J. Mol. Biol. (1978) 122, 247-253

The Size of the Bacteriophage T4 Head in Solution with Comments about the Dimension of Virus Particles as Visualized by Electron Microscopy We report X-ray diffraction results permitting calculation of the radius of the bacteriophage T4 head in solution. Isometric headed mutant particles, consisting of the two hemispherical caps of wild-type T4, were found to be spheres of radius 425 .&. Giant headed particles, an amplification of the extra capsomeres which give the T4 head its prolate shape, were found to br cylinders of radius 427 a. We use these and other small-angle X-ray diffraction results in a quantitative discussion of the distortion artefacts caused by a number of electron microscope specimen preparation techniques.

Electron microscopy is often the only available technique for determining the dimensions of molecular aggregates, and is widely used for this application even though it is general knowledge that the measurements so derived are susceptible to distortion artefacts. We describe below results of two X-ray diffraction experiments which have allowed us to calculate the solution radius of bacteriophage T4, one of the most widely studied virus particles. We then use this radius in combination with those of a number of other bacteriophages to assess the extent of distortion artefacts associated with several commonly used methods of electron microscope specimen preparation. During a study of the structural organization of DNB packaged within the heads of T4 normal (prolate), isometric and giant phages (Earnshaw et nl., 1978) we obtained small-angle X-ray diff’raction dat’a allowing us to calculate the solution radii of isometric and giant particles with great precision. All the methods used below are to be described by Earnshaw, W.. Hendrix. R. & King, J. (manuscript in preparation). Isometric headed T4 particles are produced by the mutant E920g (Eiserling et al., 1970), which has an altered major capsid protein. These particles are composed of T = 131 caps of wild-type T4 without the extra equatorial the two hemispherical capsomeres which give the head its prolate shape (Branton & Klug. 1975; Aebi et al., 1976). The radius of the isometric head variant is. therefore, the radius of these hemispherical caps which form either end of t,he wild-type head. The diffraction pattern from a purified solution of isometric headed particles shows faint rings at small angles. The rings are due to scatter from the phage head. The locations of the zero points between nine of these rings, measured using a Nikon shadowgraph, are consistent with the scatter from a solid sphere of radius 425 .& as shown in Table 1. The fact) t,hat the standard deviation was small (19/o for eight zeros measured) indicates that the solid ball approximation is a good one. We also observed small-angle diffraction rings from a concentrated solution of T4 giant phage produced by the coat protein mutant ptgl91 (Doermann et al., 1973) as shown in Figure 1. The rings allow calculation of the radius of the equatorial capsomeres at the mid-section of the T4 head (Branton & Klug, 1975), since in the greatly 247 0022.2336/78/1222-4753

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\C I978 Acaclrxllic~ Press Inc. (Lontlon)

Ltd.

LETTERS

248

TO

THE

TABLE

EDlTOR

I

The radius of T1 isometric phage heads Diffraction pattern zero number

Diffraction spacing (2 sin B/h)

3 4 5 G 7 8 9 10

Calculated radius

0.00419 0.00532 0.00645 0.00769 0.00876 0.00991 0~01102 0.01234 Average

The radius sphere is

was calculated

using

a solid

sphere

A(S) (Guinier,

1963;

Abramowit,z

& Stegun,

approximation.

= 2T2/Sjl

1965)

with

particle (A)

414 421 425 427 428 429 431 425 425 * 5

The Fourier

transform

of a solid

(2nrS) the particle

radius

given

by:

where A(S) is the transform amplitude as a function of diffraction spacing, r is the particle radius, and b is the appropriate zero of the first-order spherical Bessel function j, (Abramowitz & Stegun, 1966). The diffract,ion spacing is the radius on t,he film in units of 8-l. For both the solid sphere and solid cylinder (Table 2) approximations the radius depends on correct assignment of the diffraction pattern zero number. In both cases, if this number was shifted by 1 in either direction the calculated radius was not stable about a single mean and the standard deviation was increased 7 to B-fold. In the past where we have checked the assignment of the diffraction pattern zero number during calculation of electron density profiles we have found that the above criterion was sufficient to determine the number uniquely (Earnshaw et nl., 1976, and unpublished work). TABLE

