Sepia officinalis hemocyanin: a refined 3D structure from field emission gun cryoelectron microscopy1

Article No. jmbi.1999.3460 available online at http://www.idealibrary.com on

J. Mol. Biol. (2000) 296, 459±472

Sepia officinalis Hemocyanin: A Refined 3D Structure from Field Emission Gun Cryoelectron Microscopy Nicolas Boisset* and Fabrice Mouche Laboratoire des ProteÂines Complexes, Universite FrancËois Rabelais, Campus MeÂdecine 2 bis Boulevard Tonnelle F-37032, Tours Cedex, France

The extracellular respiratory pigment of the cuttle®sh Sepia of®cinalis was observed by cryoelectron microscopy with conventional LaB6 and ®eld emission gun electron sources at 100 and 200 kV, respectively. Each image series was used to compute one 3D reconstruction volume with correction of the contrast transfer function by Wiener ®ltering. A strong boosting of the contrast was corrected by band-pass ®ltering of the ®nal volumes, and a qualitative gain in resolution was observed when using the ®eld emission gun electron microscope. In this volume, a strong sigÊ ÿ1 and some meaningful information is nal is present down to 1/18 A ÿ1 Ê obtained down to 1/12.5 A . The complex is composed of ®ve pairs of polypeptide chains and resembles a hollow cylinder with ®ve wall oblique units and ®ve inner arches. Three types of wall-wall connections termed pillar P1 to P3 are visible in this volume and the four functional units present in the arches are each linked to the wall by two arch-wall connections. The dispositions of the functional units in the arches of Sepia and Octopus hemocyanins share no common feature. # 2000 Academic Press

*Corresponding author

Keywords: hemocyanin; cryoelectron microscopy; three-dimensional reconstruction; ®eld emission gun; contrast transfer function

Introduction The hemocyanin (Hc) of the cuttle®sh Sepia of®cinalis is an extracellular respiratory pigment ( 3.9 MDa) composed of ten polypeptide chains comprising eight domains or functional units (FUs) as demonstrated by SDS-PAGE (Gielens et al., 1983) and MALDI-MS (unpublished data). It shares common features with other cephalopod Hcs such as Octopus do¯eini (Lamy et al., 1986, 1987), Octopus vulgaris (Gielens et al., 1986; Lamy et al., 1993), or Vampyroteuthis infernalis (Mouche et al., 1999). Present address: Nicolas Boisset and Fabrice Mouche, Laboratoire de MineÂralogie Cristallographie Paris, LMCP CNRS UMR 7590, Case courrier 115, Tour 16, 4 Place Jussieu, 75252 Paris Cedex 05, France. Abbreviations used: CTF, contrast transfer function; EM, electron microscope; FEG, ®eld emission gun; FSC, Fourier shell correlation; FU, functional unit; Hc, hemocyanin; SDS-PAGE, sodium dodecylsulfatepolyacrylamide gel electrophoresis; MALDI-MS, matrixassisted laser desorption/ionization, mass spectrometry; WOU, wall oblique unit; cryo-EM, cryoelectron microscopy; SNR, signal-to-noise ratio. E-mail address of the corresponding author: [email protected] 0022-2836/00/020459±14 $35.00/0

However, Octopus Hc comprises seven FUs, while Sepia has eight, an additional one being visible in the cylinder opening (Lambert et al., 1995). A 3D Ê reconstruction of the Sepia Hc computed at 32 A resolution from frozen-hydrated specimens revealed that, as other molluscan Hcs, the molecule has a D5 point-group symmetry and comprises ®ve wall oblique units (WOU) disposed as in a short segment of ®ve-stranded right-handed helix and ®ve arches (Lambert et al., 1995). Since one WOU and one arch account for one-®fth of the whole molecule (i.e. two eight-FU polypeptide chains) and since one arch comprises four FUs, one WOU is composed of 12 FUs. Moreover, the 3D reconstruction volume reported by Lambert et al. (1995) shows that each arch is built from two types of FUs corresponding to Sod or Soe (Loncke et al., 1990), an FU absent from Octopus Hc, and Soh, the polypeptide chain C-terminal FU (Lamy et al., 1998). Here, we computed two 3D reconstructions of Sepia Hc from two series of untilted-specimen cryoelectron microscopy (cryo-EM) images recorded with LaB6 and ®eld emission gun (FEG) electron sources. Since these volumes are compared, we will refer to them from now on as the LaB6 and FEG images or volumes. # 2000 Academic Press

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Results Cryoelectron microscopy For both data sets, images of untilted specimens were recorded at two defocuses. In order to obtain a good resolution on raw images, a ®rst set of images was recorded as close as possible to focus. A second image set was then recorded at a higher defocus, carefully chosen so that the zeros of the contrast transfer function (CTF) would alternate with those of the ®rst image series. In the LaB6 electron microscope, data collection was carried out at an acceleration voltage of 100 kV with defocuses of 0.8 mm and 1.0 mm (Figure 1(a) and (b)). In the FEG electron microscope, the acceleration voltage was 200 kV with defocuses ca 3.4 and 5.4 mm. A typical ®eld recorded at 3.4 mm defocus shows the strong contrast of the particles in Figure 1(c). In cryo-EM ®elds (Figure 1), the cylindrical Hc molecules produce rectangular (white arrowheads) and circular (black arrowheads) views. Some rare intermediate views with oval shapes are also visible in micrographs. The interactions responsible for the orientation of particles within the ice layer remain unexplained, but we observed that under our experimental conditions, thin ice layer and high sample concentration were critical factors producing rectangular-side views. After digitization, 5510 and 6393 particles were interactively selected in the 12 and eight micro-

