three-dimensional reconstruction by cryoelectron microscopy of the giant hemoglobin of the...

J. Mol. Biol. (1996) 264, 111–120

Three-dimensional Reconstruction by CryoelectronMicroscopy of the Giant Hemoglobin of thePolychaete Worm Alvinella pompejana

Felix de Haas 1, Franck Zal 2, Valerie You 1, Franc ois Lallier 2

Andre Toulmond 2 and Jean N. Lamy 1*

A frozen-hydrated specimen of the hexagonal bilayer hemoglobin (HBL1Groupe d’AnalyseStructurale des Antigenes Hb) from the deep-sea hydrothermal vent polychaete worm Alvinella(URA 1334 CNRS), 2 bis pompejana, the most thermophilic metazoan known to date, was observedBoulevard Tonnelle, F-37032 in the electron microscope and subjected to three-dimensional (3D)

reconstruction by the method of random conical tilt series. At a resolutionTours Cedex, Franceof 34.6 Å by the differential phase residual method and 27.7 Å by the2Equipe Ecophysiologie Fourier shell correlation method, the 3D volume possesses a D6UPMC-CNRS-INSU, Station point-group symmetry. While in previous 3D reconstructions of annelid

Biologique, BP 74, F-29682 and vestimentiferan Hbs the vertices of the upper layer were 16° rotatedRoscoff, Cedex, France compared with those of the lower layer, in Alvinella Hb the vertices of the

two hexagonal layers are almost perfectly eclipsed when viewed along the6-fold axis. As in the HBL Hbs of Riftia pachyptila and Macrobdella decora,a central linker complex is decorated by 12 hollow globular substructures(HGS). The linker complex comprises (1) a central hexagonal toroid, (2)two internal bracelets onto which the HGSs are built, and (3) sixconnections between the two hexagonal layers. Each HGS is composed ofsix masses, which are separated when the volume is displayed at highthreshold, plus one additional mass involved in the bracelet connecting thesix HGSs in both hexagonal layers. The HGSs have a local pseudo 3-foldsymmetry and a disposition of the high-density masses different fromthose of Riftia V1 Hb.

7 1996 Academic Press Limited

Keywords: hydrothermal vent; alvinellid; hemoglobin; structure;*Corresponding author cryoelectron microscopy

Introduction

Extracellular hemoglobins (Hbs) are present in allthree annelid classes and vestimentiferan phylum.These proteins are characterized by a two-tieredhexagonal structure, clearly visible in the trans-mission electron microscope (EM), a high molecularmass ranging from 3000 to 4000 kDa (i.e. 60 S), anacidic isoelectric point, and low heme and ironcontents (Vinogradov et al., 1982; Vinogradov,1985a,b). Understanding the quaternary structureand conceiving models of these giant Hbs havebeen a challenge over the past 20 years (Chung &

Ellerton, 1979; Garlick & Riggs, 1982; Suzuki &Gotoh, 1986; Vinogradov et al., 1986, 1991; Suzukiet al., 1989; Ownby et al., 1993). However, a totalagreement has not yet been attained, except on thepoint that hexagonal bilayer (HBL) Hbs arecomposed of 12 dodecameric subunits linked by 36to 42 ‘‘linker’ chains (Vinogradov et al., 1986, 1991;Martin et al., 1996; Zal et al., 1996a,b). To date, allthe HBL Hbs studied consisted of two types ofchains, ca 70% globin chains (017 kDa) bearingheme and ca 30% linker chains (024 to 32 kDa)containing little or no heme necessary for theassemblage of the HBL structure (Vinogradov,1985a). Recently, the HBL Hb of the polychaeteannelid Alvinella pompejana was subjected toelectrospray ionization mass spectrometry (ESI-MS)and this analysis gave surprising results (Zal et al.,unpublished). Linker chains represent about 45% ofthe total mass of the molecule, compared with the

Abbreviations used: HB, hemoglobin; HBL,hexagonal bilayer; 3D, three-dimensional; HGS, hollowglobular substructure; EM, electron microscope;ESI-MS, electrospray ionisation mass spectrometry; 2D,two-dimensional; DPR, differential phase residual;FSC, Fourier shell correlation.

0022–2836/96/460111–10 $25.00/0 7 1996 Academic Press Limited

3D Reconstruction of Alvinella Hemoglobin112

30% normally found. Moreover, both globin andlinker chains possess heme group. Zal et al.unpublished hypothesized a possible thermostabil-ity function of these extra linker chains. Indeed,Alvinella lives in whitish honeycomb-like structuresecreted in the hottest parts of the hydrothermalvent ecosystem. At this level, the gradient betweenthe cold deep sea water (2°C) and the hot (350°C)hydrothermal fluid is extremely variable. Althoughit is unclear exactly where the worms reside in thestructure, observations have reported individualsliving around 50°C (Baross & Deming, 1985)and Chevaldonne et al. (1992) have observed anAlvinella specimen coiled around the tip of theAmerican submersible Alvin’s high-temperatureprobe, which simultaneously recorded 105°C.

In recent reports, 3D reconstruction volumes ofvarious HBL Hbs of annelid and vestimentiferanspecies observed in vitreous ice were described, i.e.the chlorocruorin of the polychaete worm Eudistyliavancouverii (de Haas et al., 1996a), the Hbs of theoligochaete Lumbricus terrestris (Schatz et al., 1995)and of the achaete Macrobdella decora (de Haas et al.,1996b), and the V1 Hb of the vestimentiferanRiftia pachyptila (de Haas et al., 1996c). Althoughstructural details differ, such as the presence of atoroid in the center of the molecules of Macrobdellaand Riftia instead of a flat disk as in Lumbricus andEudistylia, all the molecules have roughly the samearchitecture. Specifically, the HBL is composed of acentral linker complex decorated by 12 hollowglobular substructures (HGSs). The linker complexcomprises (1) a central hexagonal mass, (2) twointernal bracelets onto which the HGSs are built,and (3) six substructures connecting the twohexagonal layers. In addition, in all cases thevertices of the upper hexagonal layer are 016°rotated compared with those of the lower layer andthe HGSs have a local pseudo 3-fold symmetry.

