J. Mol. Biol. (1975) 93, 159-165

Electron Microscopy of Thin Protein Crystal Sections R. LtlNoEnt, CH. POPPE, H. J. SCHRAMM AND W. HOPPE Max-Planck-In&U fiir Biochemie Abteilung fiir Strwkturjorschung I, 8033 Martinsried West Germany

bei Miin.chen

(Received 15 August 1974) In continuation of sxn earlier publication (Hoppe et aZ.,1968), further experiments are described here on the preparation of thin film sections of embedded protein crystals for investigation by electron microscopy and electron diffraction. Several embedding media were compared, the best being Aquon. Periodicities were observed in electron micrographs as well aa in electron diffraction patterns. In diffraction experiments the best resolution observed was approximately 10 to 11 A.

1. Introduction Some years ago we reported methods for the three-dimensional structure analysis of protein crystals by electron microscopy, based on projection images of thin sections of protein crystals at different orientations (Hoppe et al., 1968), see also the independent and related paper on three-dimensional structure analysis of single macromolecules by De Rosier et al. (1968). The first prerequisite for this method is to prepare the crystals in such a way that they can be cut at given orientations by conventional ultramicrotome techniques; it is further necessary to avoid artifacts introduced by the vacuum in the microscope. Our general procedure consisted of first cross-linking the protein molecules in the crystals, then replacement of the salt solution by pure water, then replacement of the water (in successive steps) by a monomer of a resin, followed by polymerization of the “solvent” to a polymer, and finally cutting the crystals in an ultramicrotome. Preliminary results were reported in the above mentioned paper. This method of preparation disturbed the protein lattice significantly. The remaining X-ray resolution was determined by X-ray diftkactograms. The corresponding electron microscopical resolution (shown by electron diffraction) was found to be reduced still further. This result was partly due to the experimental technique, since the radiation dose necessary for the inspection of the thin sections at high magnifications on the fluorescent screen unavoidably leads to micrographs or diEractograms of specimens that have suffered serious damage by radiation. Today this dil%culty can be partly overcome if image intensifiers are used for inspection and focusing, especially if the final exposure is made on a specimen section which has not been exposed to radiation during the setting procedure. In spite of these d.ifIlculties we have succeeded in improving on our earlier results. t Pment

address: Fa&boobsohule

&burg,

8630 Coburg, Weet Germany. 169

160

R. LANGER

ET AL.

2. Experiments and Results (a) Equipment An LKB Ultrotome 480/A was used for sectioning of the embedded crystals ; a Buerger precession camera for X-ray diffraction; an Elmiscope IA, operated at 80 kV accelerating voltage and provided with an anticontamination device, for electron microscopy and diffraction and an optical bench for preparing diffraction patterns of the electron micrographs (Thon, 1966). (b) MC&&& All embedding materials were purchased from Serva, Heidelberg or Th. Schuchardt, Miinohen. The diimidates were prepared from the corresponding din&riles by the method of McElvain & Schroeder (1949). The din&riles were products of Fluka, Buchs, Switzerland (subericdinitrile) and Eastman Organio Chemicals, Rochester, U.S.A. (3,3’-(tetramethylendioxy)dipropionitrile). All other chemicals were of the highest purity commercially available. In the earlier experiments crystals of myoglobin, chymotrypsin and erythrocruorin were used. As it turned out that erythrocruorin crystals suffer less damage during the dehydration step than the other crystals, all subsequent experiments were performed with this protein. It was isolated, purified and crystallized by the method of Huber et al. (1969).

All steps in the preparation diffraction photographs.

