buffer and centrifuged for 5 min at 5000 revs/min in the GS3 rotor of the Sorvall centrifuge. The supernatant was centrifuged again for 10 min at 8000 revs/min and the pellet was resuspended in 100 ml TMA buffer. After two further cycles of centrifugation at 5000 and 8000 revs/min, the pellet was resuspended in 5 ml TMA buffer and used for electron microscopy of polyheads. (d) Preparation (i) Negative
for electron
microscopy
staining
Negative staining was either performed with 2% uranyl acetate (pH 4.2), or with sodium phosphotungstate (pH 7.2). All electron micrographs presented are stained the latter method. (ii)
Freeze drying
2% by
and shadowing
Carbon-coated collodion grids were glow discharged and then placed on a drop of particle suspension for adsorption, The grids were washed with several drops of distilled water and put upsidedown on a wet filter paper; thus a very thin layer of water is formed. After a few seconds the grids were removed and immediately frozen in liquid nitrogen. Freeze drying was done with a Balzers apparatus BA-511 M equipped with an electron gun evaporation source and a quartz film-thickness monitor. Sublimation was at -30°C
LAMBDA
POLYHEADS,
IN
41
I’ITRO
within a vacuum of better than 5 x low7 Torr. The dried particles were shadowed with tungsten at an angle of 30’ with a mass thickness of about 5 d. For stabilization, carbon was evaporated at an angle of 90” onto the specimen. The vacuum during evaporation was about 4 x 1O-6 Torr. (e) Electron
microscopy
and image processiny
The specimens were observed in a Philips 300 electron microscope. Optical diffraction and filtration work were done according to the prescriptions of Aebi et al. (1973). The shape of the window was chosen so that its diffraction does not, interfere with the diffraction pattern of the polyheads.
3. Results (a) In vitro aggregation of h capsid protein Petit X particles and empty X heads isolated from certain mutants of phage and host and dissolved in guanidinium *HCI constitute different types of “instant” protein
mixtures
for self-assembly
experiments
(Table
1). Thus for instance
Nu3
defective petit h protein consists of main capsid protein pE ; or head protein consists of small capsid protein pD and a set of minor proteins in addition to pE. After restoring favourable ionic conditions to the protein mixtures by dilution with the assembly buffer and concentration by pressure dialysis, the protein aggregates. About 30 to 50% of the input pE precipitates and is removed. The soluble fraction contains various types of assemblies, i.e. roundish particles of the size of petit X, smaller round&h particles (petit, petit X), a few larger roundish particles, various kinds of abnormally formed particles (monsters, spirals, sheets, long tubular structures (polyheads)) and amorphous aggregates of peanut shape (Fig. 1). All types of protein mixtures shown in Table 1 yielded these general types of assemblies in similar relative amounts, except for pNu3-containing mixtures, where the proportion of normal-sized petit h particles seemed to be higher, and for the pD-containing mixtures, where fine structures of the assembly products were different. The proportions of particles did vary, however, with ionic strength and pH of the assembly TABLE 1 Protein
composition of petit h and empty heads used as a source of protein for in vitro self-assembly experiments
Particle as source of proteins
Protein PLNU3 (16H
composition PI) (1W
Minor
proteins
Polyheads obtained
*Vu3-
petit
X
None
A> (B)f
groE-
petit
h
Unprocessed
A> (B)
Processed
:I, (B)
Processad
(1
I)
p&t
Empty
-I-
A (prehead) head
-
/-
t Molecular weight x 10 -3. 1 Minor fraction in mixture. Data from Hohn et al. (1976). The last column shows the types specific mixtures. More rarely occurring type in parentheses.
of polyheads
obtained
with
the
Figs 1 and 2. Particles obtained after self-assembly of phage h capsid protein. Electron micrographs from negatively stained preparations with 2% sodium phosphotungstate (pH 7.2). Particles are named spirals (e.g. in l(c)), petit petit X (in l(e) and 2(b)), petit A (e.g. in 2(a)),
LAMBDA
pD.pE
POLYHEADS.
IS
T’ZTRO
43
sheets (l(a)), pE sheets (l(b)), polyheads of type -4 (2(a)), type B (2(b)) and type C (2(c)), peanut-shaped particles (P(c)). Bar length represents 100 nm. Kote that the dimensions of polyheads and spherical particles cannot be directly compared, since the former ones are oriented completely flattened on the grid, while t,he latter ones are not, much collapsed.
RI. WURTZ
44
ET
.4L.
