Filamentous bacterial viruses

J. Mol. Bid. (1974) 88, 581-600

Filamentous Bacterial Viruses XII.? Molecular Architecture

of the Class I (fd, Ifl, IKe) Virion

D. A. MYARVIN, W. J. PIGRAM& R. L. WISEMANS, E. J. WACHTEL A.ND F. J. MARVIN

Department of Molecular Biophysics and Biochemistry Yale University New Haven, Corm. 06520, U.S.A. (Received 3 December 1973) The X-ray diffraction patterns of the fd, If1 and IKe strains o: filamentous bacterial viruses (class I) indicate that the arrangement of capsid prdteins in the virion approximates a left-handed helix of 15 A pitch with 4.5 units per turn. The protein molecules are each elongated in an axial direction, and also slope radially, so as to overlap each other and give an arrangement of molecules reminiscent of scales on a fish. This helix of capsid proteins is related to the class II helix by a small twist about the helix axis. The protein molecules are also perturbed (by a few Angstrom units) away from the positions that they would occupy in a simple 4.5 units per turn helix. The perturbation repeats about every five protein molecules, and is mainly axial. This arrangement of proteins forms a tube with inner diameter about 20 A and outer diameter about 60 8, encapsulating the DNA.

1. Introduction Filamentous bacterial viruses (Marvin & Hoffmann-Berling, 1963) are linear assemblies of protein subunits encapsulating single-stranded circular DNA. The bulk coat protein (B-protein) is about 5000 molecular weight (see Appendix), is largely K-helical, and is present in several thousand copies per virion. A minor capsid protein involved in adsorption (A-protein) is about 65,000 molecular weight and is present in about four copies per virion. A second minor protein may also be present in the virion (Beaudoin, 1970). Both penetration of infecting virions into the host and release of progeny virions from the host follow unusual and interesting pathways. The infecting virion attaches to the tip of a bacterial sex-pilus and is probably drawn into the bacterium by retraction of the pilus (Jacobson, 1972). The DNA and Aprotein of the infecting virion pass into the cytoplasm (Jazwinski et al., 1973), but the B-protein is deposited at the plasma membrane (Smilowitz, 1974). Viral DNA replicates in the host, producing single-stranded progeny DNA via a double-stranded intermediate. As progeny DNA is synthesized, it is coated with the product of viral gene 5, a protein with 87 amino acids in known sequence (Nakashima et al., 1974), to form a linear intracellular nucleoprotein complex (Pratt et al., 1974). One gene 5 t Paper XI in this series is Marvin et al., 1974. $ Present address: Department of Physics, University of Keele, Keele ST6 6BG, England. 5 Present address: Public Health Research Institute, New York, N.Y. 10016, U.S.A. 581

582

D. A. MARVIN

ET

AL.

protein molecule is associated with every four nucleotides in this complex (Day, 1973). Morphogenesis and extrusion of completed virions takes place at (Smiiowitz, 1974) or perhaps even within (Marvin & Hohn, 1969) the lipid bilayer that forms the plasma membrane, with two B-protein molecules displacing each gene 5 protein molecule as the virion passes out of the cell. Formation of progeny virions does not lyse or otherwise kill the host. Inhibition of viral morphogenesis, on the other hand, does kill the host by a process involving the gene 5 protein (Timmis & Marvin, 1974). (The earlier literature on filamentous bacterial viruses has been reviewed by Marvin & Hahn, 1969.) These viruses are interesting not only as systems for studying morphogenesis and transport of macromolecules across membranes, but also as models for the structure of more complicated linear assemblies of proteins. Both myosin filaments (Lowey, 1971) and neurofilaments (Day & Gilbert, 1972) are rich in a-helices, but the arrangements of cc-helices in these assemblies are not known because current diffraction patterns contain insufficient information for a detailed study. Filamentous virus diffraction patterns contain more information, and therefore can be analyzed in more detail. Indeed, a hypothetical model for myosin filaments developed by Squire (1973) is similar in several respects to the arrangement of a-helices derived experimentally for filamentous viruses (Marvin et al., 1974). By learning more about the structure of filamentous viruses we may also increase our understanding of how linear nucleoproteins are constructed, and thereby perhaps gain insight into the structure and function of chromatin. X-ray fiber diffraction patterns of filamentous viruses can be separated into two classes. Class I patterns (Marvin, 1966) are observed for the fd, If1 and IKe strains; class II patterns (Marvin et aE., 1974) are observed for the Pfl and Xf strains. Class II patterns indicate a tubular arrangement of capsid proteins, forming a helix with 4.4 units per turn and a pitch of 15 8. The proteins are largely a-helix, oriented with the long axis of the u-helix roughly parallel to the long axis of the virion. These 70 A-long protein molecules overlap each other in an axial direction like fish scales (Marvin et al., 1974). Both the size and conformation of the ooat proteins and the overall distribution of intensity on the diffraction patterns are the same for class I virions as for class II. But class I diffraction patterns have additional meridional reflexions at orders of 16 d, indicating that the structure units are spaced 16 A apart along the length of the virion. Mass per length calculations indicate that Bprotein molecules are spaced about 3 A apart along the length of the virion. Therefore the structure of the class I virion is complex, with about five B-protein molecules per structure unit (Marvin & Hohn, 1969). By making the reasonable assumption that the two classes. of virion are built according to the same general design, but differ in detail, it is possible to infer the change in the class II structure that would give rise to the class I structure, and thereby to determine the class I structure.