2

The radius of Td giant phage heads Diffraction pattern zero number

Diffraction spacing (2 sin S/X)

Calculated radius

particle (A)

-4 5 6 7 8 9 10 11

0.00493 0.00612 0.00730 0.00844 0~00968 0~01081 0.01203 0.01323 .4verage

The radius solid cylinder

was calculated is

based

on a solid A(S)

(Franklin

& Holmes,

1958)

with

cylinder

approximat,ion.

430 428 425 429 42G 428 426 425 427 --1 “ The Fourier

transform

of a

=: J, (2TrrS)/(27rrS)

the particle

radius

given

by

r = H/2&, where A(S) is the transform amplitude as a fun&on of diffraction and B is the appropriate zero of the cylindrical Bessel function

spacing, T is the particlc J1 (Watson, 1960).

radius,

T4

PHAGE

HEAD

SIZE

Frc,. 1 Diffraction pattern from a concentrated solution of ‘~4 giant bacteriophage. The sharp rings at small angles an’ due to diffraction from tho phage capsids, having the form of the diffraction from a solid cylinder of radius 427 A (Table 2). The broad outer ring at a diffraction spacing of l/23 A-l is due to diffraction from the ordered sick+-by-side packing of the collapsed DN.4 molecule packaged within the phage heads n,nd is discuasrtl in detail elsewhere (Eamshaw et al., 1978). The intense vertical stwak, and the fact that. the small-angle rings are less aharp at. the left and right of the patt,em are due to t.hn geometry of thv X-ray namcra, which is described by Earnshaw et d. (197(i). Scale: 1 cm to 0.01 -4-l.

elongated giant phage the bulk of the protein is involved in an amplification of this region (Aebi et al., 1976). When eight zero points were measured, their positions were found to be consistent with the calculated zero points in diffraction from a solid cylinder of radius 427 A (Table 2). Again, a small standard deviation (CM”&) indicates that the solid cylinder approximation is valid. The striking agreement between the two radii calculated above confIrms the generally accepted model for T4 giant phage structure, i.e. that the elongate cylinder is simply a greatly lengthened version of the cylindrical portion of the wild-type head. In the past, this conclusion had been anticipated from the model for T2 head structure proposed by Branton t Klug (1975), but was difficult to prove since the giant particles flatten when negatively stained (Aebi et al., 1976). The presoak method. whereby phage adsorbed to the carbon film are placed in saturated many1

LETTERS

250

TO

THE

El)ITOR

acetate, washed and negatively stained (Doermann et al., 1973), yielded images in which wild-type and giant particles had the same diameter. Optical diffraction analysis of phage tails showed that the presoak method gives tail spacings identical to standard negative stain methods (unpublished results). Aebi et al. (1976) showed that these particles were not flattened, and gave their radius as 372 A. Our results show that this radius is actually 9% less than the value in solution, creating the dilemma that the visualization conditions which seem optimal in the electron microscope give a smaller particle size. In view of this observation, we thought it valuable to use the above X-ray diffraction results to assess several commonly applied specimen preparation techniques. The data presented in Tables 3 and 4 complement the earlier study reported by Camerini-Otero et al. (1974), who used laser light-scattering to determine the hydrodynamic radii of a number of viruses. TABLE

Comparison

Phage T7

P22

Lambda

T2/T4a

3

of bacteriophage head sizes measured by small-angle X-ray by a variety of electron microscope techniques Radius

(A)

Visualization

technique

301b 229c 229” 235d 273c

X-rayq UrAc positive stain Fixed-sectioned Freeze dried-metal UrAc’

3078 298’ 3038

X-ray Fixed-sectioned UrAc

320” 275’ 280’ 308’ 318’ 321’ 308’