TABLE 2 Main products obtained after self-assembly of petit h protein in dependence on pH and molarity of the ammonium carbonate buffer used
6
Petit petit A, sheets
Petit petit A, amorphous aggregates, spirals
Roundish particle of various size, sheets, spirals
8
Petit petit A, spirals, sheets
Pctit A, spirals, sheets, polyheads
Spherical particles various size
Spherical particles of various size, including petit A, sheets
Yolyheads and roundish particles often in state of degradation Free capsomers?
Roundish particles, often in state of degradation Free capsomers?
10
of
The radii of self-assembled petit h particles are similar to those of preheads produced in viwo, i.e. at 2413 nm. The radii of most of the petit petit h are at 16&2 nm (for instance, Fig. 2(b)). The smallest ones observed (smaller ones in Fig. l(e)) at IO&l nm. Theoretical values for T = 7, T = 4, T = 3 and 7’ = 1 petit A-like particles are 23.7 nm (standard), 17.9 nm, 1.55 nm and 9 nm. The radius of phage heads, for comparison, is 28.6 run.
buffer employed (Table 2). Optimal conditions for polyhead formation so far were found at ionic strength I = 0.1 and pH 8, where up to a few per cent of the input protein assemblies are polyheads. These polyheads are the ones used for the most part in this study. (b) Polyheads produced in vivo Lambda polyheads have been reported to be produced in cells infected by certain phage head mutants (Kemp et al.? 1968; Kemp, 1971; Murialdo & Siminovitch, 1972). If properly enriched (see Materials and Methods), some polyheads can be detected in wild-type infected lysates too, as well as in lysates of all types of h head amber mutants, except those in gene E. Polyheads produced in vivo and polyheads produced in, vitro resemble each other in all details. (c) Three types of polyheads Three structurally different types of polyheads (type A, type B and type C) can be distinguished. They are depicted in Figures 2, 3 and 4 and their properties are listed in Table 3. Protein mixtures derived from petit h yield mainly type A polyheads (Fig. 2(a)), which are narrow tubes with a rough surface appearance built from one type of cluster. Since Nu3 defective petit h, which consists of pE only (Table I), is sufficient to provide the building blocks, it can be concluded that pE is the only protein needed to form these polyheads. In viva, type A polyheads so far have been found in Ddefective lysates only. Type B polyheads (Fig. 2(b)) are obtained in minor amounts (1 to 59/o) in the same preparations that yielded type A. They are wider and they appear smoother; clustering is not so easily visible. Their proportion in polyhead mixtures increases after prolonged storage or after treatment with 4 M-Urea (Hohn et al., manuscript in preparation) or with EDTA (Kemp, 1971).
LAMBDA
POLYHEADS,
IN
VITRO
45
and filtering of negatively stained polyheads. A, B, C: type A, type B. FIG. 3. Optical diffraction type C polyheads, respectively. (1) Electron microgrephs of samples stained as described in the legend to Fig. 2. Bar length represents 40 nm. (2) Optical diffraction patterns of (1). (3) Optical filtered images. Bar length represents 20 nm.
Jr. KURT2
ET
.4 I,
E’IG. 4. Polyheads of’ type A, B anti C as swt~ in t,he electron microscope after freeze drying untl shadowing with tungsten. One of the polyheads (AB) is half t,ype A and half t,ypo R. Bar length represents 100 nm.
F
r\’
F
s
F
A
l-3
B
c
C 22
20
10
21
27
24
No. of particles measured
3
I 1.7
56.O+
54.7&
57.3_
1.1
1.4
1.7
53.8+-‘7.”
45.5
41.8,2
Width (nm) of flattened particle (UJ)
4
94
7.8
10.4
(4
Thickness (nm) of particle
5
13+~0.2
lY.Of
12.9&0.2
13.0&0.4
0.2
10.7~1-0.2
10.8*0.14
(nm) (1)
Lattice constant,
7
59.3 & 1.4 60.5&3.3 59.0+ 1.4 60.0 i 1.3 58.5+ 1.3 59.7i2.4 59.5* 1 59.4+ 2 58.3= 1.8 58.73~ 1.3 58.2+0.6 58.0+0.6
-Wle (Y) (“) between lattice vect~ors
h polyheads
Xeasurements 6
of bacteriophage
3
10
10
10
10
10
10
(n)
8 No. of longitudinal rows around circumference the
Calculations 10
17.8
17.4
18.2
17.1
14.5
13.3
18.3
18.2
18.1
18.2
16.0
15.1
Itadius of tube (nm) calculated from columns 4, 6 and 8
9
(For
the symbols
see tho column
headings).