2. Materials and Methods Preparation of virus, formation of fibers, X-ray fiber diffraction techniques, and analysis of diffraction patterns have been described by Marvin (1966) and Marvin et al. (1974). To separate IKe from the denser flocculent contaminant (Khatoon et al., 1972), IKe is first purified by differential centrifugation and then banded in e C&l equilibrium gradient with mean density 1.3 g/cm3 at 30,000 revs./min for 60 h in the Spinoo no. 40 rotor at 15°C. The lower non-viscous contaminant is well-separated from the upper viscous virus

PLATE I. X-ray fiber pattern x 1.94. Fiber 187, film 55‘2.

of fd at 75% r.h. Specimen-to-film

distance

7.10 cm; enlargement

PLATE II. X-ray fiber pattern x 2.13. Fiber 187, film 556.

of fd at 32% r.h. Specimen-to-film

distance 7.11 cm; enlargement

PLATE III. X-ray fiber pattern of If1 at 75% r.h. Specimen-to-film x 1.44; tilt 8x5”. Fiber 1127, film 1759.

distance 9.38 cm; enlargement

PLATE IV. X-ray fiber pattern of If1 at 0% r.h. Specimen-to-film x 1.80; tilt 8.5”. Fiber 1114, film 1769.

distance

7.25 cm; enlargement

V. X-ray fiber pattern of IKe at 81% r.h. Specimen-to-film x 1.93; tilt 9.5”. Fiber 13760, film 1961.

PLATE

ment

distance

7.28 cm; enlarge-

hk

a type B crystal lattice. SpecimenPLATE VI. X-ray fiber pattern of If1 at 81% r.h., showing to-film distance 20.1 cm; enlargement x 1.15. Left: diffraction pattern (fiber 1064, film 1099). Right : corresponding reciprocal lattice rotation diagram for a hexagonal lattice with n = 59.3 A, c = 210 A. Circles represent crystalline reflexions, bars represent continuous transform or merged crystalline reflexions. Filled symbols indicate stronger intensity, for reference. Only the central region of the pattern is shown, to about 7 .& resolution.

-

f-

c

-

-

r

I-

-

-

-E -

* -

-

-

I

k

-

-

7

1

PLATE VII. X-ray fiber pattern of If 1 at 32% r.h., showing a type B crystal lattice. Specimcnto-film distance 20.3 cm; enlargement x 1.15. Left: diffraction pattern (fiber 1064, film 1013). Right: corresponding reciprocal lattice rotation diagram for a hexagonal lattice with a = 56.8 A, c = 207 A. Symbols as for Plate VI.

PLATE VIII. X-ray fiber pattern of Ml3 enlargement x 1-51. Fiber 1097, film 1945.

R5

at 75%

r.h.

Specimen-to-film

distance

9.38

cm :

STRUCTURE

OF FILAMENTOUS

583

VIRUSES

band. The contaminant, dissolved in 0.05 M-Tris (pH Sal), has a broad optical density maximum between 270 and 275 nm, and a n&imurn at 250 nm, with Emax/Emin = 1.18.

Strain Ml3 am3-H5 was obtained from D. Pratt. strain

when

grown

in a non-suppressing

host

The “polyphage”

is prepared

produced by this

as described

by Pratt

et al.

(1969). The viscous non-infectious band isolated from CsCl equilibrium gradients and clialysed against 0.1 M-NE&Cl, O-02 M-Tris (pH 7.3) has an optical density maximum at 268 nm, and a minimum at 247 run, with Emsx/Emin = 1.23. The Ml3 strain R5 was obtained from D. Pratt. This strain, a revertant of Ml3 am%Hl, is prepared as described by Pratt et al. (1969). When C&l is added to the virus in Tryptone the virus forms a flocculent band upon broth, the viscosity of the solution increases; 0.02 equilibrium centrifugation (Pratt et al., 1969). The virus dissolved in O-1 ~-N&cl, M-Tris (pH 7.3) has an optical density maximum at 268 nm and a minimum at 246 nm, with E,,,/E,i, = 1.30.

3. Class I Diffraction

Patterns

(a) Generalfeatures patterns of fd (Plates I and II; see also Marvin, 1966), If1 and IV) and IKe (Plate V) are similar in their overall distribution of intensity and in their layer-line pattern, so we group these strains of filamentous virus as having a class I structure. For these patterns, crystalline reflexions are observed on the equator, indexing on a hexagonal lattice with a minimum value of The fiber diffraction

(Plates

III

0.25

52 $523 0

0.20 :

*

46 .45

.

0

.

39

- 13

0.05 7 6

OO

I 0.05

I 0.10

’ o---.-.-d,-w-

0.15

0.20

0

0.25

Rlii-‘)

1. Dift&otion from fd: reciprooal space co-ordinates of regions of intensity on fiber diffraction patterns. The centers of the more striking regions on the patterns were measured on the film and converted to reciprocal space co-ordinates (R,{) after correcting for tilt of the fiber. Filled symbols indicate .stronger intensity, for reference. Meridional reflexions had nearly the same spacing for all films, and are therefore shown as single points. The strong 10 A equatorial and near-equatorial regions are shown as bars. Central crystalline reflexions are omitted. Some of the layer lines predicted for c = 208 A are included for reference. Circles: fiber 178B, film 617, 76% r.h., 4” tilt. Squares: fiber 178B, film 521, 66% r.h., 7” tilt. Triangles: fiber 187, film 5.50, 75% r.h., 2” tilt. Pm.