X-ray Fixed-sectioned UrAc positive stain Freeze etch-metal shadowed Air dried-metal shadowed PTA” Ur AC

426” 350’ 370m 373” 390’ 408” 405*

X-ray Fixed-sectioned Freeze etch-metal shadowed UrAc presoakt-UrAc Air dried-metal shadowed PT.4 UrAc

diffraction

Difference

and

(%)”

shadowed

-3 -1

-16 --14 -4 -. 1 -0 -4 .-2%

--15 -9 -9 -4 -5

a Phage: We assume that T2 and T4 bacteriophages have the same head dimensions (Aebi et al., 1976). b-p Radius references are b Ruark, J., Stroud, R. & Rerwer, P. (unpublished work) cited by Serwer (1977) ; O Serwer (1977) ; d Fraser & Williams (1953) ; e Earnshaw et al. (1976) ; r Lenk et ul. (1976) ; g average of values from Botstein et al. (1973) and Earnshaw (1977); h Earnuhaw, Hendrix & King (manuscript in preparation); * Eiserling et al. (1970); J Bayer & Bocharov (1973) ; k this work; ’ Favre et al. (1966); m Branton & Klug (1976); ” Aebi et al. (1976); o average of values from Boy de la Tour & Kellenberger (1966) and Favre et al. (1966); p average of values from Luftig (1968) and Eiserling et al. (1970). small-angle X-ray diffraction radius measured from the q-t Visualization techniques : 9 X-ray, location of zeros of the diffraction patterns (see Tables 1 and 2) ; r UrAc, uranyl acetate nega.tive stain; * PTA, phosphotungstic acid negative stain; t technique described in text. u Difference: (Microscope radius-X-ray radius)/microscope radius.

T4 PHAGE

HEAD

SIZE

251

We found that all of the techniques for preparation of bacteriophages for electron microscopy caused some degree of particle alteration, usually shrinkage. The magnitude of the effect for any given technique was found to vary widely from phage to phage, but among techniques a constant hierarchy of effects was observed. Techniques involving fixation of the DNA by uranyl acetate (Schreil, 1964), thin sectioning, positive staining (i.e. viewing the particle as black against a white background), and presoaking in saturated uranyl acetate, give the smallest sizes. This is not surprising, as the region of packaged DNA is 75:/, (v/v) water (using the DNA packing parameters reported by Earnshaw & Harrison (1977) and a partial specific volume of 0.50 cm3 g-l for the DNA (Tunis& Hearst, 1968)). The headvolume changes required by the linear differences calculated in Table 3 for these t’echniques (30 to 500/,) could easily be accounted for by removal of a portion of this water. Freeze-etching apparently produces variable dimensions, perhaps due to difficulty in choosing views where the particle width could by accurately measured (Branton $ Klug, 1975). Negative staining in its original form (Brenner & Horne, 1959; Huxley & Zubay, 1960) seems to give results which agree best with the X-ray data. Serwer (1977) points out, however, that T7 particles in negative stain apparently have the same volume as positively stained particles (i.e. have undergone the same degree of shrinkage), but have become flattened on the carbon film resulting in an increase in the measured particle radius. It has been clearly shown that T4 giant phage particles are flattened when observed in negative stain (Aebi et al., 1976). Distortions have also been found for other types of viruses when radii determined by negative staining and X-ray diffraction are compared. These values, listed in Table 4, show that RNA-containing viruses tend to flatten in negative stain. Our results indicate that despite the complication described by Serwer (1977), negative staining is still the method of choice for measuring molecular dimensions in the electron microscope. Caution must be applied in interpreting such measurements, however, as the distort’ion effects vary from system to system. TABLE Comparison

of the dimensions

Virus

X-ray

4

of a variety of viruses as determined and X-ray diffraction

radius (A)

Negative

stain radius (a)

by negative

Difference

staining

(%) --

R17 TYMV PM2 Sindbis Reovirus

120” 133c 300e 350% 382’

132b 14oa 300’ 350h 426’

