JMB—MS 295 Cust. Ref. No. CAM 355/94 [SGML]

J. Mol. Biol. (1995) 245, 461–466

COMMUNICATION

Three-Dimensional Reconstruction of Mammalian 40 SRibosomal Subunit Embedded in Ice

Suman Srivastava, Adriana Verschoor, Michael RadermacherRobert Grassucci and Joachim Frank

A platform-like structure, which appears equivalent to the platform or lobeWadsworth Center forLaboratories and Research structure of the 30 S subunit of the eubacterial ribosome, is observed in the

reconstruction of the small 40 S ribosomal subunit from images ofNew York State Department ofHealth, Empire State Plaza ice-embedded particles. This cup-shaped structure, 15.0 nm in side lengthP.O. Box 509, Albany and 13.5 nm wide at its rim, extends obliquely upward on the back of theNY 12201-0509, U.S.A. subunit. Other previously characterized features of the 40 S subunit can

readily be identified: the head with its prominent beak structure, the bodywith its two back lobes expressed as relatively small-scale features, and thetwo widely separated feet that comprise the base of the subunit.

Keywords: eukaryotic ribosome; 40 S ribosomal subunit;three-dimensional reconstruction; cryo-electron microscopy

The interaction of the eukaryotic 40 S ribosomalsubunit with initiation factors, tRNA and mRNA,and its association with the 60 S ribosomal subunitto form the 80 S ribosome are essential steps inprotein biosynthesis. Translation is regulated by therate of formation of 40 S initiation complexes.Determination of the three-dimensional (3D) struc-ture of the 40 S subunit therefore is essential tounderstanding the process of protein synthesis.Several models of the eukaryotic 40 S ribosome havebeen published based on specimens either nega-tively stained or shadowed with heavy metal (Lutschet al., 1979; Boublik et al., 1985; Ivanov & Sabatini,1981; Kiselev et al., 1982; Vasiliev et al., 1989).Negative staining and air drying have severaldrawbacks, including possible positive staining ofrRNA exposed at the surface, and flattening anddistortion of the structure. In contrast, in cryo-electron microscopy the specimen remains fullyhydrated in vitreous ice, thus preserving the nativestructure. We report here the first 3D reconstructionfrom 40 S ribosomal subunits in a frozen hydratedpreparation, and we describe several novel featuresof the structure.

40 S ribosomal subunits from rabbit reticulocytes(Figure 1) were examined by cryo-electron mi-croscopy (Lepault et al., 1983; Wagenknecht et al.,1988). At low magnification the 40 S particles areseen to have a sharp beak, distinct back lobes, and

widely separated feet. A total of 1441 particles wereinteractively selected from pairs of tilt and non-tiltmicrographs. The non-tilt images were aligned bycross-correlation methods to a reference particle inlateral-view orientation. A two-dimensional (2D)average, obtained from a homogeneous subset of 253particles, shows the distinctive L-view morphology,in which the head and extended beak, the back lobesand the feet are seen (Figure 2A). However, themorphology of the beak more closely resembles thatof an average from a negative-stain single-carbonspecimen (Figure 2C; Frank et al., 1982) than that ofthe average from a double-carbon preparation(Figure 2B; Verschoor et al., 1989). The 2D averagesfrom ice and the negatively stained single-carbonspecimen are quite similar, except that the latter hasa more pronounced lower back lobe. The averagefrom the negatively stained double-carbon prep-aration appears broader than either of the former twoaverages, which could possibly be due to ‘‘spread-flattening’’ from air drying and the presence of twocarbon layers.

A 3D reconstruction was performed on the cryodata by application of a weighted back-projectionalgorithm (Radermacher et al., 1987) to a set of tiltimages corresponding to the non-tilt images that hadbeen averaged. The reconstruction was refined (seelegend, Figure 2) using alignment techniques basedon Radon transforms (Radermacher, 1994). At 5.5 nmresolution the refined 3D reconstruction computedover 312 tilt images shows the body connected to thehead by a narrow neck (Figure 3A). The 40 S subunitis 29.6 nm long from head to base, 18.0 nm wide (at

Abbreviations used: 3D, three-dimensional;2D, two-dimensional; CTF, contrast transfer function.

0022–2836/95/050461–06 $08.00/0 7 1995 Academic Press Limited

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its maximum), and 16.5 nm thick. A prominentplatform-like structure projecting upward on the‘‘back’’ of the subunit resembles the well character-ized platform of the 30 S small subunit of theeubacterial ribosome. It appears roughly as aninverted cone with a side length of 15 nm and amaximum width (at the rim) of 13.5 nm (Figure 3A,5th image in 1st row to 3rd image in 2nd row). It isshallowly cupped; the P-site (Gorinicki et al., 1984;Oakes et al., 1987) is presumed to be located in thiscup. In the following we describe the principal

features of the reconstruction, as they are seen indifferent rotations around its long axis. We compareit with a previous reconstruction computed bysimilar methods from a negatively stained specimen(Verschoor et al., 1989).