Here, we present the 3D reconstruction of theAlvinella Hb that shows important architecturaldifferences with other HBL Hbs reconstructed todate.

Results

Cryoelectron microscopy

A typical untilted-specimen field of frozen-hydrated Alvinella Hb is shown in Figure 1. Theparticles appear in two well-defined orientations,termed top (white arrows) and side (black arrows)EM views. In the top view, the molecule observedalong its 6-fold axis has a hexagonal overall shapeand the distance between its diametrically opposedvertices is 31 nm. In the side view the molecule has31 nm × 21 nm bilayered rectangular pattern. Theparticles also produce approximately oval views,termed intermediate views (black dot). The imageset used for the 3D reconstruction was composed of3210 untilted-specimen images taken at a magnifi-cation of 34,400 × with a defocus of −1.5 mm. The

Figure 1. Micrograph of a frozen-hydrated sample ofAlvinella pompejana Hb observed at 0° tilt angle. The whiteand black arrows and the black dot designate moleculesin the top, side and intermediate view orientations,respectively. The scale bar represents 50 nm.

repartition of the whole image set in top, side andintermediate views is described below.

Three-dimensional reconstruction

The 6-fold rotational symmetry present in theaveraged top-view images (not shown here)suggested that, like all the annelid and vestimen-tiferan HBL Hbs so far studied, Alvinella Hb has aD6 point-group symmetry. Thus, a C6 point-groupsymmetry was first applied to all the reconstructionprocesses. Then, after having verified that the C6

symmetrized volume also possessed the 2-fold axescharacteristic of the D6 point-group symmetry, thereconstruction procedure was repeated with a D6

point-group symmetry imposed on all the 3D-re-construction volumes. Only the results of the D6

symmetrized volume are shown here.First, Eulerian angles were assigned to the 3210

images composing the image set by one cycle ofangular refinement using the 3D projection align-ment procedure with the 3D volume of the leechMacrobdella Hb, our best resolved volume at thetime of this experiment (de Haas et al., 1996b), as areference volume. The topology sphere in Figure 2aand b demonstrates that the set of 3210 images didnot suffer from a severe lack of directions so that noadditional electron microscopy was required toselect images corresponding to the missing orien-tations. The histogram in Figure 2c indicates thatthe images projecting in the zones corresponding tothe top views (in the neighborhood of the poles)and to the side views (close to the equator) are bothoverrepresented. At this stage, a quantitativeanalysis of the Eulerian angles gave the relative

3D Reconstruction of Alvinella Hemoglobin 113

Figure 2. Topology spheres showing the distribution ofthe orientations corresponding to the images used toreconstruct the molecule. Topology spheres viewed fromthe top (a, d and g) and the equator (b, e and h). c, f andi, Histograms of the number of images per direction asa function of the second Eulerian angle (u). Topologyspheres for the 3210 untilted-specimen images (a, b andc), the subset of 1430 selected images (d, e and f), and the1430 images after the three refinement cycles (g, h and i).The final 3D reconstruction volume was calculatedfrom these refined Eulerian angles corresponding to g, hand i.

Figure 3. Histogram of the absorbances (x-axis) in thefinal reconstruction volume of Alvinella pompejana Hb.The broken line on the right side of the ice peak indicatesthe normal threshold values allowing the visualization ofthe normal reconstruction volume. The black arrowindicates the threshold value corresponding to themolecular volume calculated from the molecular massand the partial specific volume.

subjected to three cycles of refinement by 3Dprojection alignment. At each cycle, the reconstruc-tion volume calculated from the images withimproved Eulerian angles and centration obtainedat the previous cycle was used as a reference.

The resolution of the reconstructed volume asdetermined by the differential phase residual (DPR)method at 45° phase difference was 34.6 A and thatobtained by the Fourier shell correlation (FSC)method was 27.7 A. The volume was finally filtereddown to a resolution limit of 27.7 A. The topologysphere and the histogram in Figure 2g to i showthat in the final reconstruction volume some of theimages, that initially had u values corresponding tointermediate views, had moved to the top-viewareas in the 0 to 10° and 170 to 180° ranges (Figure2i). However, this change was negligible because ofthe small number of directions in the neighborhoodof the poles. The cause of the u value modificationduring the refinements is not clearly understood,but it may be related to the fact that, as shownbelow, the structure of the reference volumediffered somewhat from that of the reconstructedvolume.

The volume visualized at normal threshold

The histogram shown in Figure 3 displays theabsorbance values in the refined final 3D volume,rescaled on a 0 to 1 scale. The high peak with thelowest absorbances obviously corresponds to theice voxels, while the smaller peak on the right side(density values 0.6 to 0.9) reflects the distribution ofthe protein densities. For visualization of theprotein, a surface was calculated enclosing all thedensities higher than a certain value defined as thenormal threshold. Here, the normal threshold(0.62), indicated by the vertical broken line inFigure 3, generates a surface enclosing 15,596voxels, i.e. 5.97 × 106 A3 that, in our opinion,

amounts of top and side-view images. Thus, 59images (1.8%) had a value of the second Eulerianangle (u) in the range 0 to 6° or 174 to 180° (topviews) and 532 images (16.5%) in the range 86 to 94°(side views). The highly unbalanced proportions oftop and side views indicate that the molecules arenot randomly oriented within the ice layer, aphenomenon already encountered with other HBLHbs (de Haas et al., 1996a,b,c). To prevent a possibleanisotropy due to the overrepresentation of someprojection directions, a selection of 1430 imagesproduced an excellent coverage of the Eulerianangles with at most two images per orientation(Figure 2d to f). When more than two images wereavailable for the same direction, the two for whichthe 3D projection alignment procedure gave thebest correlation coefficients were selected. Then, afirst 3D volume was calculated by back projectionof the 1430 images. Finally, the 1430 images were