Re®ned Architecture of Sepia of®cinalis Hemocyanin

graphs recorded with the LaB6 and the FEG electron sources. Despite their high concentration, most particles were suf®ciently scattered to be selected. Aggregating or partially overlapping particles (Figure 1(c), rectangle) were rejected during the interactive selection. Finally, tobacco mosaic virus (TMV) (Figure 1(a) and (c) arrows) was used to determine the magni®cation and the pixel size. Image processing and 3D reconstruction A similar strategy of image processing was applied to the two sets of digitized images. Selected images were submitted to contrast inversion and normalization (Boisset et al., 1993), and were subjected to several cycles of 3D projection alignment (Penczek et al., 1994). The purpose of this procedure was to shift each 2D projection of the particle at the center of the image and to determine its direction of projection required by the 3D reconstruction procedure. In this operation, a reference volume is projected in every direction with an angular increment of 1  . Then, each experimental image is compared by cross-correlation to the whole set of 2D projections, the best match between experimental and reference images providing the direction of projection. The low-resolution 3D reconstruction volume described by Lambert et al. (1995) was used as a ®rst reference (Figure 2, Vref1). Three alignment cycles applied to the whole set of centered images produced a

Figure 1. Cryoelectron microscopy of Sepia of®cinalis Hc. (a) and (b) Untilted-specimen images recorded at 100 kV in a Philips CM12 EM equipped with a LaB6 electron source at (a) 0.8 mm and (b) 1.0 mm defocuses, respectively. (c) Image recorded at 200 kV and 3.4 mm defocus on a JEOL 2010F EM with an FEG as electron source. White and black arrowheads point toward typical rectangular (side) and circular (end-on views), respectively. The circles mark intermediate orientations. Aggregated particles were not selected for image processing (rectangle). Tobacco mosaic virus used for calibration of the magni®cation is also present in the EM ®elds (black arrows).

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the two selected values (0.8 and 1.0 mm for LaB6 and 3.4 and 5.4 (mm for FEG sources), small defocus variations inducing noticeable CTF changes in the high spatial frequencies were detected. Therefore, Wiener ®ltering was applied to the n primary volumes with their speci®c ®lters, producing a unique CTF corrected volume (Figure 2, VCTF1). As discussed below, this volume was subjected to a band-pass ®ltering to better show its ®ner structural features (Figure 2, VCTF2). Estimation of the CTF and correction by Wiener filtering

Figure 2. Strategy of image processing. The computation of a volume is represented by a cube. Vref1 is the low-resolution reference volume (Lambert et al., 1995). It was used as reference volume for three cycles of 3D projection alignment of the whole image set. The aligned images produced a volume Vref2 which was used as a new reference for ®ve additional cycles of 3D projection alignment. Image subsets originating from given micrographs and corresponding to different defocuses were used to compute speci®c volumes VM1 to VMn. These volumes were simultaneously subjected to Wiener ®ltering and produced a single CTF corrected volume VCTF1. A band-pass ®ltering was applied to VCTF1 and produced a ®nal volume VCTF2 with enhanced visibility of small structural features.

secondary reference volume (Figure 2, Vref2). Five additional cycles of 3D projection alignment with Vref2 as reference were used to re®ne the centering and the Eulerian angles. Then, the set of centered images with re®ned Eulerian angles was split into subsets, each corresponding to one micrograph with a particular defocus and the corresponding primary 3D reconstruction volumes were computed (Figure 2, VM1 ÿ VMn). Indeed, although the micrographs were recorded at defocuses close to

The CTF parameters were determined on the one-dimensional power spectrum pro®les of the micrographs (Zhu et al., 1997). Although the 12 LaB6 and eight FEG micrographs were studied independently and provided their own CTF curve, it would be redundant to show all of them, since they correspond mainly to two defocuses. Therefore, in Figure 3(a) and (b) we plot the CTFs corresponding to the two average defocuses. For LaB6 microscope images (Figure 3(a)), the small defocus values of 0.8 mm and 1.0 mm produce very few oscillations in the CTF with ®rst zeros located at Ê ÿ1 spatial frequencies, Ê ÿ1 and 1/20 A 1/17 A respectively. Moreover, the two curves alternate along the spatial frequencies, so that when one curve reaches a value of zero, the other reaches a maximal amplitude (either negative or positive). The only region where the two curves cross the zero line next to each other is in the range of 1/ Ê ÿ1 (Figure 3(a), circle). For the FEG images, 12 A (Figure 3(b)), the high defocus values (3.4 and 5.4 mm) induce more oscillations of the CTF. Hence, the two curves cannot perfectly alternate at all spatial frequencies. One observes at least six critical zones where both CTF curves are almost simultaneously close to zero (Figure 3, circles). Therefore, one can expect local drops of the signal at these spatial frequencies. For LaB6 and FEG data sets, 12 and eight volumes were computed, respectively. Assuming an even angular distribution of the projections, the signal-to-noise ratio (SNR) of the 3D reconstruction volumes were estimated as follows (P.A. Penczek, personal communication): r SN SNR ˆ 2R where S is the data redundancy related to the point-group symmetry (here D5 gives a tenfold redundancy), N is the number of 2D projections, and R is the smallest radius (here 28 voxels) containing the reconstructed particle. Considering an average of 580 particles per micrograph, the SNR is close to 10.2. Using this value of SNR, the primary volumes were combined and subjected to Wiener ®ltering as described (Frank & Penczek, 1995).