In 0° to 80° rotations (1st to 5th images in 1st rowof Figures 3A and B) the reconstructions from bothice and negative stain are more or less cylindrical andresemble each other overall, although minordifferences in the feet and the width of the bodyexist. The feet are more widely separated in the ice

Figure 1. Typical pair of 0° and 50° fields of 40 S ribosomal subunits in ice. Arrowheads indicate 40 S subunits in typicalL lateral view. The scale bar represents 50 nm. The 40 S ribosomal subunits were isolated from reticulocyte-rich wholeblood from rabbit (Hardesty et al., 1971) and diluted in buffer containing 20 mM Tris-HCl (pH 7.5), 75 mM KCl, 2 mMMgCl2 to a concentration of 0.45 A260/ml for cryo-electron microscopy (Lepault et al., 1983; Dubochet et al., 1988;Wagenknecht et al., 1988). Cryo electron microscopy was done on a Philips EM 420 equipped with low-dose kit, GATANcryostage, and cryo-transfer device. Each specimen field was photographed twice (magnification 49,000×): the first image,taken at a tilt of 50°, has a midfield defocus of 1.5 mm; the second, untilted image was taken at 2.0 mm defocus. Eachexposure corresponds to an electron dose of about 1000 e−/nm2. Fourteen pairs of 0° and 50° micrographs were analyzedaccording to SECReT, the random conical reconstruction scheme (Radermacher et al., 1987, 1992). All of the programs usedare contained within the SPIDER image processing system (Frank et al., 1981a). A particle in L lateral-view orientationwas chosen initially as a reference for alignment of the first set of particles (N = 50). A new reference was created fromthe total average of aligned images in each cycle of the refinement and then used to refine the alignment of the particlesin the next cycle of the alignment. Comparison of 2 half-averages from a subset of 1350 aligned particles gave a reproducibleresolution of 3.2 nm (measured by the differential phase residual, DPR; Frank et al., 1981a). Correspondence analysis(Frank & van Heel, 1982), a form of multivariate statistical analysis, and hierarchical ascendant classification (Frank, 1990)were applied to the above subset in an attempt to obtain a homogeneous subset of particles. Non-linear mapping wasapplied, using the first 6 factors from the correspondence analysis; a graphic display showed a good sorting as judgedby local averaging across the map (Radermacher & Frank, 1985). Similarly, the clustering produced 2 large-membershipclusters apparently composed of particles with good L-view morphology. However, inspection of the individual imagescomprising the clusters revealed a significant proportion of ‘‘contaminant’’ images that deviated significantly from theappearance of the cluster averages. These images had to be discarded by a visual sorting. Subsequent investigation of thisproblem suggests that it may be minimized by the specification in the correspondence analysis and classification of a verylarge number of eigenvectors or factors (say, N/2 for N images, if sufficient memory is available; Pawel Penczek, personalcommunication). Finally, a homogeneous subset of 253 particles was obtained.

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Figure 2. A, A 2D average calculated over the set of 253aligned images of 40 S particles embedded in ice, low-passfiltered to 2.8 nm resolution, showing characteristic L-viewmorphology. B, Average of aligned L-view particles fromnegatively stained (uranyl acetate) double carbon prep-aration (reproduced from Verschoor et al., 1989). C, Amatching average of 40 S particles from a single carbonregion of a negatively stained preparation (reproducedfrom Frank et al., 1982). b, beak; h, head; f, feet; bl, backlobes. Scale bar represents 10 nm.

The tilt images corresponding to the images averaged inA were centered and labeled by their azimuthal angles inthe random-conical tilt geometry. 3D reconstruction wasperformed on these 253 particles by a weightedback-projection algorithm (Radermacher et al., 1987). Theresolution of the first reconstruction was calculated to be6.5 nm by the DPR method.