Other
(column
explanations
r = t
are given
9) or r =
in the text.
n .I . cos a 2rr (column
lo),
ci -
30”
Particle thickness (column 5) as moasurrd from the length of the shadow according to the evaporation angle and the orientation of the particle. Lattice crxlstant (column 6) : values mediated from the three dire&ions of t,he lattice are given. (column 7) Two of the angles, namely the ones between the lattice vectors and the long axis of the particle, are given. The radius of the polyheads was e&her calculated according to
N
(‘9
Negative staining (N)/freeze dryingshadowing
2
A
Sample
1
Propertkr
TABLE
48
11. WUH’I’Z
F2’ I
.1I 2.
Type C polyheads (Fig. 2(c)) are obtained whenever pD is present in addition to pE (Table 1) and, if the amount of pD is sufficient, they are the only species observed. They are as wide as type B and t’heir rough surface appearance is made up of two types of clusters. In viuo bhey are found in wild-type lysates and in all types of headdefective lysates, except t’hose mutant in genes D or E.
(d) Fine structure of polyheads A survey to determine structural details of the three types of polyheads involved observation of at least 20 well-preserved species of each kind. One series was studied by negative staining and another one by freeze drying and shadowing. After optical diffraction, a minimum of five selected micrographs was used for image reconstruction. Structural details are already discernible on some of the untreated electron micrographs (Fig. 2). Images, however. can be improved by optical filtering (Figs 3 and 5). Usually the polyheads were found to rest as flat double ribbons on the grid (Fig. 4). Assuming that the polyheads in solution are cylindrical, their mean radii (Table 3, column 9) can be calculated from the width (Table 3, column 4) of the ribbon. Wall thickness can be determined from the freeze dried specimens. Type A polyheads have the thickest walls and type B polyheads the thinnest ones. Optical diffraction patterns of all three types of polyheads indicate hexagonal lattices (Fig. 3 A(2), B(2) and C(2)). Lattice constants are 10.8 nm for type A and 13.0 nm for type B and C, and pitch angles are 30” & 2” in each case. In the diffraction patterns of type B polyheads the intensity of low-order spots is very low (Fig. 3 B(2)), indicating a relatively fine structure on t’he polyhead surface. Negatively stained micrographs of type A polyheads show rings with approximately 65 nm diameter between the centres of mass concentration (Fig. 3A(l)). On the other hand, relatively compact, protuberances. which seem to be connected by bridges, are revealed in shadowed preparations (Fig. 5A( 1)). The bridges are of little mass thickness and lie deeply within the surface structure, since they can hardly be seen in negative staining and are only weakly shadowed. In type B polyheads, ringlike structures can be discerned also. However, they have a smaller diameter (4.5 nm) and can be seen both in negatively stained and freeze dried preparations (Figs 3B( 1) and 5B( 1)). The surface is rather smooth and the ring structures protrude only slightly from the surrounding matter. Type C polyheads clearly show two types of clusters, confirming the results of Williams & Richards (1974). Rings of approximately the same size as in type B polyheads are surrounded by six clusters of a different type, each lying symmetrically between three of the ringlike clusters. They appear very bright in negative stain (Fig. 3C(l)) and are strongly shadowed in freeze dried preparations (Fig. 5C(l)). The arrangement of the clusters on the polyheads can be described in terms of a helix. Ten of the clusters in type A and type B polyheads and ten of the ringlike clusters in t*ype C polyheads are needed for one t’urn of the helix with a pitch angle of 30”. Helices of this type show longitudinal rows. which indeed are easily discernible on all types of X polyheads. The lattice constant and the number of clusters per turn allow another way of calculation of the radius of the tube (Table 3, column 10). The values obtained are in agreement with the ones described above, which were obtained from ribbon width (Table 3, column 9).