Figure 4. Surface representation of the 3D reconstruc-tion volume of Alvinella pompejana Hb viewed at normalthreshold. a, Top-view orientation. The 6-fold axis ofsymmetry is indicated by a small open hexagon. b,Intermediate view resulting from a 45° rotation arounda horizontal axis. The open triangle shows the location ofthe local pseudo 3-fold axis present in the HGS. c and d,The two types of side-view orientations seen along their2-fold axes of symmetry. c1 and c2, Intrahexagonal layercontacts between neighboring HGSs. c4, Connectionbetween HGSs superimposed in the two hexagonallayers. c5, Contacts between the hexagonal toroid and theHGSs. The numbers in broken-line circles mark thelocation of the six HGS masses visible at high threshold.

As shown below (Figure 7), raising the thresholdreveals that the HGS is composed of six globularmasses termed mass numbers 1 through 6. Thepositions of these masses are indicated in Figure 4by broken-line circles. In the top view (Figure 4a),mass numbers 1 through 3 and the contacts, termedc1 connections, linking mass numbers 2 and 3belonging to neighboring HGSs are exposed to theobserver. In the intermediate view of the HBL(Figure 4b), the central HGS of the upper layer isviewed along a local pseudo 3-fold axis ofsymmetry materialized by a triangle. This viewshows (1) the hexagonal toroid connected by thinconnections, termed c5, to the HGS face regardingthe lumen of the cylinder, (2) mass numbers 4, 5and 6 located in the part of the wall closest to theequatorial plane, and (3) a second interHGSconnection type, termed c2, involving massnumbers 4 and 6. Figure 4c and d shows the twoside views of the volume seen along their 2-foldaxes. These orientations are particularly favorableto the observation of the c4 connections between thetwo hexagonal layers and of the c1 and c2connections.

The hollow globular structures

A single HGS was extracted from the whole 3Dreconstruction by careful masking of the neighbor-ing structure (Figure 5). When displayed at athreshold showing 40% of the normal volume, theHGS looks to be composed of interconnectedglobular masses numbered according to thepreviously described system (de Haas et al.,1996a,b,c). However, due to the better resolutionand the particular architecture of Alvinella Hb,several new features are visible. When the HGS isseen along the HBL 6-fold axis in the top vieworientation (Figure 5a), the fore plane contains massnumbers 1, 2 and 3, and the back plane containsmass numbers 4, 5 and 6 plus the remains of the c3connection body (behind mass number 1). Figure 5bshows the HGS in the same orientation but seenfrom behind. At this threshold, the c3 connectionbody is clearly linked to mass number 6, but not tomass number 4. The appendages on mass numbers4 and 6 are the beginning of the c4 connection withthe HGS of the opposed hexagonal layer. The sameHGS viewed along the local pseudo 3-fold axisfrom above and below is shown in Figure 5c and d,respectively. One distinctly sees the large linkbetween mass numbers 3 and 5, and the inter-mediate position of the c3 connection body betweenmass numbers 1 and 6. Figure 5e and f shows theexternal and luminal faces of the HGS in the sideview orientation, and Figure 5g and h shows theorganization of the lateral walls. The reasons for thedifference in the aspect of these two walls areobvious: the group of mass numbers 3, 4 and 5 isdenser than that of numbers 2 and 6, and the c3connection body is linked to mass number 6 but notto 4.

corresponds to a not too swollen or not too erodedvolume. We refer to this volume as the normalvolume. The molecular volume of Alvinella Hb,calculated from the molecular mass of 3833(214) kDa determined by multi-angle laser light-scattering (Zal et al., unpublished) and the partialspecific volume of 0.734 ml/g obtained for Macrob-della Hb (Weber et al., 1995) represents 12,207voxels, i.e. 4.671 × 106 A3 (black arrow in Figure 3).This value, smaller than the normal volume,corresponding to an obviously too eroded volumemay be due to the fact that at 27 A resolution smallcavities in the protein are not resolved and aretherefore interpreted as mass.

When, for the first time, we observed the 3Dreconstruction volume along its 6-fold axis ofsymmetry at normal threshold, we were surprisedto see that the vertices of the two hexagonal layersare almost perfectly eclipsed (Figure 4a). Indeed, inall the other annelid and vestimentiferan Hbs so farstudied, an angle of 016° was obtained betweenthe vertices of the two hexagonal layers. As in otherannelids and vestimentiferans, the half-molecule ofAlvinella Hb comprises six HGSs. Also visible in thecentral part of Figure 4a is a ring-like structuresimilar to those already described in Macrobdellaand Riftia Hbs, that we term the hexagonal toroid.


Figure 5. The structure of the HGS of Alvinellapompejana Hb observed at a threshold displaying 40% ofthe normal volume. a and b, The HGS viewed along theHBL 6-fold axis (top-view orientation) from top (a) andbottom (b). c and d, The HGS viewed along the localpseudo 3-fold axis from outside (c) and inside (d) themolecule. e and f, The HGS viewed along one of the2-fold axes (side-view orientation) from outside (e) andinside (f) the molecule. g and h, The HGS viewedperpendicularly to its lateral walls. In g and h the externalwall is located on the left and on the right, respectively.The numbers mark the location of the six high-ab-sorbance masses visible at high threshold. c3, Thec3-connection body.

Figure 6. Shaded surface representation and sectionsthrough the reconstructed volume of Alvinella pompejanaHb. a1 to a6, Levels of the sections perpendicular to the6-fold axis shown in (b(1 to 6)). c1 to c6, Levels of thesections perpendicular to the 2-fold axis shown in (d(1 to6)). The thickness of each section is one voxel, i.e. 7.26 A.In a and b to simplify the description, A through F andA' through F' designate the various HGSs of the upperand lower hexagonal layers, respectively. In b3 and b6the white hexagons allow comparison of the respectiveorientations of the two hexagonal layers. The scale barrepresents 10 nm.