A refinement of the reconstruction was obtained usingalignment techniques based on Radon transforms(Radermacher, 1994). The reconstruction obtained withSECReT was Radon-transformed and used as a reference.All images extracted from the tilt micrographs wereRadon-transformed and cross-correlated with the 3Dreference. The orientation search was carried out with anincrement of 12° in all 3 Euler angles and a translationradius of 3 pixels. The resulting cross-correlation functionswere searched for the highest peaks, and only thoseorientations were accepted where the highest peak was atleast one standard deviation higher than the secondhighest peak. This criterion led to the selection of 312images out of the original 1441. To a large degree this imageset contained the images used for the SECReTreconstruction, yet approximately 50 images from therandom conical set did not meet the above criterion. The312 images were used for a new reconstruction, which wasused in turn as reference for a second refinement step ofthe alignment within a range of 214° and 2° increments.A second iteration of the alignment was done with a refinedreference and 21° within a 28° range. In both refinementsteps a translational alignment was done separately fromthe orientation search. The optimally aligned image set wasused for a final refined 3D reconstruction. The resolutionof this final reconstruction was assessed to be 5.5 nmthrough comparison of reconstructions from 2 indepen-dent subsets of the image set, via the DPR calculation overspherical shells in the 3D Fourier transforms. The final 3Dvolume was low-pass filtered to this value using aGaussian filter with a fall-off parameter of (1/5.5) nm−1.The results were displayed using the surface shadingtechnique described by Radermacher & Frank (1984).

other, causing the view to resemble a view of theeubacterial 30 S model proposed by Shatsky et al.(1991). A shallow notch marks the merging of theplatform with the body (Gorinicki et al., 1984). Asmall ledge (sl) as seen in the eubacterial 30 S subunitis present on the ‘‘front’’ of the body, opposite to theplatform (Stoffler & Stoffler-Meilicke, 1984).

In the views at 100 to 140° the structure appearsquite different from the negative-stain reconstruc-tion. The body of the subunit, which was roughlycylindrical in negative stain (Figure 3B, 1st image in2nd row), is strongly conical and appears to bedivided into two unequal parts in ice, owing to acentral linear feature. With further rotation (es-pecially, 160 to 180° views) this linear feature resolvesinto a central concavity of the body, in strikingcontrast to the convex appearance of the body innegative stain. In fact, the whole body-feetorganization in ice appears as flat and sheet-like, withthe ‘‘edges’’ curled to form the lower back lobe, theback foot, the small ledge, etc. We cannot completelyexclude as an explanation possible edge-enhance-ment artifacts from the contrast transfer function(CTF) of the cryo micrographs (relatively lowdensities are seen in the central part of the subunitin the 2D average; see Figure 2A); however, theimplicit high-pass filtration due to the CTF has beenpartially offset by use of a Gaussian low-pass filter.

The distinctive body structures of the 40 S smallsubunit, the back lobes (bl), can be seen clearly in the180 to 220° views (Figure 3A, 4th to 6th images in 2ndrow). In a comparison with the classical L view in thenegative-stain reconstruction (180° view; Figure 3B,4th image in 2nd row) the upper back lobe appearsreduced in size. However, the lower lobe at the baseof the platform is pronounced, and both lobes appearto be suprimposed on the underlying platformstructure. In the negative-stain reconstruction, thereis no differentiation between upper lobe andplatform; the lower lobe is again distinct. In ice, thehead has a well defined crest (Verschoor & Frank,1990) extending orthogonally to the trend of thebeak, whereas in negative stain the crest was almostcoplanar with the beak. Overall, the base of thesubunit is wider in ice, owing to the separation ofthe feet. In contrast to the broad, sheet-like back foot,the front foot is conical in ice.

The comparison in Figure 3A and B points up aninteresting discrepancy, which may be seen from the2D averages in Figure 2A and C. If we consider thatFigure 2C represents the classical L-view projectedmorphology, it is clear that the averaged view inFigure 2A is not precisely the same. To wit, the feetare closer together, and the upper back lobeprotrudes further than the lower. Thus, in the cryopreparation, the preferred orientation is not preciselythe L-lateral orientation that was characterized in1981 (Frank et al., 1981b). The amount of rotation ofthe averaged view away from the lateral view wasfound to be about 20° (see legend, Figure 3). Thus, theviews of the rotated structure shown in Figure 3Athat actually correspond to the 2D average are thoselabelled 340° and 160°. This departure from a strict

reconstruction than in the negative stain one, givinga broader appearance to the particle.

A surprising result was that, at a rotation of about120° (Figure 3A, 1st image in 2nd row), the 40 Ssubunit resembles the eubacterial 30 S subunit in its‘‘left featured’’ orientation (Verschoor et al., 1984). Inthis orientation the back lobes of the 40 S subunit aredirectly facing the viewer and the feet overlap each

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(A)

(B)

Figure 3. Comparison of surface views of (A) the refined reconstruction in ice and (B) a previous negative stainreconstruction (Verschoor et al., 1989) shown at 5.5 nm resolution. The negative stain reconstruction was originally obtainedat 3.85 nm resolution, but was further low-pass filtered for this comparison. The matching of the surface views in ice andnegative stain was initially done visually, but a 20° rotational discrepancy was confirmed by aligning the 0° ice projectionto the negative-stain 3D reconstruction using Radon transforms (see legend to Figure 2). To bring the new cryoreconstruction into register with the negative stain structure, a rotation of 20° around the long axis has been applied tothe cryo volume before computation of the surface views. Thus, the view marked 160° was originally the 180° view, orthe view showing the face of the particle that adsorbs to the carbon film in the cryo preparation (marked by an asteriskin A). Surfaces shown with an axial rotational increment of 20°. Marked are b, beak; c, crest; f, feet; h, head; n, neck; p,platform; bl, back lobes; sn, small notch; sl, small ledge; L, L view; R, R view. Scale bar represents 15 nm.