584

D. A, MARVIN

d, 0.15 z . 0.10-

-"-g--o *CD

ET

AL.

n

26

LL A-a

-20 19

n

13

I

0.05*-

-32 .33 u

7 6 0' 0

0.05

' a-a--o--n0.10 0+5 0.20 0.25 R(8--')

FIG. 2. Diffraction from Ifl: reciprocal space co-ordinates of regions of intensity on fiber diffraction patterns. Details as for Fig. 1. Squares : fiber 1065, film 1136, 75% r-h., 11.5” tilt. Triangles : fiber 1127, film 1746, 75% r.h., 11’ tilt. Ciroles: fiber 1114, film 1769, 0% r.h., 14’ tilt.

a (at lowest water content) of about 55 A (Marvin, 1966). A series of meridional reflexions is observed at orders of a 16 A spacing, with the first and third orders appearing strongest; near the position of the fifth order a broad diffuse meridional region of intensity is observed for suitably tilted fibers. Off-meridional intensity is observed about half-way between the layer lines defined by the meridionals, at R about 0.04 to 0.05 A-l. Very strong intensity appears on the equator at R about 0.10 d-l, and at similar R for t; about 0.03 8-l. Strong intensity also appears at (R, 5) about (O-10, 0.18). These features are best seen by comparing Plates I to V with Figures 1, 2 and 3. (b) Crystalhe

re$exions

Crystalline equatorial reflexions can be indexed on a hexagonal lattice with a between about 55 and 65 A, depending on water content. Crystalline reflexions are seen out to the fourth or fifth order for intermediate water content, where a is between about 58 and 62 A. For very wet fibers, the lattice becomes more disordered and crystalline reflexions broaden. Since crystahine reflexions are observed out to O-10A-l, and reflexions separated by 0.005 A-l can be resolved (Plates VI and VII), a = b and y = 120” to within 5%. Therefore at the resolution defined by the crystalline equatorial reflexions, about 10 A, the virions pack laterally in one of the hexagonal plane groups. Sharp reflexions are also observed on the meridian, at orders of about 0.062 8-I. These reflexions are sharpest for IKe, less sharp for Ifl, and still less sharp for fd. Sharp reflexions near these (O,O,Z)reflexions are also often observed, indexing as (1,OJ) based on the equatorial lattice.

STRUCTURE

OF FILAMENTOUS

585

VIRUSES

59 58

46 45

0.10 ci-

20 19

0

0.05*-I

13

-lo

L

0.25

OO

R (iv’) FIG. 3. Diffraction from IKe: reciprocal space co-ordinates of crystalline reflexions and diffuse regions of intensity. Positions were measured and converted to reciprocal space co-ordinates (R,<) after correcting for tilt of the fiber. Filled circles indicate regions of stronger intensity, for reference. The square indicates the meridional reflexion due to the contaminant. Crystalline reflexions at 5 g 0.21 8-l are represented by arcs with the measured reciprocal space radius of the reflexion, and with length indicating the limits of error in the (a,() measurements. The equatorial reflexions can be indexed on a hexagonal unit cell with a = 59.9 A. Some of the row lines predicted for this lattice are indicated in the region of the crystalline reflexions at < g 0.21 A-1. Some of the layer lines predicted for c = 213 A are included for reference. Fiber 1367, film 1820, 75% r.h., 10” tilt.

Much of the intensity

off the meridian and off the equator must be attributed to the breadth of the intensity maxima compared with the breadth of the equatorial reflexions. When crystalline reflexions are observed in this region, they are neither as consistent nor as striking as the equatorial reflexions, and they seldom are seen beyond 5 = O-062 8-l. One exception is IKe (Plate V; Fig. 3), which shows crystalline reflexions at 5 z 0.21 A-l, although not on most of the lower layer lines. This failure to find conditions for perfectly crystalline fiber patterns suggests that the virion does not have true trigonal or hexagonal symmetry in three dimensions. A mixture of virions randomly oriented both “up ” and “down ” in the fiber cannot be truly crystalline unless the virion has a dyad perpendicular to its long axis. Nevertheless, some insight into the symmetry of the virion may be gained by considering the three types of semicrystalline patterns that have been observed. continuous

transform,

considering

Type A. Semicrystalline fd fibers show rather fuzzy crystalline reflexions on layer lines with 5 = O-062 8-l and 5 = O-031 A-l, as well as on the equator (Plates I and II; Fig. 4). The reflexions on the O-031 A-l layer line can be indexed on the basis of a unit cell three times the area of the cell defined by the equatorial lattice: the true unit cell dimension is a’ = a1/3. Even so, these reflexions do not fit perfectly

586

D. A. MARVIN

ET AL.

on the layer line with 5 = 0.031 8-l. Th’IS may indicate a tendency towards the layer-line splitting that is seen more clearly for other t,ypes of pattern. Type B. A few fibers of If1 show crystalline reflexions on layer lines with 5 = 0.062 8-l and 5 = O-034 8-l (Plates VI and VII) that can be indexed on the basis of the equatorial lattice. The layer lines can be assigned indices 1 = 7 and 1 = 13 with c =

0 I---I

-.-J-o 2

hk 20 30 22 II

21

41

30 33

22 31 40 6052 44 26

III 0.04

0 600

006

ma-9 hk 20 I 22

(b)

21 41

30 33

22 31 60 52 26

c I oL-----J0

0.02

R(#-‘) FIG. 4. Diffraction from fd: reciprocal space co-ordinates of type A crystalline reflexions. Coordinates of crystalline reflexions near the center of the pattern were measured and converted to reciprocal space co-ordinates (R,<). Reflexions can be indexed (bottom rows of hk) on a hexagonal unit cell with a’ = ~43, where a is the unit cell side detied by the equatorial reflexions (top row of hk); and on 1 values with the first meridional on 2 = 26 for c g 416 A. Each reflexion is represented by an arc indioating measured reciprooal space radius of the reflexion, with arc length indicating the limits of error in the (R,<) measurements. Fiber 187. (a) Film 550, 75% r.h., cb = 59.2 A, n’ = 102.4 A, o = 416 A. (b) Film 566, 32”/0 r.h., a = 56.2 A, a’ = 97.4 A, c = 410 A.