(c) Preparation of the specimens of the protein crystals were monitored

by taking

X-ray

(i) Cross-linking First, the protein crystals were cross-linked with glutaraldehyde to stabilize the lattice. The routine conditions were 0.5% glutaraldehyde in 3.0 M-phosphate buffer, pH 6.5, for 4 h. Cross-linked erythrocruorin was stable in distilled water. For instance after 90 h soaking in water the crystals still showed the usual X-ray diffraction pattern although their life-time in the X-ray beam was reduced from about 120 to 60 h. Cross-linking with 0.03% glutaraldehyde by the method of Labaw & Davies (1971) was not suflloient for erythrocruorin: crystals treated in this way dissolved slowly in water. It is also possible to use diimidoesters for the cross-linking of the protein crystals. For instance dimethyl suberimidate and dimethyl 3,3’-(tetramethylendioxy)dipropioimidate were found to cross-link erythrocruorin crystals very well at pH 7-O or higher. It is necessary to add the diimidates 2 or 3 SUCCeSSiVt3 times. (ii) DehyoWion This step was performed with graded solutions of either acetone or ethanol followed by propylene oxide, or, in the case of water-soluble resins, with aqueous solutions of the monomers. Dehydration with ethanol or acetone gave rise to considerable distortion of the crystals when the resulting water content had been reduced to about 10% or leaa. An initial water content of 70%, instead of 30% aa usually described, was routinely employed but still did not prevent damage during the final steps. The resulting crystal X-ray diffraction patterns generally lacked all reflections of spacings less than 7 to 9 A. A continuous dehydration method suggested by Sitte (1962) was also attempted; however, it gave results no better than the stepwise procedure. If water-miscible resins were used the state of the crystals after dehydration was much better. With monomers of glycol methacrylate, Durcupan R or Aquon, complete dehydration could be accomplished with relatively small changes in the X-ray diffraction patterns. An exception was a mixture of monomeric polyampholytes which destroyed the crystals even when the water content was still as high as 60 to 70%. (iii) Polymerization The polymerization conditions were generally those given in the literature references cited. In all cases some further deterioration oocurred to give the final values of X-ray resolution listed in Table 1.

@ w

Acetone Acetone or ethanol (from 70% water), propylene oxide Monomer Acetone or ethanol (from 70% water), propylene oxide Ethanol

Myoglobin Myoglobin, erytbrooruorin Erythrooruorin Erythrocruorin

Erythrocruorin

t Reimer

(1967) gives a wrong

Monomer

Erythrocruorin

Reimer, 1967 (P. 493)

(8) Aquon

Monomer

Erythrooruorin

Fluke Informationt

(7) Duroupan

@

Erythrocruorin

& Singer,

McLean 1964

composition

Mixture of monomers

Dehydration

Protein crystals

(6) Polyampholytes

@ 4206

1969

Spurr,

(6) ERL

(4) Mare&s

Reimer, 1967 (P. 494) Reimer, 1967 (P. 491)

Reimer, 1967 (P. 492) Reimer, 1967 (P. 496)

Literature

(3) Glycol metha0Qdat.e

(2) Epon @I S/2

(1) Vestopal

Embedding material

1

4r8 4d, 60°C

mixture!

7-8 46, 39V

of the embedding

Destruction dehydration

-

during

No satisfactory hardening 76-9

no spots

12h, 39°C or 12h, 60°C -

Shrinkage, diffraction 8.6

-

Good sectioning properties

Poor infusion of monomer into crystals?

Remarks

17”, 62°C

up to 34, 60°C or 44, 37°C U.V.

a-9

up to 3d, 60°C or 2d, 37”C, U.V.

resolution (4 12

X-ray

36, 60°C

Polymerization

Embedding of cross-linked protein crystals

TABLE

162

R. LANCER

ET

AL.