F and A' through F', as indicated in Figure 6aand c.

Sections perpendicular to the 6-fold axis.

The structural relationships between the massescomposing the HGS are well understood from theobservation of the sections of Figure 6a. The firstslice (Figure 6b1) contains the upper portion ofmass numbers 1, 2 and 3 of the upper HGS layer.Figure 6b2 shows the second section cutting the sixHGSs of the upper layer near the top of the cavities.Notice the irregular pentagonal contour of the HGSand the high density of mass number 1 on itsluminal side. The dense nucleus on the externalside corresponds to mass number 3 and the fuzzyzone on the right side (when viewed from thecenter) to mass number 2 and its contact with massnumber 3 of the neighboring HGS. The second andthird sections (Figure 6b2 and b3) look similar,

Distribution of the densities within the volume

While the surface representation of a 3D volumestrongly depends on the threshold value, in slicescut through the volume the densities are indepen-dent of the threshold. Therefore, understanding theinternal structure is more reliable when studiedfrom thin slices integrating the absorbances ratherthan from surfaces. Here, we present two series ofslices through the 3D volume cut perpendicular tothe 6-fold (Figure 6B (1 to 6)) and 2-fold (Figure 6d(1 to 6)) axes. The positions of the cutting planesand the directions of observation correspondingto Figure 6b and d are represented in Figure 6aand c, respectively. To shorten the descriptionof the structure, the HGSs of the upper andlower hexagonal layers are labelled A through


except that the third section plane, passing throughthe lower third of the upper HGSs, cuts massnumbers 4, 5 and 6 on the external side and the c3connection body on the luminal side. The followingsection (Figure 6b4) through the floor of the HGSshows the lower part of mass numbers 4, 5 and 6,and of the c3 connection body as well as the faintc5 connections with the upper portion of the toroid.The central section of the volume shows thehexagonal toroid and the kidney-shaped c4connection area obviously composed of twoglobular masses (Figure 6b5). Each of these twomasses connects mass numbers 4 and 6 ofsuperimposed HGSs belonging to the two hexago-nal layers. This pattern seems to be completelydifferent from that recently observed in Riftia Hb(de Haas et al., 1996c). Finally, because of the HBL2-fold axis of symmetry, Figure 6b6 shows a slicethrough HGSs A' to F', homologous, but mirrorinverted, when compared with slice 6b3. The whitehexagons circumscribing the sections of Figure 6b3and b6 show that the two layers of HGS areeclipsed.

Sections perpendicular to the 2-fold axis.

In slice 6d1, cutting HGSs B, B', A, A', F and F',the two dark circular zones correspond to theinternal cavities of HGSs A and A'. Notice that theyare almost perfectly superimposed and that no shiftoccurs between the upper and lower layer, asoccurs in the volumes of Eudistylia, Macrobdella andRiftia Hbs (de Haas et al., 1996a,b,c). It is obviousfrom Figure 6d1 that the 2-fold axis passes exactlyin the center of the slice. The lower left and upperright areas show the external parts (mass numbers2, 5 and 6) of HGSs B' and F, while in the upper leftand lower right corners only mass number 4 of Band F' is sectioned. The complex pattern of the sliceof Figure 6d2 shows (1) the central cavities of HGSsB' and F (the dark zones in the lower left and upperright corners), (2) the external wall of HGSs B andF' reduced to mass number 5, (3) the ceiling ofHGSs B and F' reduced to mass number 1, (4) theluminal face of the wall of HGSs A and A' (the twodense nuclei correspond to mass number 1 and thefuzzy part to the 3 connection), and (5) the linkbetween mass numbers 4 and 6 of the upper andlower layers in the c4 connection area near the endof the median horizontal line. Slice 6d3 passesthrough the internal cavities of four HGSs (B, B', Fand F') producing four dark spots. In addition, nearthe vertical median line one sees three high-absorbance spots corresponding, from top tobottom, to the luminal face of HGS A, the sectionof one vertex of the hexagonal toroid, and theluminal face of HGS A'. The structure of thehexagonal toroid is also easily understood fromFigure 6d4, where the four c5 connections linkingthe toroid to the HGSs located at the four cornersare visible. In the HGSs located in the upper leftand lower right areas, the three high-absorbance

nuclei correspond to mass numbers 1, 2 and 6. Thec5 connections between the toroid and the c3connection body of the four HGSs are perfectlyvisible. Figure 6d5 shows a section containing the6-fold axis. The two densities located near thecenter of the slice on the median horizontal lineobviously belong to the hexagonal toroid and thegroups of three densities at the four cornerscorrespond to the c1 and c2 connections (on theexternal side) and to the bracelets of c3 connections(on the luminal side). The sixth slice (Figure 6d6) isthe mirror-inverted replica of Figure 6d3. Themirror inversion due to the D6 point-groupsymmetry is particularly apparent in the center ofthe slice in the area of the toroid vertex. Animportant difference with the same slice cut in RiftiaHb is that no shift between the upper and lowerlayers is visible and that the four HGS cavitiesappear similar (de Haas et al., 1996c).

Discussion

Differences from other species

Alvinella Hb, as all the other HBL Hbs so farstudied, is made up of 12 HGSs and a linkercomplex comprising a central hexagonal piece, twobracelets of c3 connections, and six interlayers c4connections (de Haas et al., 1996a,b,c). Of thepreviously studied species, two groups emerged,one possessing a central flat hexagonal piece(Lumbricus and Eudistylia) and the other ahexagonal toroid (Macrobdella and Riftia). From thispoint of view, the presence of a hexagonal toroidassigns Alvinella Hb to the second group. However,this molecule can be distinguished from Macrobdellaand Riftia Hbs, and from all HBL Hbs andchlorocruorin analyzed to date by two importantcharacters, the disposition of the two hexagonallayers and the respective locations of the HGSmasses.