STRUCTURE

OF FILAMENTOUS

VIRUSES

587

210 A. Diffuse weak diffraction is seen on I= 7 extending to the meridian on Plate VII, and also on 1 = 6. However no crystalline reflexions are observed on 1 = 6, over a range of unit cell dimensions. These patterns illustrate most clearly the layerline splitting characteristic of many class I patterns. Although a meridional reflexion layer lines are observed at O-034 AL-l at 0.062 8-l is typical for class I patterns, and O-028 A-l, “split” slightly from the O-031 A-l half order of O-062 4-l. Higher layer lines are also split, with the lower half of the pair absent in the R = 0.04 to 0.05 8-l region. The crystalline reflexions can also be indexed on 1 = 8 and 1 = 15 with c = 242 A, rather than 1 = ‘i and 1 = 13 with c = 210 A, or on intermediate values. As emphahk

(al

IO

II 20

II

30

I 0.06’-

Z.k cn

0.04 -

21

30 33

22 31 60 52

40 44

>,

IO

20

21

\

\

0.02 -

-.-

/TR-‘) FIG. 5. Details cu (a) Fiber (b) Fiber

Diffraction for Fig. 4. 1127, film 1114, film

from Ifl: reciprocal space co-ordin&tes of type c crystalline Reflexions can be indexed on I values with the first meridioml 1745, 75% r.h., a = 58.2 A, CZ’= 100.9 A, c = 206 A, 1769, 0% r.h., a = 55.3 A, a’ = 95.7 .& c = 205 $.

reflesmrs. on E = 13.

588

D. A. MARVIN

ET

AL.

sized by Ramaohandran (1960), integral layer lines are no more than a convenience; non-integral layer lines are structurally just as meaningful in describing a helix diffraction pattern. Type C. Crystalline reflexions on 1 = 6 can only be indexed on type C patterns of If1 (Plate IV; Fig. 5) on the basis of a unit cell three times the area of the cell defined by the equatorial lattice: the true unit cell dimension is a’ = ad3, as for type A. With this choice of lattice, the observed reflexions are consistent with the selection rules h - k = 3q (q any integer) for I= 0 and 1 = 13, and h - k # 3q for I= 6, Reflexions with h - lc = 3q are systematically absent on I= 6, and not just fortuitously located at positions of low molecular transform. For instance, the (22,s) reflexion is absent on Fig. 5(b) (Plate IV), where it is predicted to ooour at R = O-042 A-l, even though the (3,1,6) reflexion is observed at R = 0.042 A-’ on Fig. 5(a). At least one h - k = 3q reflexion, (4,1,7), is observed on 1 = 7. 0.065

0.060 t

FIG. 6. Variation of parameters of the If 1 type B helix with water content of the fiber. Positions and limits of error were measured for the crystalline reflexions at (R,<) positions about (0.0 to 0.05, 0.062); for crystalline reflexions at (O-06 to 0.10, 0.034); for the diffuse regionat (0.00 to 0.02, 0.034); and for the diffuse region at (0.03 to 0.05, 0.029). Several crystalline reflexions at 6 # 0 for each diffraction pattern were indexed on the equatorial lattice to determine the best 5. For diffuse regions, (R,Q were measured at several points along the streak and an average 5 was determined. For the 5 = O-034 A-r region, limits of error are shown only for the crystalline reflexions. The points represent the mean positions for the diffuse region, with no limits of error shown. The error for this region is in fact about the same as for the crystalline reflexions. Eaoh set of measurements was made on a series of diffraction patterns of fiber 1064, taken at different relative humidities, and plotted using the measured a* as an index of water content. From right to left: 0% r.h., film 998; 32% r.h., film 1013; 57% r.h., film 1051; 75% r.h., lilm 1077; 81% r.h., film 1099. A line is drawn to fit the points near < = 0.062 A-l, and lines with 5 calculated for 6/13 and 7/13 of this value are drawn through the other two regions. Top: crystalline I = 13; middle, crystalline and diffuse I = 7; bottom, diffuse I = 6.

STRUCTURE

OF FILAMENTOUS

VIRUSES

589

Although the 5 position of the 16 A meridional reflexion varies slightly with water content of the fiber, the split layer lines of If1 can be indexed as 6/13 and 7/13 (+2%) of the meridional 5 over the range 0% to 81% r.h.t (Fig. 6). (0) Comparison

of strains

All class I patterns show hexagonal reflexions on the equator, with minimum a about 55 A, and all show meridional reflexions at orders of 16 A. Layer lines are apparent roughly midway between the 16 A meridional reflexions. Where precise measurements are possible, on semicrystalline Ifl, the layer lines are apparent at 6113 and 7113 of the meridional spacing, indexing as 1 = 6 and I= 7 for c = 210 A. On the basis of this repeat, sharp meridional reflexions appear on 1 = 13, 26, and 39 ; and diffuse meridional intensity appears near 1 = 63. Off-meridional regions of intensity at R z 0.04 8-l appear on or near 1 = 6, 20, and 33. Although all class I strains are similar, they can readily be distinguished from one another. The fd and If1 patterns can be distinguished on the basis of the intensity distribution at (RJ) of (O-10, 0.18). For fd there are striking maxima at (O-10, 0.19) and (O-12, 0.16); whereas for If1 there are maxima at (0.09, O-17) and (0.10, 0.19). Therefore a glance at this region enables one to distinguish fd, with a ridge of intensity leading out and down on the pattern, from Ifl, with a ridge leading out and up. The IKe pattern is more like If1 than fd in the (0.10, O-18) region. Fibers prepared from unpurified IKe solutions show a sharp meridional reflexion at about 11 A (indicated as a square on Fig. 3). Fibers prepared from IKe preparations which had been freed of the flocculent dense contaminant by CsCl equilibrium centrifugation showed no such meridional reflexion (Plate V). The disappearance of this meridional reflexion was not due to a structural change in IKe caused by high CsCl concentration, since incubation of unpurified IKe in CsCl without centrifugation gave no change in this reflexion. The flocculent contaminant was purified free of IKe by equilibrium centrifugation as described in Materials and Methods, a fiber was prepared, and the diffraction pattern obtained. Sharp meridional reflexions at the first and second order of 22 A were observed, but the strong 10 A equatorial reflexion typical of filamentous viruses was absent. Therefore the meridional reflexion at 11 A on diffraction patterns of unpurified IKe is almost certainly due to the presence of a contaminant that diffracts strongly at this spacing, probably pili (Mitsui et al.: 1973). Diffraction patterns of wild-type Ml3 are virtually indistinguishable from those of fd. The Ml3 strain am3-H5, which carries an amber mutation in gene 3, the gene thought to control the minor coat protein (A-protein), produces non-infectious “polyphage” when grown in a restrictive host (Pratt et al., 1969). Diffraction patterns of polyphage fibers (not shown) looked broadly similar to those of wild-type fd or M13, but had the same 11 A meridional reflexion that was seen on unpurified IKe. Since polyphage are not infectious, it was difficult to identify precisely the polyphage region in a CsCl equilibrium gradient. It is probable that the 11 A meridional on polyphage diffraction patterns was also due to pili. The Ml3 strain R5 carries a mutation in the B-protein gene that makes the virion insensitive to antiserum against wild-type virions (Pratt et al., 1969). However, there 7 Abbreviation