Clyool methacrylate, whioh had given fuie dehydrated orystals with an X-ray solution of 3.5 A, could not be polymerized without complete destruction of the protein crystal. This was probably due to shrinkage. Moreover it produced insuflicient hardening, a problem common in some degree to all water-soluble embedding materials. In summary we found that Aquon gave the best results. Unfortunately the preparation of Aquon by water extraction of Epon R could not be repeated in our most recent attempts, It appears that the composition of Epon R has been changed by the manufaoturer so that it now contains a lower percentage of watersoluble constituents. To our knowledge commercial sources of Aquon do not exist. The best substitute for Aquon is Durcupan R. (iv) Alignment The embedded crystals were freed from resin with a razor blade, fixed on a glass fibre and aligned by taking X-ray photographs. They were then embedded a second time in the same resin to preserve the alignment, and the polymerization blooks were used for cutting thin sections. (d) Andy&q by electrons (i) Di@z&on It should first be mentioned that electron diffraction spots could not be obtained in every experiment. This may be mainly due to stressing of the crystals during the cutting process and to the rapid radiation damage by the electron beam. The best results were obtained with Aquon-embedded crystals, which also gave the best X-ray resolution. Plate I is a typical X-ray diffraction pattern of such an erythrocruorin crystal before outting. The strong reflections in the central region extend out to about 8 A, the weak ones to about 4 A. Compared with these values the resolution in electron diffmction patterns was reduced to 10 to 11 A or more (see Plate V). The sensitivity to radiation damage varied appreoiably even if the same embedding material was used. While many specimens gave no refleations at all after irradiation for a few seconds, others gave high resolution reflections (out to 10 A) for several minutes. This means that some crystals resisted an electron bombardment of 1 ooulomb/cma or more! (ii) Miorogro&3 The contrast of periodic structural details in electron micrographs of unstained crystals was weak and often the periodicity was oompletely concealed by noise. The light optical diffraotion pattern of the micrographs revealed the periodic part of the image structure (Plate II(a) and (b)). The transfer function (Hoppe, 1970) is well pronounced. Plate III(a) shows that periodicities become more visible in the electron micrographt when some sections of the same Aquon-embedded crystal are stained with uranyl acetate (2% in water, 15 min). To avoid misunderstanding at this point it must be emphasized that when applying negative staining techniques to crystals the resolution limit is determined by the staining material, not by the crystal itself. The orystal surface is not uniform as a whole, but small uniform regions can be recognized. The light diffractogram shows very clearly the transfer function and the “periodicity spots.” In Table 2 the spacings found in the micrographs of the specimen are compared with those of wet crystals and those determined by the electron diffraction pattern of the same specimen (Plate IV). It was cut parallel to its a and c crystal axes, so the spacings correspond to the strong reflections (110) and (TIO) (cf. Plate I). The great difference between the value found in the electron micrograph of the unstained se&ion and the other values may be explained by the fact that this section, unlike the stained one, was stretched over a hole in the supporting carbon foil. Finally, attention is drawn to the second-order reflections in the light optical diffraction pattern of the micrograph of the stained crystal (Plate III(b)). As the electron diffraction pattern of a seation of the same crystal does not show these reflections (Plate IV), this may be due to non-linear effeots in the imaging process in the electron microscope. The negatively stained crystal section is no longer a so-called “weak phase object” and one should take note of this fact when carrying out image reconstructions of such objects. t As is well known thecaontraat mmy be enhanced by underfoousing.

PLATE I. X-ray (MO) plane.

diffraction

pattern

of erytbrocruorin

embedded

twice in Aquon,

showing

[.fa&lg

the

p. 162

PI‘f LTE II. (a) Electron micrograph, near focus, of an unstained trysts tl embedded in Aquon (cut parallel to the n and c axw). pattern of the micrograph. (b) Light optical diffraction

thin section of orythrocrw

k’L4TE 111. (a) Electron micrograph, near focus. of tt dact iuti stained wilh urit11yl a(‘~ cfirystal as in Plate II. (b) Light. optjicel diffraction pattern of tho same micrograph.

PLATE

reflections

IV. Electron diffraction pattern of a thin section, same crystal as in Plate II. The two correspond to a spacing of 24 A (calibration with ammonium sulphate crystals).

I-'LATE V. Electron diffraction pattern 1xt,c,nd to approximately 10 to 11 d.

of a thin section, orientation

not, known,

wcxk reflect

t Mean value determined

wet arystals Aquon embedded orystal Unstsined s8otion Se&ion stanied with uranyl Unstained section

specimen

by light optioal

acetate

dilTraotion

di&aotion

of various

X-ray diffraction Electron miorosoopy Eleotron microscopy Electron d.i&a.otion

x-ray

Method

I III Iv

II

-

l?late no.

parts of the electron

24

1Qt w

27.12 26-6

d/A

et al. (1969)

Remarks

micrograph.

Se&ions of the same Aquon embedded orystsl, out parallel to a; and c-axes

R. Huber

(llO)-8~ings in erythrocruorin cyst& (hexagonal; wet crystals: a = b = 54.24 4; c = 35-53 A (4))

TABLE 2

104

R. LANQER

ET AL.