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Figure 3. CTF correction and resolution limit. (a) and (b) CTF curves deduced from the power spectra of the micrographs according to the method of Zhu et al. (1996). (a) CTF curves corresponding to average defocuses of 0.8 mm and 1.0 mm used in the Philips CM12 EM with a LaB6 electron source at 100 kV. (b) CTF curves corresponding to the average defocuses of 3.4 mm and 5.4 mm applied in the JEOL 2010F EM at 200 kV. (a) and (b) Critical spatial frequencies where both CTF curves are simultaneously close to zero are marked by circles. (c) and (d) FSC curves of the 3D reconstruction volumes after CTF correction (VCTF1) obtained from (c) LaB6 and (d) FEG data. The resolution limit is estimated either using a threshold value of 0.5 (FSC0.5) or when crossing the broken 3s noise curve (FSC3s). (d) The FSC curve shows ®ve local minima in spatial frequencies close to the critical zones of the CTF curves circles in (b).

Estimation of resolution limits The Fourier shell correlation (FSC) criterion (Saxton & Baumeister, 1982; van Heel, 1987) used here, corresponds to the normalized cross-correlation coef®cient between two 3D volumes as a function of spatial frequencies. Two types of threshold values may be chosen as the resolution limit. On the one hand, a threshold cutoff of 0.5 for the FSC curve is commonly used (BoÈttcher et al., 1997; Conway et al., 1997; Malhotra et al., 1998). On the other hand, other groups prefer to de®ne the resolution limit as the crossing point between the experimental FSC curve and the calculated ``3snoise curve'' displaying FSC values equal to three times the expected standard deviation re¯ecting uncorrelated random noise (Orlova et al., 1997). For the sake of convenience, we will refer to these two measures as FSC0.5 and FSC3s, respectively. For the LaB6 volume (Figure 3(c)), since the FSC curve drops with a steep slope, both criteria are in Ê ÿ1 and the good agreement (FSC0.5 is 1/19.6 A ÿ1 Ê FSC3s is 1/18.4 A ). For unknown reasons, after Ê , the FSC reaching a minimum value at 1/18 A curve increases anew at high spatial frequencies. This phenomenon, observed for all the CTF-corrected 3D reconstructions that we have calculated

so far, is independent of the shape and size of the mask imposed to the volumes to remove surrounding noise. For the FEG volume, due to the absence of steep slope in the FSC curve (Figure 3(d)), the values Ê ÿ1 and FSC3s of disagree (FSC0.5 of 1/18.1 A Ê ÿ1). As proposed by Orlova et al. (1998), 1/10.6 A the FSC3s should be restrained to two-thirds of the Ê ÿ1), but this still leaves Nyquist frequency (1/12.4 A a large gap between the two estimations. Moreover, the FSC curve shows several local minima (labeled 1 to 5 in Figure 3(d)) coinciding with the critical zones of the CTF plots in Figure 3(b) (circles 1 to 5). Since in these critical zones the two sets of CTF curves have simultaneously a value close to zero, they correspond to spatial frequencies where the signal is weak. Band-pass filtering of CTF corrected volumes The surface representations of the CTF-corrected volumes VCTF1 from LaB6 and FEG data look disappointing (data not shown). Similar slices extracted from these volumes (Figure 4(a) and (e)) resemble binary images with bright and dark areas corresponding to protein and vitreous ice, respectively. The structural details appear smoothed by a

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fuzzy background noise. The outer cylindrical wall and the inner arches are hardly distinguishable, and local high-density zones are faint. All the features that were visible in the primary volumes before CTF correction are now dif®cult to observe, even when magni®ed (Figure 4(b) and (f)). To rescue the high-resolution information we tried to enhance the contrast in the high spatial frequencies by applying a correction similar to the temperature factor (BoÈttcher et al., 1997), but all attempts in this direction failed and the background noise was enhanced (data not shown). Another approach was to reduce the contrast in the low spatial frequencies by applying a bandpass ®lter (see Discussion) designed to apply a tenfold attenuation of the signal in the low spatial frequencies (asterisk) and a complete low-pass ®ltration beyond the resolution limits estimated with the FSC3s criterion (Figure 4(i)). The ®lters of Figure 4(i) were applied to the LaB6 and FEG CTF-corrected volumes VCTF1 (Figure 4(a) and (e)) and produced the volumes VCTF2 (Figure 4(c) and (g)). In these volumes, the outer wall and the inner arches became easily recognizable. When comparing the magni®ed areas of the VCTF2 (Figure 4(d) and (h)) and VCTF1 volumes (Figure 4(b) and (f)), small high density zones (P1P3) were hardly visible, but clearly appear after band-pass ®ltering (Figure 4(d) and (h), arrows). Among these spots, P2 appears as a faint arch-wall connection in the LaB6 volume (Figure 4(d)), but is more resolved in the FEG volume (Figure 4(h)) and produces an S-shaped structure. Despite this small difference concerning P2, the redundancy of these elements indicates that they correspond to authentic structural features rather than band-pass ®ltering artifacts. The only noticeable side-effect of the band-pass ®ltering is the increase of noise around the particles. This is particularly visible in the FEG volume (Figure 4(g)), outside the particle and within its central cavity. However, the density values of this noise (ca 0.35) remain much smaller than those related to the reconstructed particle (from 0.55 and up to 1.0) and they can be eliminated by thresholding, as shown in Figure 7(g) and (h) where all the densities lower than 0.35 are set to zero. Clearly, Figure 4(c) and (g), provide the same structural information but in Figure 7(g) and (h), the noise is replaced by a dark background. FU nomenclature of the LaB6 volume The LaB6 volume VCTF2 resulting from the Wiener and the band-pass ®ltering was subjected to surface rendering using a threshold density leaving apparently 100 % of the expected molecular volume. The volume observed along its 5-fold axis appears as a circular wall with ®ve arches in its central cavity (Figure 5(a)). In an intermediate orientation (Figure 5(b)), ®ve WOUs disposed as in a ®ve-stranded right-handed helix are separated by large grooves (broken lines in Figure 5(c)-(f)). Since the symmetry order is odd, when the molecule is