used: r.h., relative

humidity.

4590

D. A. MARVIN

ET

AL.

was no apparent diff’erence between diffraction patterns of R5 (Plate VIII) and those of Ml3 or fd. Therefore the serological distinction between R5 and the wild-type probably reflects a change in the detailed configuration of the B-protein, rather than a change in the organization of B-proteins relative to each other.

4. Relation between Class I and Class II (a) General comparison The existence of several features common to both class I and class II diffraction patterns suggests that the two classes of virions may be related structurally, if not genetically. In both classes, the virions pack laterally in a hexagonal plane lattice with minimum dimension a z 55 A. On both types of pattern, there is a strong region of intensity at about 5 A in the meridional direction, and a strong region of intensity at about 10 a in the equatorial direction. In fact, it. is difficult to distinguish a poorly oriented class I from a poorly oriented class II patt.ern by qualitative inspection. TABLE

1

Comparison of 5 for elms I (c = 210 A) and cl4h9sII (c = 75 A) layer lines

1

1 1;@-*1

0 6 7 13 19 20 26 32 33 39 45 46 52

0.0 0.029 0.033 0.062 0,090 0.095 0.124 0.162 0.167 0.186 0.214 0.219 0.248

C18SS

1

chbss II i (-k-1)

0 2 3 5

0.0 0.027 0.040 0.067

7 10

0.093 0,130

12 15

0.160 0.200

17 20

0.227 0.260

Detailed comparison of the layer-line positions reveals significant differences. Layer lines on class I patterns fit a selection rule for a 13/i helix with c = 208 d, whereas layer lines of class II patterns fit a selection rule for a 22/? helix with c = 75 A (Marvin et al., 1974). Nevertheless there are several areas of correspondence between the layer lines of the patterns (Table 1). Layer lines 6, 20, 33, and 46 of class I are similar to layer lines 2, 7, 12, and 17 (the m = 1 set) of class II, both with respect to the 6 co-ordinate of the layer line, and with respect to the distribution of intensity along the layer line. Layer lines 5, 10, and 15 (the m = 0 set) of class II can be transformed to layer lines 13, 26, and 39 of class I by a decrease of about 10% in < and an imaginary rotation of the pattern by 10 to 15” around the origin to bring the first maxima of J,, J,, and J, of class II onto .the meridian for class I. That is,

STRUCTURE

OF FILAMENTOUS

VIRUSES

591

the pitch of the class II helix corresponds to the meridional (asymmetric unit) spacing of the class I helix. For both class I and class II patterns, there is a diffuse meridional refiexion at 3.1 to 3.4 A. These relations lead us to conclude that the class I and class II structures are built according to the same general design, but that the class I structure is related to the class II helix by two small transformations. The first transformation is a change in the helix parameters from 4.4 units per turn for class II to 4.5 for class I. The second transformation is a perturbation, superimposed on the simple 45 units per turn helix, that repeats about every five units. The effect that this perturbation has on the class II diffraction pattern is reminiscent of the effect that the introduction of a coiled-coil conformation has on a simple u-helix (Crick, 1953), although the similarity of a for class I and class II shows that any supercoiling in class I has only a small radial component, In the case of the u-helix, the layer line at 5.4 A arising from the pitch of the unperturbed helix is transformed to a meridional reflexion at 5.1 d in the coiled-coil, just as the 15 A layer line (1 = 5) in class II is transformed to a meridional reflexion at 16 di in class I. The fact that the spacing defined by the meridional reflexion is larger than the pitch of the unperturbed helix suggests that the major helix has the same sense as the minor helix (they have opposite senses in the u-helix coiled-coil). In subsequent discussion we shall refer to the structure

n FIG. 7. The (n,Z) plot for the helix selection rule I = - 14n + 6.7772 (left-handed helix) is indicated by large open circles. This 63/n helix with c = 210 d is equivalent to a 9/z helix with c = 30 A. Solid lines are drawn through (ra,Z) values of constant era. The same (n,Z) plot can be generated by the helix selection rule Z = 49rr + 63m. The dashed line is drawn through (n,Z) values with 112= 0 for this 63/49 helix. Small filled circles give the (n,Z) plot for the helix selection rule Z = - 14n + 13m. 39

592

D. A. MARVIN

ET

AL.

. .

.

240

3 3

+ (degrees) 8. Surface lattice for a 63/n helix, with c = 208 A. This helix is equivalent to a 912 helix c = 30 tf. The surface lattice can be described either in terms of a left-handed helix with 63 in 14 turns, with pitch 15 A; or in terms of & right-handed helix with 63 units in 49 turns, pitch 4.3 A. The horizontal scale corresponds roughly to a surface at radius 16 b. Some directions of the 63/i& helix a,re indicated. The projection is viewed from outside the helix.