LAMBDA
POLYHEADS,
IAX VI!l’lZO
FL h in infected cells (Hohn ef al.. 1975,1976). (1) Similar clusters. i.e. hexagonally packed rings are seen on “squashed” preheads and on type A polyheads (compare Figs 6(a) and (b) and 2(a)). The same is true for squashed heads and type C polyheads (compare Figs 6(c) and (d) and 2(c)). (2) Type A polyheads are 20 9 0 narrower than types B and C. A similar size difference is observed between preheads on one side: and enlarged preheads and heads on the other side (Hohn et al.. 1947.1976) (Figs 2, 3, 4: 5 and 6). (3) Treatment with urea (Hohn et al.. 1976 and manuscript in preparation) transforms both the prehead and the type A polyhead int,o their enlarged forms, i.e. enlarged prehead and type B polyhead. The enlarged forms can be transformed to heads and type C polyheads. respectively. by adding protein PD. (4) Assuming similar lattice constants for polyheads and their icosahedral analogues, one can calculate the radius of the approximated spheres according to the relation
+ 4rr P = 20 TT12. where r is the radius, T is the triangulation ~nmber (= 7) and 1 is the lattice constant. Radii thus obtained are 23.7 nm for the prehead and 28.6 nm for enlarged prehead and head. These values are in excellent agreement with those obt,ained by light scattering (P. Kiinzler, this Institute, unpublished measurements), by low-angle X-ray scattering (W. Earnshaw, personal communication) and by negative staining (Eiserling et al.. 1970 and own measurements). There is however one interesting deviation: the circumference of the polyheads corresponds to the circumference of an icosahedron of the triangulation number (Caspar & Klug, 1962) T =- 3. Jn contrast’ the T numbers of the h prehead and head are T = 7 (Williams & Richards, 1974; Hohn ef al., 1974). Thus the polyheads are not simply elongated versions of their icosahedral analogues. After either negative sta,ining or freeze drying. the polyheads are completely flattened and appear as double ribbons. In the first case, t’he flattening can be explained by surface tension (Anderson. 1962) cxcrtcd cvhen sucking off t’lre fluid. In the second case, it is probably an effect’ of t~hrrnmal oscillat,ion of the polyhcad walls, which start’s when the ice has sublimed and t hta particle lies free on the grid. As a consequence, the tubular form collapses and rests as a ribbon because of the stickiness of proteins in vacuum (thermal collapse) (Anderson, 1954). All three types of X polyheads (t’ypes A. B and C) show a helical arrangement in a hexagonal lattice. which is defined by the lat,tice constant, (I) and thri pit’ch angle (a). In addition an orientation angle (6) of tlrcl capsorner in respect to the lat’ticc, line
FIG. 6. Subunit and (d)). Clusters Xopntire staining
clustering correspond as in Fig.
on squashed petit h ((a) ant1 (b)) and on squashed to those in polyheads type A4 and t,ypr C. Compare ‘7. Bar length reprcsrnt,s 100 urn.
phage heads ((c) with Fig. 2 or 3.
52
RI.
~VUlZTZ
El’
_-lL .
has to be taken into account (Pig. 7). In all three cases the pitch angle was det,ermined to be 30”. At this angle the two sets of diffraction spots are superimposed. which indicates some unknown synthesis of both the motifs of the upper and lower polyhead wall. As a consequence, the orientation angle of the capsomers cannot be determined from negatively stained specimens. The superposition of the diffraction spots does not necessarily mean a superposition of the lattice points (i.e. the capsomers) of the upper and lower wall. Figure S(a),(b) shows two extreme models of orientation of the polyhead walls. In case (a) t,he capsomers of the upper and lower wall coincide. In this case, along the polyhead four rows of capsomer pairs are visible, flanked by two slender rows of single capSomers, either broken or bended. These flanking capsomers alternate in position right or left. This is the appearance of nearly all of the negatively stained type A polyheads (Fig. 2(a)) and some of the freeze dried ones. In case (b) the capsomers of the upper and lower wall are displaced laterally and each capsomer of the upper wall is located between t,wo of the lower wa,ll. In this case five unflanked rows are visible and for each capsomer on the left side there is a corresponding one on the right side. This is the appearance of most of the freeze dried type A polyheads (see the type A polyhead part of Fig. 4 AB). Figure 8(c) shows a model for coincidence for a composite lattice as it corresponds to the appearance of most of the type C polyheads. In bacteriophage T4 polyheads the pitch angle differs from 30” and accordingly in diffraction diagrams of negatively stained preparations two sets of spots are observed corresponding to the upper and the lower wall. In this case the images of the upper and the lower wall can easily be separated by optical filtration and the orientation angle determined (De Rosier & Klug, 1972; Yanagida et al., 1972; Steven et al., manuscript in preparation). In order to overcome the difficulties resulting from the superimposed diffraction spots in h polyheads one can follow three approaches: (i) selection of one-sided stained preparations, (ii) screening for rare polyheads with a pitch angle different from 0” and 30”, and (iii) applying freeze drying-shadowing techniques. where only the upper layer will contribute information to the image. So far, we have taken the third approach; micrographs of shadowed preparations (Fig. 