observed along a 2-fold axis, two different rectangular views are obtained depending on the observation direction. To simplify the interpretation of these sometimes confusing views we use two arti®ces. First, starting from the center of the particle, each half 2-fold axis is labeled with a white or a black ellipse when passing through an arch (Figure 5(c) and (d)) or between (Figure 5(e) and (f)) two arches, respectively. Second, we cut the molecule by a plane perpendicular to the 2-fold axis and containing the 5-fold axis and we display separately their external (Figure 5(c) and (e)) and luminal (Figure 5(d) and (f)) surfaces. As shown in Figure 5(c) and (d), a half 2-fold axis labeled by a white ellipse always passes through the middle of a large groove (broken line) and a half 2-fold axis labeled by a black ellipse passes between large grooves (Figure 5(e) and (f)). This disposition indicates that each arch bridges neighboring oblique wall units over a large groove. Figure 5 also shows that the large groove is composed of a pair of large holes through the wall, related by the 2-fold symmetry, (Figure 5(c), open circle). As previously observed by Lambert et al. (1995), within the WOU a line of smaller holes draws a smaller groove parallel with the large groove (Figure 5(e), broken ellipse). The location of the eight FUs Soa to Soh within the polypeptide chain has been studied by Lamy et al. (1998). Their nomenclature is completely independent of the FU sequence in the polypeptide chain and is shown in Figure 5(e) and (f). Brie¯y, the FUs located in the wall are termed W1 to W6 and those located in the arches A1 and A2. Since 12 FUs are present within each WOU, those related by the 2-fold symmetry have the same names. However, to arti®cially distinguish identical FUs related by the symmetry, those located in the right part of the WOU are suf®xed with a prime (e.g. W1 and W10 ). We also use interchangeably the terms FUs and high-density zones to describe the spheroid masses composing the 3D reconstruction volume. New features visible in the FEG volume The volume VCTF2 resulting from the Wiener and band-pass ®ltering shows a strong resemblance to the LaB6 volume. However, in surface representations displaying 100 % of the expected molecular volume, some structural details are better de®ned or appear slightly different (Figure 6). Selected slices extracted at similar levels in the two volumes provide a way of comparison independent of threshold density (Figure 7). Pillars in the cylindrical wall In Figure 6, the reconstructed particle is sharply de®ned and many holes and bridges are visible. Such an abundance of details makes the volume dif®cult to interpret. Seen from the outside, homologous FEG (Figure 6(a)) and LaB6 volumes

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Figure 4 (legend opposite)

Re®ned Architecture of Sepia of®cinalis Hemocyanin

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Figure 5. Surface representations of the LaB6 3D reconstruction volume VCTF2 of Sepia of®cinalis Hc, at a threshold displaying 100 % of the expected molecular volume. (a) Whole volume in the circular end-on view orientation. The half 2-fold axes passing through and between arches are labeled by open and ®lled ellipses, respectively. (b) Whole volume in an intermediate orientation corresponding to a 45  rotation around the X axis. Half-volumes viewed along the half 2-fold axes passing (c), (d) through and (e), (f), between arches, seen from (c), (e) outside and (d), (f) inside the volume. The broken lines follow the large grooves between the WOUs. Within the wall, the circles in (c) and (d) label the deep cavities located along the large groove. Smaller cavities appear along the small groove (broken ellipsoid in (e)). (d) and (f) Arches are marked by broken parallelograms. (d) and (f) The two FUs located along the small diagonal are labeled A2 and A20 , and the two other two FUs are labeled A1 and A10 . (e) The FUs of the WOU are termed W1 to W6 and W10 to W60 according to Lamy et al. (1998).

(Figure 6(e)) share many features. The main difference is the presence in the FEG volume of several bridges within the wall, that partially ®ll the cavities forming the large and small grooves. Since these bridges are approximately parallel to the cylinder axis and seem to support the architecture of the wall, we term them ``pillars''.

Within the large groove holes (Figure 6(a), open circle), two pillars termed P1 and P2 are visible. Pillar P1 (Figure 6(a)) is located near the external surface of the wall, while P2 is closer to the lumen (Figure 6(a), broken arrow). Therefore, pillar P1 connects FUs W60 and W3 (or W6 and W30 ) of two neighboring WOUs. Similarly pillar P2 bridges FUs

Ê above the central plane of Figure 4. Band-pass ®ltering. A slice perpendicular to the 5-fold axis was extracted 33 A each volume. (a)-(d) LaB6 volumes. (e)-(h) FEG volumes. (a) and (e) Volume VCTF1 before band-pass ®ltering and (c) and (g) VCTF2 after band-pass ®ltering. (b), (d), (f) and (h) Images correspond to magni®ed areas in (a), (c), (e) and (g), respectively. (d) and (h) Arrows point to high-density zones, termed pillars P1 to P3. (i) Plots of the band-pass ®lters applied to the LaB6 and FEG volumes after CTF correction. The tenfold Gaussian attenuation of the contrast in the low spatial frequencies is marked with an asterisk. The cut-off in the high spatial frequencies correspond to the Fermi low-pass ®lter Ê for LaB6 and 1/10.6 A Ê for FEG volumes, respectively). set to the FSC3s resolution limits (1/18.4 A

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Figure 6. Surface representations of the FEG 3D reconstruction volume VCTF2 of Sepia of®cinalis Hc, at a threshold displaying 100 % of the expected molecular volume. Two types of half-volumes seen from (a) outside and (b) inside. Filled and open ellipses mark the 2-fold axis and follow the same convention as in Figure 5. Deep grooves are marked by broken lines. (a) Two pillars P1 (full arrow) and P2 (broken arrow) are visible within a large opening in the wall (circle). A third pillar P3 is present in a cavity of the small groove (broken ellipsoid). (b) One arch (broken parallelogram) comprises FUs A1, A2, A10 , and A20 . Asterisks mark a new bridge linking the left and right sides of the arch. The arch-wall connections A1-W2 and A10 -W20 are marked by black dots. (c) Enlarged portion of (b) with the section plan containing pillar P2 and connections A20 -W3 and A20 -W50 (arrows).