FIG.

with units with (n,a)

that gives the class II diffraction pattern as the simple helix, and the structure that gives the class I diffraction pattern as the perturbed helix. (b) Change in the helix parameters A simple 2215 helix with c = 75 A has a rotation per asymmetric unit (unit twist) of --8l+l2”, and a z axis advance per asymmetric unit (unit rise) h = 3.409 A, giving 4.4 units per turn. If this structure were given a slight twist about its helix axis, to change the unit twist to -8OWY’ and the unit rise to 3.302 A, giving 4.5 units per turn, the structure would become a simple 63/n helix with c = 208 A. The (n,l) plot of a 63/n helix is shown in Figure 7. As a result of the small twist, the (n,Z) values (9,l) and ($,3), that describe the strong near-equatorial intensity on the simple 2215 helix with c = 75 A, are transformed to (9,O) and (5,7) on the simple SS/lJ helix with c = 208 8. That is, the class I helix need be only very slightly different from the class II helix in order to explain the observed change in 5 position of the near-equatorial Js and JF contributions from 1 = 1 and 1 = 3 (c = 75 A) in class II to 1 = 0 and 1 = 7 (c = 208 A) on class I. The surface lattice of a 63/n helix with c = 208 A is shown in Figure 8; it is quite similar to the surface lattice of a 2215 helix with c = 75 A (Fig. 6 of Marvin et al., 1974; see also Fig. 9(a)). The orientation of the B-protein molecules associated with each surface lattice point must change as a result of this small twist, in order to retain acceptable contacts between neighboring B-proteins. The change that is required can be seen by comparing Figure 9(a) with Figure 9(b). The axis of the B-protein molecule lies between the (‘%,5) and (i&,4) lines on the surface lattice of the class 11 helix, following a helix with pitch about -300 A (Marvin et al., 1974; Fig. 9(a)). If the corresponding B-protein in the class I structure bears the same reIation to its neighbours, it

STRUCTURE

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b, (degrees) Pm. 9. Radial projections of class I and olass II structures. The points represent the surface la&ice, viewed from outside the helix. Each B-protein is represented by an elongated wedge, to indicate that the B-protein extends from radius T = 15 L%(thin end of the wedge) to r = 29 ip (thick end). (a) Class II helix, 22/c, c = 75 A. The surface lattice is identical to Fig. 6 of Marvin et ~2. (1974), except that it is viewed from outside rather than inside the helix. (b) Class I helix, 63/i& unperturbed, c = 208 A. The dashed line passes through the points to which the thin end of the B-protein was translated parallel to the u-helix axis in the trial perturbed model.

would lie between the (%,7) and (a,7) lines, following a helix with pitch about -500 H (Fig. 9(b)). Sections perpendicular to the axis of the helix are then virtually the same for both the 2215 and the 63/14 helices (Fig. IO), and the intramolecular contacts remain qualitatively acceptable. (c) Perturbation at

Class I diffraction patterns also differ from class II in having meridional reflexions orders of 16 8. These meridional reflexions can be explained by a periodic

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180

FIG. 10. Se&ion through the ckss I 63/f4 unperturbed helix, perpendicular to the s axis. Each circle represents a section through one of the a-helical B-proteins, with diameter 9 A. The section at z = 0 A (see Fig. 9(b)) would pass through the axis of a B-protein at r = 15 A, Q = 0”, z = 0 A (labeled 0 on the diagram); it would pass through the clxis of another B-protein with origin at 4 = 80°, s = -3.302 A, that has co-ordinates r = 15.6 A, 4 = 77’ at x = 0 A (labeled - 1 on the diagram); etc. A similar section through the class II helix would appear &no& identical to this.

perturbation (Crick, 1953; Lang, 1956; Johnson, 1959; Ramachandran, 1960; Caspar & Holmes, 1969) of the 63/B helix. In a perturbed helix, the asymmetric units of the helix are moved away from the positions that they would occupy in the simple helix. Any perturbation can be described in terms of a periodic function of r, $, and Z. Perturbation along r (radial displacement) changes the position of intensity on layer lines; perturbation in 4 changes the helix parameters and thereby the layer-line positions; and perturbation in z introduces new reflexions on the meridian. The effect of a perturbation can be visualized by drawing a continuous straight line (genetic helix) through all points on the surface lattice of a simple helix. Now replace this straight line by a sine curve having the straight line as abscissa (Fig. 9(b)). Move all surface lattice points from the straight line, parallel to x, onto the sine curve. The result is the surface lattice of one kind of perturbed helix. The periodic function can be more complicated than a sine function (in general, it can be represented by a Fourier series in r, 4 and z) ; and it need not repeat after an integral number of surface lattice points. The 16 A meridional reflexion of class I diffraction patterns falls on 1 = 13 for c = 208 A. Therefore the x component of the perturbation repeats in a distance corresponding to 63113 = 4.85 units of the simple helix. The perturbed helix ret’ains the 14 turns in 208 A of the 63/n helix, but has only 13 periods of the perturbation, giving the perturbed helix some of the characteristics of a 13/n helix. A 13/n helix predicts additional layer lines on the diffraction pattern (Fig. 7). However, small perturbations contribute little diffracted intensity to most of these layer lines; a small change in physical structure gives only a small change in the diffraction