To highlight these differences, in Figure 7we compare the architectures of the Hbs of A. pom-pejana, a polychaete annelid, and R. pachyptila, avestimentiferan, both species strictly endemic to thehydrothermal vent ecosystem. To show thedifferent disposition of the hexagonal layers themolecules are viewed along their 6-fold axis at athreshold displaying 40% of the normal volume. InFigure 7a and b, the orientation of the two Hbs wasobtained by aligning their 6-fold and 2-fold axes ofsymmetry. It immediately appears that while inAlvinella the number 1 masses are eclipsed (Fig-ure 7a), in Riftia they are shifted away from thetoroid vertex (Figure 7b), thus producing the 16°rotation between the two hexagonal layers. As aresult of these different organizations, the c5connections between the toroid and the HGSs aredifferently disposed and oriented. Thus, as shownin Figure 7c and d, the two c5 connections linkingeach toroid vertex to the nearest HGS (black


Figure 7. Comparison of the HBL Hbs of Alvinellapompejana (a, c and e) and Riftia pachyptila (b, d and f). aand b, Surface representation viewed along theHBL 6-fold axis. c and d, A thin section cut perpendicularto one of the 2-fold axes and passing through thetoroid vertex. The black arrows show the locations ofthe c5 connections on one toroid vertex and the brokenarrows the binding point of the c5 connections onthe HGSs. e and f, Disposition of the Alvinella and RiftiaHGS masses viewed from outside the HBL along theirlocal pseudo 3-fold axis (triangle). The alignment hasbeen processed by manual adjustment in such a waythat mass numbers 1, 4 and 6 (back plane) aresuperimposed best in 3D. The threshold leaves apparent40% of the normal volume in a and b, 60% in c and d, and20% in e and f. In e and f the broken and full lines linkthe masses located in the back and fore planes,respectively.

symmetry of the two HGSs. To produce the patternshown in Figure 7e and f, the HGs were firstoriented in such a way that they were viewed fromoutside the HBL along their pseudo 3-fold axes.Then, mass numbers 1, 4 and 6 (in the back plane)were aligned and the disposition of massnumbers 2, 3 and 5 (in the fore plane) wasexamined. Clearly, mass numbers 2, 3 and 5 of Riftia(Figure 7f) appear clockwise rotated comparedwith their counterpart of Alvinella Hb (Figure 7e).Thus, although in both Hbs the six HGS masses arerelated through a pseudo D3 point-group sym-metry, the positions of mass numbers 2, 3 and 5 aredifferent when compared with those of massnumbers 1, 4 and 6.

Repartition of the polypeptide chains in theAlvinella 3D volume

Recently, Zal et al. (unpublished) determined thepolypeptide composition of Alvinella Hb. Theirstudy revealed that this HBL Hb contains only 108globin chains distributed among 72 monomericglobin chains a and 12 disulfide-linked trimers(each composed of globin chains b, c and d). Inaddition, the molecule contains an unusually highnumber (86) of linker chains (48 L1 + 12 L2 + 2L3 + 24 L4), among which the most abundant, L1and L4, contain a heme group. These observationsraise an interesting problem. Specifically, thenumber of 108 globin chains does not allow theconstruction of 12 dodecameric HGSs, as in otherspecies, but at most of 12 nonamers composed eachof one trimer and six monomers of globin chains.This model is compatible with the existence of thepseudo 3-fold symmetry. If all the globin chains areused for the construction of the nonamers, then allthe other substructures of the HBL must be madeup of linkers. If, in addition, we admit that L3 is anartifact or an underestimated chain, there remainsa group of 14 or 15 × 6 linker chains to account forthe toroid, the c3 and c4 connections. The toroidcannot be composed of less than six linker chains,but 12 (one per c5 connection) may be a convenienthypothesis. The c3 connection comprises at least 12linker chains (one per HGS), but most likely 24 or36 (two or three linker chains per HGS). The c4connection most likely corresponds to one linkerchain per mass number 4, i.e. 12 linker chains forthe whole HBL. This makes a total of 6 or 12 + 24or 36 + 12 = 42 or 60 linker chains. In this model, 26to 44 linker chains are not used. Unless certaincomponents of the linker complex were stronglyunderestimated in the model, the only possibilitywould be to assign the remaining linker chains tothe HGSs and to suppose that the HGS contains adodecamer composed of nine globin chains andthree linker chains, i.e. 3 × 12 = 36 for the wholeHBL. This model looks plausible and com-patible with the data, but it is clearly in conflict withthe dodecameric concept initially proposed byVinogradov et al. (1991).

arrows) are shifted apart from the vertex inAlvinella (Figure 7c), while they are superimposedin Riftia (Figure 7d). The HGS area where the c5connection binds is also different in the two species(broken arrows).

The location of the six HGS masses with respectto each other is particularly well visible when theHGSs are viewed along the HBL 6-fold axis. Thus,mass numbers 1 and 3 are in close contact inAlvinella (Figure 7a) but distant from each other inRiftia (Figure 7b). Similarly, mass number 5 is closerto 6 than to 4 in Riftia, while the reverse pattern ispresent in Alvinella. Beside this difference, thedistances between mass numbers 1 and 5, 1 and 2,and 4 and 6 are similar in both species. Thesedispositions are visible also when comparing the


What can explain the unique structure ofAlvinella HBL Hb?