Re®ned Architecture of Sepia of®cinalis Hemocyanin

Figure 7. Selected slices extracted from (a), (c), (e), (g), (i) and (l) LaB6 and (b), (d), (f), (h), (j) and (l) FEG volumes. (a) and (b) Levels of the cutting planes are represented by horizontal lines between the top (T) and the Ê, center (C) of the volumes. Slices were cut (c), (d) 66 A Ê , (g), (h) 33 A Ê and (i), (j) 16.5 A Ê above the (e), (f) 49.6 A (k), (l) central plane. (d) and (f) Arch-wall connections A1-W1 and A1-W2 are marked by arrows. (h) and (j) Pillar P2 and arch-wall connections A20 -W3 and A20 -W50 are marked by arrows. (h) Open circles surround highdensity spots corresponding to pillars P1 and P3. The space bar represents 40 nm.

W50 and W3 (W5 and W30 ) of two neighboring WOUs. These pillars also produce circular highÊ above the density zones in the slice located 33 A central plane (Figure 4(h) and (g), white circles). Similarly, the small groove holes (Figure 6(a), broken ellipsoid) contain one pillar termed P3 located near the inner side of the cylinder wall

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(Figures 4(h) and 7(h)) linking FUs W1 and W40 belonging to the same WOU. The presence of pillars P2 and P3 in the LaB6 volume after (Figure 4(d)), and before CTF correction (data not shown), supports the interpretation that they do not result from band-pass ®ltering but are genuine structures. Arch-arch connections When observed from the 5-fold axis, the LaB6 (Figure 5(d)) and FEG (Figure 6(b)) volumes look somewhat different. In the LaB6 volume the FUs of the arches are poorly de®ned and appear as oblong masses. Conversely, in the FEG volumes many small features and new connections between the subunits composing the arch are visible. Thus, in Figure 6(b) (asterisks) small connections link FUs A20 and A1 (A2 and A10 ). Similarly, one connection occurs between FUs A10 and A20 (A1 and A2). In the selected slices extracted from the LaB6 volume, arches appear as an almost continuous blurred central annulus (Figure 7(e), (g), (i) and (k)). Conversely, in the FEG volume (Figure 7(f), (h), (j) and (l)) the arches look separated and in the central slice, arches appear as pairs of hollow masses corresponding to FUs A2 and A20 (Figure 7(l), arrow). Arch-wall connections In the LaB6 volume, arch-wall connections are not visible in surface representations (Figure 5) and look fuzzy in selected slices (Figure 7(c), (e), (g), (i) and (k)). Conversely, the FEG volume provides a clear signal in these areas. Thus, in the section Ê above the central plane of the mollocated 49.6 A ecule FUs A1 are connected to the wall by two connections (Figure 7(f), open arrows). One of these connections links the arch FU A1 to the wall FU W1 and is termed for this reason the A1-W1 connection. Similarly, the second connection is designated as A1-W2. Connection A1-W1, slightly more distant from the central plane than A1-W2 is partially visible in the top slice (Figure 7(d), arrow). In the surface representation of Figure 6(b), the section plane passes exactly through connections A1W1 (black dots at the upper-right and lower-left corners of the half-volume). These data demonstrate that FUs A1 (or A10 ) are in contact with the upper (or lower) tier of the wall, but never with the central tier. Another characteristic of A1 and A10 is that their connections to the wall are located far from the large grooves (Figure 5(f), broken lines). Therefore, each A1 FU is linked to a single WOU. When seen from the 5-fold axis, the FUs A20 (or A2) stretch over the large grooves (Figure 6(b), broken line) and are linked to the upper (or lower) and central tiers of the wall by two connections designated as A20 -W50 and A20 -W3 (Figure 6(c)). In the small portion of the section plane enlarged in Figure 6(c) one observes the relationship between connections A20 -W50 and A20 -W3 and pillar P2.

This intricate architecture gains another level of complexity when observed in the slices of Figure 7. Indeed, connections A20 -W50 and A20 -W3 both converge to pillar P2, but A20 -W50 is bent to the right (Figure 7(h), arrow), while A20 -W3 is bent to the left (Figure 7(j), arrow). Such redundancy between pillar P2 and arch-wall connections A20 -W50 and A20 -W3 produces a strong anchorage between two neighboring WOUs.