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view orientation in the cryo preparation isinteresting, given the stability and generallyoverwhelming preponderance of this view innegative-stain preparations.

In the ice reconstruction the 40 S subunitpreferentially adsorbs to the carbon in an orientationapproximately 20° away from the classical L-vieworientation in negative stain. The surface of thesubunit that faces the carbon has the crest, backlobes and the small ledge as protruding features(Figure 3A, 4th image in second row). The pref-erence for this orientation is thus clearly not due toany maximization of surface contact; charge inter-actions are more likely to be involved. The stabilityof this range of orientations, if not the precise L-lateralorientation, in different preparations is remarkable.

The views at 220 to 240° are comparable for the tworeconstructions, except that while in ice the back footis separated from the front foot, in negative stain theyoverlapped each other. Further rotation (280 to 340°views, 3rd to 6th images in 3rd row) brings theplatform to the right side of the subunit in ice,showing an appearance quite different from that innegative stain. The platform, whose rim extends upto the level of the beak, merges at its base with thesubunit body at a sharp angle, marked by a smallnotch (sn). The neck appears as a constricted featureseparating the platform and the upper body from thehead. In contrast, the neck in negative stain wasformed by two apparent connections of the head tothe body, to either side of a large central hole. Becausethese two morphologies would appear to representessentially opposite mass distributions, we suggestthat the central hole in the neck region of thenegative-stain structure may reflect positive stainingof the near-surface 18 S rRNA. The upper body in iceis wider than in negative stain owing to the extensionof the platform. A small lobe below the beakprotrudes upward on the side of the subunit oppositeto the platform. In the characteristic R view (definedto be about 160° from the L view; i.e. the 340° view)the only notable difference between the ice andnegative stain reconstructions is in the neckconnection between head and body.

If we examine the view 100° from or roughlyorthogonal to the L view (Figure 3A and B, 5th imagein 1st row) we see that in the negative-stainreconstruction the form of the 40 S subunit appearedroughly cylindrical, with no features projecting. Inthe cryo reconstruction, however, the platformprojects as a diagonal feature with respect to the longaxis of the subunit. If we consider that in thenegatively stained specimen the presence of thecarbon layers on either side may have compressedthese features to a more coplanar organization, wepossibly account for the lack of the stronglyprotruding platform structure that we see in the newcryo reconstruction.

A model proposed by Ivanov & Sabatini (1981)based on critical point drying and metal shadowingshowed a platform-like structure most visible intwo typical views (45° and 225° projections). Thehandedness of this model was derived from

observing the changes caused by tilting the specimenand from the surface features of the subunitsrevealed by metal shadowing. The frontal view (45°)of the model showed a platform-like structureprojecting upward from the right-hand side of thecentral part of the subunit. However, this model didnot show a beak, back lobes, or feet. A ‘‘right-handed’’ model of the 40 S subunit was published byVasiliev et al. (1989). Besides the characteristic beakand feet features, this model showed the body witha hump. The hump consisted of two lobes, the upperlobe forming a protuberance on one of the lateralsides of the subunit revealed only at certainorientations relative to direction of shadowing. Thismodel was similar to that proposed by Lutsch et al.(1979) but had the opposite handedness. Thus,several of the visually derived models anticipated theprominence of the platform feature that is seen in ournew cryo reconstruction.

This morphology of the upper body brings intoquestion the assumption in the literature based onnegatively stained specimens (e.g., Lake et al., 1985)that the two back lobes of the 40 S subunit representa simple bifurcation of the platform structure of the30 S subunit of the eubacterial ribosome. The backlobes in the new 40 S structure appear as relativelyminor features distinct from, and in fact super-imposed on, the large platform structure. In anexperiment where a 236-base yeast 18 S RNAexpansion sequence was inserted into the 16 S RNAin the platform region of 30 S subunits, electronmicroscopy showed an additional structure to theplatform (Oakes et al., 1990). Conceivably, the lobesare the morphological expression of the majorinsertions (relative to the 16 S RNA sequence of the30 S ribosomal subunit) in the 18 S RNA of the 40 Ssubunit.

Acknowledgements

Supported by NIH GM29169 (to J.F.). We acknowledgePawel Penczek for assistance and discussion at earlierstages of this work.

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Edited by M. F. Moody

(Received 2nd August 1994; accepted 26 October 1994)