We have seen above that the 3D volumes ofAlvinella are different from those of all other HBLHbs studied to date. The observed differences arelocated both in the HGS (the different positions ofthree of the six masses related by the local pseudo3-fold symmetry) and in the linker complex (thedifferent locations of the HGSs around the 6-foldaxis), resulting in a unique eclipsed position of thetwo hexagonal layers. These differences may berelated to the unusually low globin-to-linker ratio(55/45) of this Hb compared to those of otherspecies (ca 70/30). Linker chains are rich in cysteineinvolved in intra-disulfide bridges (Green et al.,1995; Weber et al., 1995; Martin et al., 1996; Zal et al.,1996a,b) and this is the case for Alvinella HBL Hb(Zal et al., unpublished). Since in Alvinella Hb 84%of the linker chains possess a heme, an enrichmentin linker chains could strengthen the structure ofthe whole native molecule without impairing itsoxygen-binding capabilities. This could explain theelevated thermostability found for this Hb (stableup to 50°C, Terwilliger & Terwilliger, 1984), afeature that may prove adaptive considering thehigh temperature (mean 30 to 40°C, up to 100°C) atwhich this species lives (Baross & Deming, 1985;Chevaldonne et al., 1992).

Conclusion

The 3D reconstruction of A. pompejana producesa 3D volume with an architecture both similar anddifferent from those of other annelid and vestimen-tiferan HBL Hbs. This volume is particularlyinteresting. Indeed, on the one hand, it confirms thegeneral organization of HBL Hbs in 12 HGSs builton a linker complex comprising one central flatpiece, two bracelets of interHGS connections, andsix interhexagonal layer connections. The betterresolution (34.6 A by the DPR method and 27.7 Aby the FSC method) obtained for this volumeallows the observation of new structural detailssuch as the intraHGS links of the c3 connectionbody with mass numbers 1 and 6 or the localpseudo 3-fold symmetry of the HGS. On the otherhand, unexpected differences between Riftia andAlvinella Hbs, such as the intramolecular location ofthe HGSs with respect to the toroid and therespective positions of the six masses within theHGS, may be related to a special resistance to hightemperatures.

Materials and Methods

Animal collection and sample preparation

The polychaete annelid Alvinella pompejana(Desbruyere & Laubier, 1980) used in this study werecollected from 2600 m depth at 13°N sites (12° 46'N, 103°56'W and 12° 50'N, 103° 57'W) at the East Pacific Rise

during the HERO’92 expedition in April 1992. Wormswere picked using the manipulator of the Americansubmersible Alvin, and maintained at collection tempera-ture (2.4°C) in a thermally insulated container during thetrip to the surface (two to three hours). On board theanimals were opened dorsally, the blood, uncontami-nated with coelomic fluid, was withdrawn from the mainvessel into glass micropipettes and pooled on melting ice.The blood was centrifuged at low speed for a fewminutes and the supernatant was frozen in liquidnitrogen until the purification step.

In the laboratory, the defrosted sample was centri-fuged for ten minutes at 10,000 rpm (Zmax = 9 cm) at 4°C.Hb solutions were purified by gel-filtration on a1 cm × 30 cm Superose 6-C column (Pharmacia LKBBiotechnology, Inc.) using a low-pressure FPLC system(Pharmacia). The column was equilibrated with a salinebuffer as described (Zal et al., 1996a). The flow rate wastypically 0.5 ml per minute, and the eluate wasmonitored with a UV detector (Pharmacia). The elutionproduct corresponding to the HBL Hb was collected andconcentrated with a 10 kDa cut-off microconcentrator,Centricon-10 (Amicon).

Cryoelectron microscopy

To prevent the aggregation of the molecules on thesample grid, a 0.1 mg/ml dilution of the Hb wasprepared in 0.1 M Tris-HCI (pH 7.2), 10 mM CaCl2,10 mM MgCl2. A droplet of 5 ml was applied onto acopper grid covered with holey carbon film and a thinliquid film was allowed to form within the holes of thecarbon film by blotting the excess of solution. Frozen-hy-drated samples were prepared by rapidly plunging thegrid into liquid ethane (Adrian et al., 1984; Dubochetet al., 1988). Then, the frozen samples were transferredusing the Gatan 626 cryotransfer system into a PhilipsCM12 microscope equipped with a Gatan CCD cameraand a Gatan 651N anticontaminator. With the specimenat a temperature of −175°C, micrographs were recordedon Kodak film SO163 at an accelerating voltage of 100 kVand a magnification of 34,400 ×. Micrographs of theuntilted specimen were recorded, with −1.5 mm underfo-cus, at low dose (<10e−/A2) and developed for 15 minutesin full-strength Kodak D 19 developer.

Image processing

Digitization, windowing and alignment of the images

Micrographs of the untilted specimen that showed nodrift or blurring due to charge build-up in the specimenwere selected for digitization. An Optronix P1000 drumdensitometer was used to digitize the selected images at25 mm scan step resulting in a pixel size of 0.726 nm.Images of single particles, with a background free fromcontamination artifacts or overlapping molecules, wereselected from the micrographs. They were windowed,normalized, and rescaled so that their noise statisticswould match a reference distribution corresponding to aportion of micrograph free of particles and artifacts(Boisset et al., 1993). Finally, their contrast was invertedto render the protein material white and the backgrounddark. All programs used for image processing are part ofthe SPIDER (Frank et al., 1981a) and SIGMA (Taveau,1996) softwares.


3D reconstruction

The 3D reconstruction was carried out with the methodof random conical tilt series (Radermacher et al., 1987a,b)adapted for cryoelectron microscopy by Penczek et al.(1992, 1994). Here, since most of the projections of theparticle were present in untilted-specimen micrographs,we used a different method from that used for thereconstruction of Eudistylia, Macrobdella, Riftia andLumbricus Hbs (de Haas et al., 1996a,b,c; unpublishedresults). First, Eulerian angles were assigned to untilted-specimen images by a single cycle of 3D projectionalignment (Penczek et al., 1994), using as a reference the3D volume of a structurally related HBL Hb. Second, foreach direction of projection (defined by the first twoEulerian angles, f and u, in a 2°-spaced set of directions),the two images having the best correlation coefficientswith the projection of the reference 3D volume wereselected. Third, a 3D reconstruction was carried out onthe image subset resulting from the selection step asdescribed by Penczek et al. (1992, 1994). In this process,to take advantage of the symmetry present in themolecule, a modified version of the 3D reconstructionalgorithm (Penczek, 1992) was used. Finally, the 3Dreconstruction volume was refined with several cycles of3D projection alignment.