STRUCTURE

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pattern. The main differences between the observed diffraction pattern and that predicted for the 63/14 unperturbed helix are the introduction of new meridional reflexions, indicating an axial component; and in some cases splitting of the layer lines half-way between the meridionals, indicating an azimuthal component. In order to confirm these general ideas about the perturbation, we have constructed trial molecular models, calculated their Fourier transforms, and compared the calculated transforms with the observed intensity distribution. One such perturbed model is illustrated in Figure 9(b), where the B-proteins were each translated parallel to the u-helix axis by multiples of one amino acid. The Fourier transform of this perturbed structure has meridional reflexions on 1 = 13, 26, and 39, and strong intensity on 1 = 0 and 1 = 7 at the positions observed on class I diffraction patterns. The slight. differences in helix parameters that are observed for different class I strains or different relative humidities indicate slight variations between class I structures. The breadth of the meridional reflexion near 1 = 63 can be attributed to the fact that diffraction in this region arises not only from the axial spacing between B-proteins (itself variable, because of perturbation) but also from the periodic spacing of the nucleotides. These periodicities need not bear an integral relationship to each other. (d) Crystal and molecular symmetry When the unit cell contains more than one virion, some of the crystalline reflexions may be systematically absent. The nature of the systematic absences can give information about the crystal symmetry, even though not all regions of the fiber diffraction pattern are sampled by the crystalline reflexions. As in class II, the offmeridional reflexions on some class I diffraction patterns (type C, Fig. 5) can only be indexed on a unit cell with an area three times that defined by the equatorial reflexions. For this larger unit cell with side a’ = ad3, equatorial reflexions are only observed for h - k = 3q, q any integer. This means that the larger unit cell must contain three virions at co-ordinates x, y, z, $ (113, 113, zl, 9~ (213, 0, x2, 46,~ (0, 213, z3, 43) where x, y, and z are fractions of the unit cell edges, and $ is a fraction of 2~ rotation of the molecule about its x axis. These relative positions are only possible for trigonal space groups (Marvin et al., 1974). In the c-axis projection the plane-group symmetry is therefore 233, p3m1, or p31m. The plane-group symmetry must still be one of these three if the unit cell of side a, containing only one molecule, is chosen to describe the projection. The information about crystal symmetry gives information about the relative translational and rotational positions of virions in the unit cell. But in addition the translation z and rotation + about the z axis of each virion are related to each other by the helix parameters. These two relations can sometimes be combined to predict relative molecular positions for known (n,l) or to predict (n,Z) for molecules of known position. For crystalline fibers that show non-equatorial reflexions only for h - k # 3q, such as type C, 1Ax - nA$ = p/3 where p is an integer not equal to 3q (Marvin et al., 1974). The (n,Z) relations for class I are complex, but for simplicity consider the 63114 unperturbed helix. In the

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unperturbed helix, Jz and J, are predicted on the same layer line (I = 7), whereas the layer line in this region is observed to be split for type C. The approximation involved in assuming a 63114 unperturbed helix should not invalidate packing arguments. The 63/a helix is identical to seven repeats of a 912 helix with c = 30 A, and we choose only one of these repeats to discuss packing. Thus the observed 1= 6 and 1= 7 for c = 210 A become I= 1 for c = 30 A in our approximation. Then AZ - 413 = p/3 and AZ is restricted to 0 or -l/3 (-10 8). The virions in the unit cell are related by either a rotation triad or a left-handed screw triad (Table 2 of Marvin et al., 1974). The 912 helix itself has a right-handed screw triad (Fig. 8). Some diffraction patterns of IKe (Plate V; Fig. 3) have crystalline retlexions around 5 = 0.21 8-l (on or near 1 = 46), but not on most lower layer lines except the equator. Indexing of reflexions near 1 = 0.21 8-l is difficult because 4 nearly equals p, so a small error in 5 introduces a large error in R = l/(p2 - c2). Nevertheless these reflexions are clearly crystalline, not continuous transform. The surface lattice of a 63/B (left-handed) helix can equally well be described by a 63149 (right-handed) helix. For the 63149 genetic helix, 1 = 49 belongs to the m = 0 set of Bessel functions. Therefore it is possible that the crystalline reflexions near 5 = 0.21 A-l for IKe arise from screw disorder (Klug et al., 1958). A random screw along the 63149 genetic helix (that is, along the (1,49) directions in Fig. 8) would permit crystalline reflexions on 1 = 49 but forbid them on most other layer lines. A screw along the 63149 helix is structurally reasonable, since the axes of the m-helical B-proteins are perpendicular to this line, so overlapping B-proteins create a ridge along this helix. Crystalline reflexions may be observed on 1 = 46 rather than 1 = 49 because ‘u may be 60 rather than 63 for IKe, giving a SO/46 helix; or because the perturbation of the 63149 helix may slightly change the line along which the virions screw. Explanation of these crystalline reflexions in terms of screw disorder lends further support to the choice of zc = 22 as the number of units/turn in the simple class II helix (Marvin et al., 1974). No other possible choice of u would predict m = 0 on or near 1 = 46 for the related class I helix.

5. Discussion The similarity between class I and class II diffraction patterns indicates that the two structures have similar electron density distributions. Chemical evidence shows that the B-protein molecules are about the same size and largely u-helical for the two classes. Therefore it is probable that both classes are built according to the same general design, but differ in the detailed position and orientation of the B-proteins within the virus helix. The strong intensity at about 10 A in the equatorial direction falls on two separate layer lines, at much the same R for the two classes. Therefore we conclude that the strong intensity on these layer lines can be attributed to the same Bessel functions (Jg and J;j) for class I as for class II. Inspection of the helix selection rule shows that the 5 positions predicted for J, and Js move from those observed for class II to those observed for class I if the number of units per turn is changed from 4.4 to 45. The meridional reflexions observed on class I patterns at orders of 16 A are not predicted for the simple 4.5 units per turn helix derived by this argument, but are predicted if an axial perturbation, repeating about every fifth subunit, is superimposed on the simple helix.