3. Discussion The experiments described show that the most damaging part of the procedure is the polymerization. In some examples studied it was possible to retain the undistorted crystal structure up to the point of total replacement of the solvent water by the (water-miscible) monomer. This leads to the question whether polymerization might be avoided. One possible method would be freezing of the monomer-containing crystal, and cutting and taking micrographs at very low temperatures (provided that no crystallization of the liquid in the crystal takes place). It is evident that such a scheme would not be restricted to monomers of a resin since any liquid which does not crystallize and which does not destroy the crystal during cooling could be chosen. The choice of the best liquid would depend on the protein. In this connection an interesting new approach may be mentioned. It has been shown that very thin natural protein crystals, examined in hydration cells by electron diffraction, show reflections of the non-distorted lattice (in some cases to a resolution of 2 8) (Matricardi et ccl., 1972). One difficulty in high resolution microscopy with hydration cells is the background caused by diffraction from the vapour in the cells (diffraction from window foils can be avoided by using differentially pumped cells). The use of structure-preserving media with a low vapour pressure at room temperature instead of water leads therefore to advantages. The pressure in the cell can be decreased further by cooling the specimen and liquid container. The same procedure certainly can also be applied with water. But ice crystallization may lead to major difficulties. In another context we have shown that cooling at pressures of 2000 atm produces a modification of ice which does not destroy the crystal lattice (Thomanek et al., 1973), but this technique cannot easily be adopted to electron microscopy. The question whether sectioning should be replaced by the less damaging procedure of preparing very thin crystals cannot be answered generally. One of the experiments reported above (Plate III(a)) shows that the cutting did not lead to a very uniform surface, so natural surfaces oould be more uniform. The accidental orientation of natural crystals is no longer necessarily a limitation. Tilting stages can be used. The shape of the transfer function (needed for the analysis) can be determined either from light diEra&ograms or by computer reconstructions (Hoppe, 1970) from the unavoidable non-periodic part of the crystal (see e.g. Plate II(b)). But on the other hand cutting of macroscopic crystals gives more flexibility for the experiments. Summarizing we can say that the possibility of using water or other structurepreserving liquids leads to a greater overall flexibility of the method.

REFERENCES

De Rosier, D. J. & Klug, A. (1968). N&we

(Lo&m), 217, 130. Hoppe, W., Langer, R., Knesch, G. C Poppe, Ch. (1968). Natutiaenach. 55, 333. Huber, R., Epp, 0. 81 Formanek, H. (1969). Natzcrwissensch. 56, 362-367. Labaw, L. W. & Davies, R. (1971). J. Biol. Chem. 246, 3760-3762. Matriacardi, V. C., More& R. C. & Parsons, D. F. (1972). Science, 177, 268-270. McElvaiu, S. M. & Schroeder, J. P. (1949). J. Amer. Chem. Sot. 71, 40-46. McLean, J. D. t Singer, S. J. (1964). J. Cell. Bid. 20, 518-520. Reimer, L. (1967). EEektronenmikroakopi8c~ Unterauchunga- und Prtiparatiowmethoden, 2nd edit., Springer-Verlag, Heidelberg.

ELECTRON

MICROSCOPY

OF PROTEIN

Sitte, P. (1962). Natutisenach. 49, 402-403. Spurr, A. R. (1969). J. U&a&r. Rec. 26, 31-43. Thon, F. (1966). 2. Nahrforechg. %a, 476-478. Thomanek, U. F., Parak, F., Mossbauuer, R. L., Formanek, (1973). Acta Cryetallogr. sect. A, 29, 263-265.

CRYSTALS

165

H., Schwager, P. & Hoppe, W.

Note added in proof: In the meantime it has been demonstrated (Taylor, K. A. & Glaeser, R. M., Eighth International Congr. on Electron Microecopy, Canberra (1974), vol. II, p. 64; Science, vol. 186, p. 1036 (74)) that even freezing at 1 atm of small crystals of catalase can lead to preparations, which show electron diffraction patterns with a resolution of 3.4 A.