5) provide a three-dimensional aspect’ of the polyhead surface and render interpretations of the structural transitions much easier. Nevertheless, thermal collapse effects during freeze drying and the limited resolution of shadowing material do not allow the protomers (i.e. monomeric subunits) to kcome visible, which would be vital for determining the orientation angle (Fig. 7). Thus on one hand the technical procedure still has to be improved. On the other hand, imaging of the inner polyhead surface could probably provide the necessary information. Freeze dried preparations of structurally related sheets, like the one shown in Figure 1 would help to overcome this problem and give further insight into the surface relief at the same time. In spite of these technical difficulties and with the caution necessary in interpreting negatively stained electron micrographs, but in relation to the biological data (Hohn et al., 1975,1976), one arrives at a model for the polyheads and the morphological transitions between them (Figs 7(b) and 9). In type A polyheads pE clusters emerge from the surface and seem to be connected by bridges at the base. Comparison of shadowed and negatively stained pictures indicate that these clusters are hollow on the inner side. Enlarged polyheads (type B) still consist of pE only. They appear much smoother and might be derived from type B by flattening the
LAMBI).%
POLYHEADS,
IN
VITRO
(b)
Pro. 7. Hypothetical lattices of preheads and type A polyheads (top line), expanded preheads and type B polyheads (middle line), and heads and type C polyheads (lower line). The white areas on the subunits represent the protruding parts of the molecule, whereas the thin lines represent, the molecule contours and thus the intermolecular contacts, Pitch angle of polyheads (a), subunit orientation angle (p), lattice vector angle (y) and lattice constant (1) are drawn in the left upper lattice for illustration of these parameters. The orientation angle (j3) is arbitrarily chosen, the pitch angle, lat,tioe angle and relative lattice constants correspond to the measured values.
FIG. 8. (a), (b) Ten TOWVIof plastic caps arranged on a flattened tube folded from a hexagonal net, with a pitch angl? of 30”. The flattening of the tube i* done in two ways. (a) One row of subunits on each side of the ribbon is tilted 90’ with respect. t,o those in the plane. In t.hiy case the caps on the upper aud lower ribbon coincide and the caps on the left. and right flanking rows alternate. A similar situation (nut shown) would OPCIII’ if the flanking POWPof caps were cut in half with uric half remaining with the upper and the other with the lower ribbon. (b) The tube is folded between the rows. In this case caps of the upper and the lower rihbon do not coincitle, and for each cap of the left rim thwr is a corrwponding one for the right rim. The two models correspond to the appearance of type A polyheads on the electron microscope grid. (c) A model as in (a) but built on a composite lattice with caps and triangles as used in Fig. 7(b). This model corresponds to the appca~~~~rcc of tFpt% (’ polyheads on the electron microscope grid.
LAMBDA
POLYHEADS,
I
/
IN
VITRO
55
pE monomer I
TYPE
FIG. 9. Hypothetical sections through type A, type B and type C polyheads along the lattice line (i.e. at 60” to the long axis of the polyhead lattices, as in Fig. 7, right). Half-hexamers of pE are shown that flatten during transition of type A to type B polyheads. This makes binding sites for pD trimers accessible. Shaded parts are located back in the plane. The drawing does not account for the structural shifts of the capsid subunit which is likely to cause the reorientation. capsomers and pushing them apart. That type A and type B polyheads are related is also shown by the existence of polyheads that are partially type A and partially type B (Fig. 4 AB). Type C polyheads are derived from type B by adding protein pD trimers around the centre of the pE hexamers. The pD clusters emerge far from the polyhead surface and are thus nearly covering the pE central rings from shadowing. Negatively stained pictures show these surrounding structures to be very bright, indicating a high mass concentration due Do the addition of the masses of the pD molecules to the masses of the pE bases. This interpretation stands in contradiction to the alternative model (Williams & Richards, 1974; Hohn et al., 1974). according to which, trimers of pE protein are surrounded by hexamers of D protein (Fig. 7(a)). From the structural point of view this model cannot be excluded. It is unlikely, however, since the transition of type B to type C lattices for this case would involve considerable rearrangements of the main capsid protein. Further biochemical studies by chemical crosslinking of protein subunits, and electron microscopical ones by imaging the inner polyhead side (or by imaging one-layer “sheets”. as shown in Figure 1) will help to clarify this question. The expert technical assistance of Hcndrikje Flick is gratefully acknowledged. We thank Elisabeth Leutlrardt for typing that manuscript and Regina Oetterli for the photographic work. We thank Barbara Hohn and Edward Kellenherger for many discussions throughout tlie work and A. Steven for critical reading of the manuscript. This work was funded hg a grant (30650.73) from the Swiss National Foundation for scient,ific research.
56
M. WURTZ
El’
AL.
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