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597

Several different explanations for this periodic perturbation are conceivable. The energetically optimum packing relations between B-proteins may be incompatible with the same helix parameters at different radii, as found for the dahlemense strain of tobacco mosaic virus (Caspar & Holmes, 1969). Alternatively, periodic perturbation of the protein positions may be a consequence of a non-integral ratio of nucleotides to B-proteins (Marvin & Hohn, 1969), leading to quasi-equivalent interactions between DNA and protein and thence to quasi-equivalent positions for the B-protein molecules in the virus helix. Differences between class I and class II structures may also be related to differences in the process of morphogenesis in the respective hosts, Whatever the reason for the differences between the two classes, detailed comparison of the class I with class II structures should give more insight into the structure and morphogenesis of filamentous viruses than the study of either alone. The class I and class II structures are similar but distinct: we have found no circumstances in which fd gives a class II diffraction pattern, or Pfl gives a class I diffraction pattern. Nevertheless it is easy to imagine a structure which does have two states, and can be converted from one to the other by external agents. The possibility of a transition between two states of a tubular structure composed of rod-like structure units suggests a general model for transport by tubular structures. A structural transition could be triggered at one end of the tube and propagated along its length, as suggested for some models of flagellar motion (reviewed by Bode, 1973). If this transition involves a transient change from state I to state II and then back again to state I, a travelling wave of state II would pass down the length of the tube. For rod-like structure units, a small change in helix parameters would yield a large change in orientation of the structure unit. For instance, in the transition from class I to class II, if we ignore the axial perturbation and consider only the change from 4.5 to 4.4 units per turn (Fig. 9), a motion of 1” in azimuth at one end of the Bprotein is accompanied by a 30” motion at the other end. A periodic wave could function in transport like peristalsis, except that the wave of altered state need not be a wave of contraction, but could be a wave of exposed charged groups passing down an uncharged structure ; a wave of exposed hydrophobic groups passing down an otherwise hydrophilic structure; or a wave of more specific molecular structure. Such a wave could, for example, transport flagellin molecules through hollow bacterial flagella to the distal ends of the flagella, where they polymerize; could transport material down microtubules;

or could transport

molecules

through

pores across membranes

(pores can be considered as short lengths of tube). Periodic structural changes of this sort could also transport myosin filaments relative to actin : in this case the prime mover would be not just the myosin head group, but the whole length of the myosin molecule. We are indebted to J. Lapointe for help with some of the diffraction experiments. One of us (W. J. P.) was a Seessel Fellow of Yale University (196%1970), and another of us (E. J. W.) was a postdoctoral fellow of the U.S. Public Health Service (1971-1973). This investigation was supported by research grant no. GB20819 from the U.S. National Science Foundation, by U.S. Public Health Service research grants nos. AI06524 and CA13094, and by computing grants from Yale University. REFERENCES Beaudoin, J. (1970). Ph. D. Thesis, University Bode, W. (1973). Angew. Chern. Internat. Edit.

of Wisconsin. 12, 683-693.

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Caspar, D. L. D. & Holmes, K. C. (1969). J. Mol. Biol. 46, 99.-133. Crick, F. H. C. (1963). Acta Crystallogr. 6, 689-697. Day, L. A. (1973). Biochemistry, 12, 5329-5339. Day, W. A. & Gilbert, D. S. (1972). Biochim. Biophys. Acta, 285, 503-506. Jacobson, A. (1972). J. Viral. 10, 835-843. Jazwinski, S. M., Marco, R. & Kornberg, A. (1973). Proc. Nat. Acud. Sci., U.S.A. 70.

205-209. Johnson, C. K. (1959). Ph.D. Thesis, Massachusetts Institute of Technology. Khatoon, H., Iyer, R. V. & Iyer, V. N. (1972). Virology, 48, 145-155. H. W. (1958). Acta CrystaZZogr. 11, 199-213. Klug, A., Crick, F. H. C. & Wyckoff, Lang, A. R. (1956). Acta Crystallogr. 9, 436-445. Lowey, S. (1971). In S&units in Biologicd Systems (Timasheff. 8. N. & Fasman, G. D., eds), part A, pp. 201-259, Marcel Dekker, New York. Marvin, D. A. (1966). J. Mol. Biol. 15, 8-17. Marvin, D. A. 8.~Hoffmann-Berling, H. (1963). Nature (London), 197, 517-518. Marvin, D. A. & Hohn, B. (1969). Bacterial. Rev. 33, 172-209. Marvin, D. A., Wiseman, R. L. & Wachtel, E. J. (1974). J. Mol. Biol. 82, 121-138. Mitsui, Y., Dyer, F. P. & Langridge, R. (1973). J. Mol. Biol. 79, 57-64. Nakashima, Y., Dunker, A. K., Marvin, D. A. & Konigsberg, W. (1974). FEBS Letters,

40, 290-292. Pratt, D., Tzagoloff, H. & Beaudoin, J. (1969). Virology, 39, 42-53. Pratt, D., Laws, P. & Griffith, J. (1974). J. Mol. BioZ. 82, 425-439. Ramachandran, G. N. (1960). Proc. Ind. Acad. Sci. 52, 240-254. Smilowitz, H. (1974). J. Viral. 13, 94-99. Squire, J. M. (1973). J. Mol. BioZ. 77, 291-323. Timmis, K. & Marvin, D. A. (1974). Virology, 59, 293-300.

APPENDIX

Reinvestigation of a Region of the fd Bacteriophage Coat Protein Sequence Y. NAKASHIMA Department

AND

W. KONIGSBERG

of Moleculaar Biophysics and Biochemistry Yale University New Haven, Conn. 06520, U.X.A. (Received 25 March 1974)

We report here the results of an experiment designed to check the amino acid sequence of a region of the coat protein (the B-protein) of the bacteriophage fd. The amino acid sequence of the coat protein was originally determined by Asbeck et al. (1969). According to their work, the protein was 49 residues long and the section from residues 24 to 29 had the following structure: Tyr-Ala-Trp-Met-Val-Val. The possibility that the sequence in this region might be incorrect was suggested by Sanger et al. (1973) when they synthesized a deoxyribooligonucleotide sequence complementary to a position of the fd DNA that would match the amino acid sequence (Trp-MetVal) that had been found in the coat protein. They hoped that this complex would