Symmetry, resolution

The presence of an authentic D6 point-group symmetrywas checked by a previously described test based on thecorrelation coefficients between the 3D volume in itsoriginal position and after rotations around its 2-fold and6-fold axes (Lambert et al., 1995).

For the resolution limit estimation of 3D volumes, twosubsets were randomly drawn and subjected to separate3D reconstructions. The corresponding pairs of 3Dvolumes were compared in reciprocal space on increas-ing spherical shells with the DPR (Frank et al., 1981b)using a phase residual threshold of 45° and the FSCcriteria (Saxton & Baumeister, 1982).

The topology sphere, a method for displaying thedirection of projections corresponding to the images

In the back projection procedure, obtaining an isotropic3D reconstruction volume requires that the variousdirections of projections are evenly represented. Toensure that no over- or underrepresentation of certaindirections occurs, we designed a graphical representationin terms of Eulerian angles of the directions of projectionof all the images used for the 3D reconstruction. In thisrepresentation, which we term the topology sphere, the3D volume is first placed at the center of the sphere withits 6-fold axis oriented along the axis passing by thepoles. Then, we project the volume in all the directionsof the sphere defined by the first two Eulerian angles (fand u), each direction of projection producing a point atthe intersection with the sphere. If the image set containsat least one image corresponding to a given projectiondirection (i.e. having the same first two Eulerian anglesdefined by the 3D alignment projection procedure), ablack dot is drawn on the sphere. Thus, empty areas onthe sphere correspond to the underrepresented directionsof projection. In addition, the overrepresented directionscan be detected by a histogram displaying the averagenumber of images per direction of projection as afunction of the second (u) Eulerian angle.

AcknowledgementsF.d.H. thanks Region Centre for the grant that enabled

him to work in Tours as a post-doctoral fellow. We thankthe members of the Alvin groups as well as the captainand crews of the R/V Atlantis II and R/V Vickers. Weare indebted to the chief scientists of the research cruiseHERO’92, H. Felbeck and J. J. Childress, who allowed usto conduct this work. We express our gratitude toProfessor Serge Vinogradov for very stimulating discus-sions and encouragement to carry out this work, and toDr Nicolas Boisset for reading the manuscript. This workwas supported by research grants from CNRS (UPR9042), INSU, UPMC, IFREMER (URM no. 7) and theDorsales program (CNRS, INSU, CNRS-SDV, IFREMER,BRGM, ORSTOM).

ReferencesAdrian, M., Dubochet, J., Lepault, J. & McDowall, A. W.

(1984). Cryoelectron microscopy of viruses. Nature,308, 32–36.

Baross, J. A. & Deming, J. W. (1985). The role of bacteriain the ecology of black smoker environments. Bull.Biol. Soc. Wash. 6, 355–371.

Boisset, N., Penczek, P., Pochon, F., Frank, J. & Lamy,J. N. (1993). Three-dimensional architecture ofhuman a2-macroglobulin transformed with methyl-amine. J. Mol. Biol. 232, 522–529.

Chevaldonne, P., Desbruyeres, D. & Childress, J. J. (1992).Some like it hot . . . and some even hotter. Nature,359, 593–594.

Chung, M. C. M. & Ellerton, H. D. (1979). Thephysico-chemical and functional properties of extra-cellular respiratory hemoglobins and chlorocruorins.Prog. Biophys. Mol. Biol. 35, 53–102.

de Haas, F., Taveau, J.-C., Boisset, N., Lambert, O.,Vinogradov, S. N. & Lamy, J. N. (1996a). Three-dimensional reconstruction of the chlorocruorin ofthe polychaete annelid Eudistylia vancouverii. J. Mol.Biol. 255, 140–163.

de Haas, F., Boisset, N., Taveau, J. C., Lambert, O.,Vinogradov, S. N. & Lamy, J. N. (1996b). Three-dimensional reconstruction of Macrobdella decora(leech) hemoglobin by cryoelectron microscopy.Biophys. J. 70, 1973–1984.

de Haas, F., Zal, F., Lallier, F. H., Toulmond, A. & Lamy,J. N. (1996c). Three-dimensional reconstruction ofthe hexagonal bilayer hemoglobin of the hydrother-mal vent tube worm Riftia pachyptila by cryolelectronmicroscopy. Proteins: Struct. Funct. Genet. in thepress.

Desbruyeres, D. & Laubier, L. (1980). Alvinella pompejanagen. sp. nov., Apharetidae aberrant des sourceshydrothermales de la ride Est-Pacifique. Oceanol.Acta, 3, 267–274.

Dubochet, J., Adrian, M., Chang, J.-J., Homo, J.-C.,Lepault, J., McDowall, A. W. & Schultz, P. (1988).Cryoelectron microscopy of vitrified specimens.Quart. Rev. Biophys. 21, 129–228.

Frank, J., Shimkin, B. & Dowse, H. (1981a). Spider—amodular software system for electron image process-ing. Ultramicroscopy, 6, 343–358.

Frank, J., Verschoor, A. & Boublik, M. (1981b). Computeraveraging of electron micrographs of 40S ribosomalsubunits. Science, 214, 1353–1355.

Garlick, R. L. & Riggs, A. (1982). The amino acid sequenceof a major polypeptide chain of earthwormhemoglobin. J. Biol. Chem. 257, 9005–9015.


Green, B. N., Suzuki, T., Gotah, T., Kuchumov, A. R. &Vinogradov, S. N. (1995). Electroscopy ionizationmass spectrometric determination of the completepolypeptide chain composition of Tylorrhynchusheterochaetus hemoglobin. J. Biol. Chem. 270, 18209–18211.