Discussion Estimation of the resolution limit For 3D reconstructions of single particles the estimation of the resolution limit has always been a matter of discussion. As shown in the results section (Figure 3(d)), depending on the selected criterion the estimated resolution limit can almost double when the slope of the FSC curve is weak. The comparative 3D reconstructions of the same particle from LaB6 and FEG images give a unique opportunity to check the FSC0.5 and FSC3s resolution criteria from a practical standpoint. According to the FSC0.5 criterion, the resolution limits of the LaB6 and FEG volumes correspond to 1/ Ê ÿ1, respectively (Figure 3(c) Ê ÿ1 and 1/18.1 A 19.6 A and (d)). With such a small difference of resolution, the two volumes should be identical if one considers that below an FSC threshold value of 0.5 the SNR is too weak to be signi®cant (Penczek, 1998). As visible in Figure 7, from a qualitative point of view the FEG volume shows more details than the LaB6 volume. Therefore, one must recognize that despite the low SNR, information is present Ê ÿ1 in the FEG volume. The resolbeyond 1/18.1 A Ê ÿ1 found with the FSC3s resution limit of 1/10.6 A olution criterion seems optimistic. In a similar situation Orlova et al. (1997) arbitrarily decided to cut down the resolution limit to two-thirds of the Ê ÿ1 in present conNyquist frequency (1/12.4 A ditions) to take into account interpolation errors and loss of information through the processing chain. The question is how can one rely on this new rule-of-thumb limit? To address this problem, we looked back to the experimental data concerning the micrographs and their corresponding primary volumes. When considering the rotational averages of a diffractogram computed from one of the eight FEG micrographs, the Thon rings are visible up to Ê ÿ1 (Figure 8(a), a spatial frequency of 1/10.5 A arrow). The CTF plot corresponding to the experimental pro®le of Figure 8(a) is given in Figure 8(b). In these Figures the minima of the experimental pro®le correspond to spatial frequencies where the CTF value is null and where all signal is lost. Such local loss of information is found also when considering the FSC plot of the primary volume computed from images extracted from the same micrograph. In this plot (Figure 8(c), circles), the FSC curve has six local minima in good agreement

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Re®ned Architecture of Sepia of®cinalis Hemocyanin

Hence, for this primary volume, the signal that one can expect to retrieve by CTF correction is truncated in six spatial frequencies (circles) and is signi®cantly higher than the 3s noise curve up to 1/ Ê ÿ1. The FSC curves of the eight primary 12.5 A volumes were similarly checked and all of them converged toward the same limit. Thus, our interpretation is that the FEG volume possesses Ê ÿ1, signal up to a spatial frequency of 1/12.5 A which is in agreement with the practical limit proposed by Orlova et al. (1997). Band-pass filtering of the CTF corrected volumes

Figure 8. CTF and resolution limit of an FEG primary volume. (a) Experimental (E) and modeled (M) pro®les deduced from the average power spectrum of a selected micrograph, according to the method described by Zhu et al. (1996). (b) Corresponding deduced CTF curve. (c) FSC curve of the primary 3D reconstruction volume VM1 computed from particle images selected in this micrograph. The circles show the good agreement of the zeros of CTF with the minima of the FSC curve while asterisks mark a discrepancy in the low spatial frequencies. In between minima, the FSC curve climbs back above the 3s noise curve up to a spatial frequency of Ê (®lled arrow). 1/12.5 A

with the zeros of the CTF curve (Figure 8(b), circles). After each local minimum the FSC curve increases above the 3s noise curve, but beyond the sixth one (Figure 8(c), arrow) the FSC, although still oscillating, remains below the noise curve.

Why did the Wiener ®ltering boost the contrast in the low spatial frequencies? A ®rst explanation is that we originally underestimated the signal in this particular area and therefore wrongly enhanced it through CTF correction. It is indeed dif®cult to measure the amplitude contrast from the power spectra of the micrographs, since this area corresponds to a high peak. For example, the plot of the experimental 1D pro®le in Figure 8(a) had to be truncated in the low spatial frequencies to show the rest of the curve. If we plot the experimental (E) and modeled (M) power spectrum pro®les, they always diverge in low spatial frequencies. A possible indication for such discrepancy can be found when comparing the CTF and FSC plots of Figure 8(b) and (c). Obviously, the local minima of the FSC coincide with the zeros of the CTF (circles). When considering the low spatial frequencies, if the contrast was as low as predicted by the CTF plot (Figure 8(b), asterisk), one should ®nd the same agreement in the FSC curve. However, this is not the case, since the FSC reaches a maximum in low spatial frequencies (Figure 8(c)). Alternatively, the contrast in the low spatial frequencies may be enhanced by the 3D reconstruction. Indeed, at low defocus the visibility of particles, which re¯ects the amplitude contrast, is different in the original micrographs and in the primary volumes (data not shown). The CTF parameters were all derived from the power spectra of the micrographs (Figure 1), where particles are dif®cult to see and the amplitude contrast is low. However the Wiener ®ltering was not performed on the raw images but on the primary 3D reconstruction volumes where the SNR was greatly enhanced, especially in the low spatial frequencies. Therefore, the contrast in low spatial frequencies might have been boosted twice, ®rst through 3D reconstruction, and second through Wiener ®ltering. Two factors may explain this phenomenon. First, the raw images were subjected to a simple histogram normalization (Boisset et al., 1993), but no high-pass ®ltering was applied to them before 3D reconstruction. Second, the reconstruction algorithm used here (SIRT), is an iterative method performed in real space. It does not provide the user with the possibility to alleviate at will the signal in