9 5 0 0 10

R17 is a single-stranded RNA-containing bacteriophage. The references are : a average of values from Overby et nl. (1966) and Vesquez et al. (1966) ; b Zipper et ~1. (1973). TYMV, turnip yellow mosaic virus, is a single-stranded RNA-containing plant virus. The references are : c Finch & Klug (1966) ; d Schmidt et al. (1954). PM2 is a double-stranded DNA-containing bacteriophage with a lipid bilayer. The references are : e Silbert et al. (1969); I Harrison et al. (1971a). Sindbis is a single-stranded RNA-containing animal virus with a lipid bilayer. The references are: g von Bonsdorf & Harrison (1975); h Harrison et al. (1971b). Reovirus is a complex double-stranded RNA-containing animal virus. The references are : ’ Luftig et al. (1972); J Harvey. J., Bellamy, R., Schuff, C. & Earnshaw, 1%‘. (unpublished work).

LETTERS

252

TO THE

EDJTOH

The result with the uranyl acetate presoak method raises another important cautionary note. Visualization techniques which opt’imise certain aspects of specimen preservation may adversely affect others. We are very grateful to S. C. Harrison, who gave much valuable advice and in whose laboratory the X-ray experiments were done; to Sue Sheby for help in preparing the mutant phage particles; and to R. A. Crowther and L. A. Amos for their critical reading of the manuscript. The work was supported by grants from the National Science Foundation (GB13117) and the National Institutes of Health to one author (F. A. E.) and by National Institutes of Health grant GM17980 to Jonathan King. Department of Biology Massachusetts Institute of Technology Cambridge, Mass. 02139, U.S.A. Molecular Biology Institute and Department University of California Los Angeles, Calif. 90024, U.S.A. Received 25 January

WILLIAM c!. EARNSHAWt JONATHAN KING

of Bact,eriology

FREDXRICK

A. EISERLING

1978

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Doermann, A. H., Eiserling, F. A. & Boehner, L. (1973). J. Viral. 12, 374-385. Earnshaw, W. C. (1977). Ph.D. thesis, Massachusetts Institute of Technology. Earnshaw, W. C. & Harrison, S. C. (1977). Nature (London), 268, 598--602. Earnshaw, W. C., Casjens, S. & Harrison, S. C. (1976). J. MoZ. Biol. 104, 387-410. Earnshaw, W. C., King, J., Harrison, S. C. & Eiserling, F. A. (1978). Cell, in the press. Eiserling, F. A., Geiduschek, E. P., Epstein, R. H. & Metter, B. (1970). J. ViroZ. 6, 865-876. Favre, R., Boy de la Tour, E., Segre, N. & Kellenberger, E. (1965). J. Ultrastruct. Res. 13, 318-342. Finch, J. T. & Klug, A. (1966). J. Mol. BioZ. 15, 34P364. Franklin, R. E. & Holmes, K. C. (1958). Acta Crystallogr. 11, 213-220. Fraser, D. & Williams, R. C. (1953). J. Bacterial. 65, 167-170. Guinier, A. (1963). X-Ray Diffraction in Crystals, Imperfect Crystals, and Amorphous Solids, W. H. Freeman & Co., San Francisco. Harrison, S. C., Caspar, D. L. D., Camerini-Otero, R. D. & Franklin, R. M. (197 la). Nature New BioZ. 229, 197-201. Harrison, S. C., David, A., Jumblatt, J. & Darnell, J. E. (19716). J. Mol. BioZ. 60,523%528. Huxley, H. E. & Zubay, G. (1960). J. Mol. Biol. 2, 10-18. Lenk, E. V., Casjens, S., Weeks, J. & King, a. (1975). Virology, 68, 182-199. Luftig, R. B. (1968). J. UZtra.struct. Res. 23, 178-181. Luftig, R. B., Kilham, S. S., Hay, A. J., Zweerink, H. Press, London. Zipper, P.. Kmtky, O., Herrmwn. K. & Hahn, T. (197 I ). Eur. J. I