Lambert, O., Boisset, N., Taveau, J. C., Preaux, G. &Lamy, J. N. (1995). Three-dimensional reconstructionof the aD and bC-hemocyanins of Helix pomatiafrom frozen-hydrated specimens. J. Mol. Biol. 248,431–448.

Martin, P. D., Kuchumov, A. R., Green, B. N., Oliver,R. W. A., Braswell, E. H., Wall, J. S. & Vinogradov,S. N. (1996). Mass spectrometric composition andmolecular mass of Lumbricus terrestris hemoglobin: arefined model of its quaternary structure. J. Mol. Biol.255, 154–169.

Ownby, D. W., Zhu, H., Schneider, K., Beavis, R. C.,Chait, B. T. & Riggs, A. (1993). The extracellularhemoglobin of the earthworm Lumbricus terrestris.Determination of subunit stoichiometry. J. Biol.Chem. 268, 13539–13547.

Penczek, P., Radermacher, M. & Frank, J. (1992).Three-dimensional reconstruction of single par-ticles embedded in ice. Ultramicroscopy, 40, 33–53.

Penczek, P. A., Grassucci, R. A. & Frank, J. (1994). Theribosome at improved resolution: new techniques formerging and orientation refinement in 3D cryo-electron microscopy of biological particles. Ultra-microscopy, 53, 251–270.

Radermacher, M., Wagenknecht, T., Verschoor, A. &Frank, J. (1987a). Three-dimensional structure of thelarge ribosomal subunit from Escherishia coli. EMBOJ. 6, 1107–1114.

Radermacher, M., Wagenknecht, T., Verschoor, A. &Frank, J. (1987b). Three-dimensional reconstructionfrom a single-exposure, random conical tilt seriesapplied to the 50 S ribosomal subunit of Escherichiacoli. J. Microsc. 146, 113–136.

Saxton, W. O. & Baumeister, W. (1982). The correlationaveraging of a regularly arranged bacterial cellenvelope protein. J. Microsc. 127, 127–128.

Schatz, M., Orlova, E. V., Dube, P., Jager, J. & Van Heel,M. (1995). Structure of Lumbricus terrestris hemo-globin at 30A resolution determined using angularreconstruction. J. Struct. Biol. 114, 28–40.

Suzuki, T. & Gotoh, T. (1986). The complete amino acidsequence of giant multisubunit hemoglobin from thepolychaete Tylorrhynchus heterochaetus. J. Biol. Chem.261, 9257–9267.

Suzuki, T., Takagi, T., Furukohri, T. & Ohta, S. (1989). Thedeep-sea tube worm hemoglobin: subunit structureand phylogenetic relationship with annelid hemo-globin. Zool. Sci. 6, 915–926.

Taveau, J.-C. (1996). Presentation of the Sigma software.Software of Imagery and Graphics for MolecularArchitecture. J. Struct. Biol. 116, 223–229.

Terwilliger, N. B. & Terwilliger, R. C. (1984). Hemoglobinfrom the ‘‘pompeii worm’’ Alvinella pompejana anannelid from a deep sea hot hydrothermal ventenvironment. Mar. Biol. Letters, 5, 191–201.

Vinogradov, S. N. (1985a). The structure of erythro-cruorins and chlorocruorins, the invertebrate extra-cellular hemoglobins. In Respiratory Pigments inAnimals (Lamy, J., Truchot, J. P. & Gilles, R., eds)pp. 9–20, Springer Verlag, Berlin.

Vinogradov, S. N. (1985b). The structure of invertebrateextracellular hemoglobins (erythrocruorins) andchlorocruorins. Comp. Biochem. Physiol. 82B, 1–15.

Vinogradov, S. N., Kapp, O. H. & Ohtsuki, L. (1982). Theextracellular hemoglobins and chlorocruorins ofannelids. In Electron Microscopy of Proteins (Harris, J.,ed), vol. 3, pp. 135–163. Academic Press, New York.

Vinogradov, S. N., Lugo, S. D., Mainwaring, M. G., Kapp,O. H. & Crewe, A. V. (1986). Bracelet protein: aquaternary structure proposed for the giant extra-cellular hemoglobin of Lumbricus terrestris. Proc. NatlAcad. Sci. USA, 83, 8034–8038.

Vinogradov, S. N., Sharma, P. K., Qabar, A. N., Wall, J. S.,Westrick, J. A., Simmons, J. H. & Gill, S. J. (1991). Adodecamer of globin chains is the principalfunctional subunit of the extracellular hemoglobin ofLumbricus terrestris. J. Biol. Chem. 266, 13091–13096.

Weber, R. E., Malte, H., Braswell, E. H., Oliver, R. W. A.,Green, B. N., Sharma, P. K., Kuchumov, A. &Vinogradov, S. N. (1995). Mass spectrometriccomposition, molecular mass and oxygen binding ofMacrobdella decora hemoglobin and its tetramer andmonomer subunits. J. Mol. Biol. 251, 703–720.

Zal, F., Lallier, F. H., Green, B. N., Vinogradov, S. N. &Toulmond, A. (1996a). The multi-hemoglobin systemof the hydrothermal vent tubeworm Riftia pachyptila.II. Complete polypeptide chain composition investi-gated by maximum entropy analysis of mass spectra.J. Biol. Chem. 271, 8875–8881.

Zal, F., Green, B. N., Lallier, F. H., Vinogradov, S. N. &Toulmond, A. (1996b). Quaternary structure of theextracellular haemoglobin of the lugworm Arenicolamarina. A multi-angle laser light scattering andelectrospray ionisation mass spectrometry analysis.Eur. J. Biochem., in the press.

Edited by P. E. Wright

(Received 12 April 1996; received in revised form 9 September 1996; accepted 13 September 1996)

three-dimensional reconstruction by cryoelectron microscopy of the giant hemoglobin of the...

Documents