469

Re®ned Architecture of Sepia of®cinalis Hemocyanin

low spatial frequencies by changing ®lter parameters is as usually done with techniques using the properties of reciprocal space. To counterbalance this effect we applied a bandpass ®lter, an operation routinely done on raw images of individual macromolecules to suppress the disturbing very low and very high spatial frequencies (Schatz et al., 1997). Although this ®ltering is mainly intended to reduce ramp effect and noise, it also improves the sharp features of the images. To estimate the shape of the band-pass ®lter, we compared a CTF corrected volume of GroEL obtained under the same conditions as those of the present LaB6 volume with the corresponding volume downloaded from the Protein Data Bank (Braig et al., 1995). This comparison indicated that an approximate tenfold reduction of the signal in the EM volume was needed in the low spatial frequencies to match the contrast of the PDB volume. Since a discrepancy is observed in the low spatial frequencies of CTF corrected volumes, two options are possible: (i) a correction of the CTF without changing the amplitudes in the low spatial frequencies; and (ii) an addition in the sample solution of another particle known at atomic level to adjust the Wiener ®ltering in the low spatial frequencies. Shape of FUs and path of the eight-FU polypeptide chains Here, we described several tiny structures between neighboring FUs that we called pillars or connections. The problem is to know whether they correspond to the passage of the polypeptide chain between contiguous FUs or to simple contact points between FUs close in the volume but not necessarily adjacent in the sequence. To solve this question, immunological approaches were used to study the binding sites of antibodies speci®c for each FU (Wichertjes et al., 1986a,b; Lamy et al., 1998). Among the 80 conceivable models, only four meet the restrictions imposed by the various observations of immunocomplexes so far studied, but the path of the polypeptide chain is not completely solved. A possible way of elucidating this problem would be to ®t the X-ray crystallographic structure of an FU to our 3D reconstruction volume. Actually, the small structural details visible in the re®ned volume (Figure 7) are probably suf®cient to align a high-resolution volume ®ltered down to Ê . This concept is based on the important 12.5 A sequence homology of all the cephalopodan Hcs known to date (Xin et al., 1990; Lang & van Holde, 1991; Miller et al., 1996), so that the knowledge of the 3D structure of a single building block is suf®cient to model the whole structure. However, the coordinates of the only cephalopodan FU solved at high resolution (Cuff et al., 1998), have not yet been put in the PDB. In this X-ray crystallographic structure, one clearly sees that FU g of Octopus (Odg) comprises a large copper-containing

N-terminal domain composed mainly of 15 a-helices and a small ®ve-stranded antiparallel b-sandwich C-terminal domain. The two domains are stacked in a pear-shaped structure with N and C termini located close to the ends of the long axis. The 3D reconstruction volume of Octopus Hc possesses seven FUs, the ®rst six being located in the wall, and the seventh (Odg) in the arches. In Sepia Hc, the architecture is quite similar but the polypeptide chain is composed of eight FUs, six of them forming the wall (Soa, Sob, Soc, Sod (or Soe), Sof, Sog), and the remaining two (Sod (or Soe) and Soh) the arches. From these data, we expected that C-terminal FUs of Sepia (Soh) and Octopus (Odg) Hcs would have similar shapes and orientations in the two molecules and that the additional FU present in Sepia Hc (Sod or Soe) would be located in a loop coming from and returning to the wall where Soc and Sof are certainly located. When considering the shapes and dispositions of the FUs located in positions A1 (A10 ) and A2 (A20 ) of the FEG volume, the results are ambiguous. First, the two FUs have different contours. A1 has a massive ovoid shape resembling the X-ray structure of Ovg (Cuff et al., 1998), while A20 has a bent rodlike outline (Figure 6(b)). Second, the connections between A1 and the wall (A1-W1 and A1W2) are located in a single WOU. Conversely, for A20 the arch-wall connections form an intricate system between two neighboring WOUs, with the pillar P2 and the curved bridges A20 -W50 and A20 -W3 (Figure 6(c)). These results tend to indicate that A1 (or A10 ) corresponds to FU (Soh), while A2 (or A20 ) corresponds to (Sod or Soe). However, when comparing the disposition and the orientation of A1 and A10 FUs with those present in the arches of Octopus, the situation is unclear. In Octopus Hc, the arches are composed of two pear-like shaped antiparallel FUs in a vertical orientation. Their main domain is connected to the central tier of the molecule, while their small domain is connected to the upper or lower tiers (Mouche et al., 1999). Here, the FUs A1 and A1' do not have a vertical orientation and they are linked by two connections either to the upper or to the lower tiers (Figure 6(b)). The FUs A2 and A20 do have a vertical orientation, but their location is different from those of the Octopus FUs. When comparing the arches of Octopus and Sepia Hcs, there is no direct correspondence of the FUs. Neither A1 (A10 ) nor A2 (A20 ) can be directly superimposed on the arches of Octopus. It seems that the additional pair of FUs of Sepia triggered a complete relocation of FUs within the arches. Conclusion The parallel 3D reconstructions of Sepia Hc from LaB6 and FEG cryo-EM images provide a comparison of the information that one can get on single particles with these two types of electron sources. Although the FSC0.5 and FSC3s disagree in the estimation of resolution, the FEG volume is indis-

470 putably better resolved than the LaB6 volume. Despite the presence of local minima in the FSC curve, revealing the low SNR in critical spatial frequencies, meaningful signal is present beyond the 0.5 threshold value of the FSC, possibly up to Ê ÿ1. The strong imbalance induced by the 1/12.5 A CTF correction by the Wiener ®ltering technique remains unexplained and is corrected by band-pass ®ltering. Considering the architecture of Sepia Hc, new small features such as the pillars P1 to P3 in the large and small grooves, or the arch-wall connections A1-W1, A1-W2, A20 -W50 and A20 -W3 are visible in the FEG volume. The speci®c shape and the disposition of these connections indicate that A2 or A20 FUs stretch over the large groove and form bridges between two neighboring WOUs while A1 or A1' FUs are connected to a single WOU. As no direct correspondence can be found in the arches between Octopus and Sepia Hc, the location of the C-terminal FU remains uncertain. However, as soon as atomic coordinates of FU g of Octopus Hc (Odg) are available from the Protein Data Bank (Cuff et al., 1998), a ®tting will be attempted with the present reconstruction volume.

Materials and Methods Sample preparation and cryoelectron microscopy For cryo-EM, 400-mesh copper grids were coated with a thick holey carbon ®lm, and a thin carbon ®lm was added on top of them to allow a clear visualization of the Thon rings in the power spectra of experimental images. The sample was prepared by ultracentrifugation as described by Gielens et al. (1986) and diluted to a concentration of 3 mg/ml in 50 mM Tris-HCl buffer (pH 7.65), 10 mM CaCl2, 50 mM MgCl2. To test the exact magni®cation of the microscope, TMV was added to the sample at a ®nal concentration of 700 mg/ml. The sample solution was applied to glow discharged grids and, after blotting the excess of solution, the grid was rapidly plunged into liquid ethane (Adrian et al., 1984). Observations were carried out in two electron microscopes equipped with LaB6 or FEG electron sources. LaB6 Cryo-EM images were recorded in a Philips CM12 electron microscope using a Gatan 626N cryoholder at a magni®cation of 43,141 and an accelerating voltage of 100 kV, with condenser and objective apertures of 200 and 70 mm, respectively. Each untilted-specimen ®eld was imaged at defocuses close to 0.8 mm or to 1.0 mm. FEG CryoEM images were collected in a JEOL JEM 2010F electron microscope using a Gatan 626DH cryotransfer kit. Images were recorded at a magni®cation of 60,491 and an acceleration voltage of 200 kV, with condenser and objective apertures of 150 and 60 mm, respectively. Each untilted-specimen ®eld was imaged at defocuses close to 3.4 or 4.5 mm. Both image sets were recorded under low-dose conditions on Kodak SO163 electron microscope ®lms developed in full-strength Kodak D19 developer for 12 minutes.

Re®ned Architecture of Sepia of®cinalis Hemocyanin Digitization and estimation of the magnification and pixel size Micrographs were digitized with an Optronics P1000 microdensitometer, using a 25 mm square scanning aperture. Individual particles were interactively selected onscreen and windowed into 120  120 pixels images. When a TMV particle was visible in the cryo-EM ®eld (Figure 1, arrows), it was subjected to the following calibration procedure. (i) The TMV was windowed in a 512  512 pixels image and oriented vertically. (ii) The power spectrum of the image was computed, revealing Ê layer-lines of its central rows. (iii) The half disthe 23 A tance between the two layer-lines was measured in pixels and used for the estimation of the magni®cation. Such internal calibration provided consistent results on all our micrographs, whenever a TMV was visible in the Ê striations. Using the same approach, ®eld with its 23 A the LaB6 images were estimated to correspond to a magÊ and the ni®cation of 43,141, and a pixel size of 5.79 A FEG images to magni®cation of 60,491 and a pixel size Ê. of 4.13 A Image processing The CTF was estimated on each micrograph by the method described by Zhu et al. (1997), using the SPIDER image processing software (Frank et al., 1996). The 2D power spectrum of each digitized micrograph was determined by averaging partially overlapping periodograms, and a rotational average 1D pro®le of this estimate was computed. The presence of the thin carbon ®lm below the ice layer produced readily visible Thon rings on the ®nal periodogram. After removing the noise contribution from the pro®les (Zhu et al., 1997), the locations of minima allowed for each micrograph an accurate determination of defocus. Finally, the source size, amplitude/ contrast ratio, and half-width of the envelope function were estimated from the whole series of 1D pro®les as de®ned by Frank (1973), Wade (1992) and Wade & Frank (1977). Using the volume reported by Lambert et al. (1995) as a ®rst reference volume, the untilted images were subjected to three cycles of ``3D projection alignment'' (Penczek et al., 1994). The resulting images were centered and their directions of projections with respect to the reference volume were determined. A primary volume was then computed from the image set and used as a second reference for ®ve additional cycles of 3D projection alignment. At this stage, several volumes were computed with the simultaneous iterative reconstruction technique according to Penczek et al. (1992) from the centered images and their accurately de®ned eulerian angles. Aligned image subsets windowed from each micrograph, and therefore corresponding each to a given defocus value, were then used to compute primary volumes. CTF correction and merging (see the Results) were carried out by Wiener ®ltering of the primary volumes (Frank & Penczek, 1995) and a ®nal band-pass ®ltering was applied to reduce the contribution to the contrast of the low spatial frequencies. For the resolution limit estimation of 3D reconstruction volumes, two classes were randomly drawn from the image set. The corresponding 3D volumes were then compared in reciprocal space on increasing spherical shells using the FSC technique (Saxton & Baumeister, 1982; van Heel, 1987). The resolution limit was either set at the spatial frequency where the FSC falls below a threshold value of 0.5 (FSC0.5) (Malhotra et al., 1998;

Re®ned Architecture of Sepia of®cinalis Hemocyanin Penczek, 1998), or below the 3s noise curve (FSC3s) (Orlova et al., 1998). Because of the D5 point-group symp metry, the noise curve was multiplied by 10 to account for the tenfold redundancy of the data. The interpretation of the reconstructed 3D volumes was carried out with the help of SIGMA software (Taveau, 1996) for computation of solid-body surface representations and examination of selected slices. For solid-body surface representations, a threshold displaying the molecular volume was computed on the basis of a partial speci®c volume of 0.73 ml/g and a global molecular mass of 3,901,660 Da calculated by MALDI mass spectrometry, corresponding to a molecular volume of 66988 voxels or 4728.6 nm3.

Acknowledgments We are grateful to Dr Penczek and Dr BoÈttcher for fruitful discussions and advice on CTF correction and band-pass ®ltering, to Dr Gilbo and Dr Dubuisson for letting us use their JEOL JEM 2010F electron microscope at CEA/Saclay, to Mr Ravel-Chapuis and Mr Cailler (JEOL S.A., France), to Dr Ambroster (Gatan) for technical assistance, and to Miss Audouin (Fondation Paul MeÂtadier). This work was ®nancially supported by grant CNRS 98N60/0642 (to N.B.) and by Comite d'Indre et Loire de la Ligue Nationale Contre le Cancer (to N.B.).

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Edited by W. Baumeister (Received 11 October 1999; received in revised form 8 December 1999; accepted 8 December 1999)