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Fibrinogen structure in projection at 18 Å resolution
J. Mol. Biol. (1991) 222, 89-98
Fibrinogen
Structure in Projection
at 18 A Resolution
Electron Density by Co-ordinated Cryo-electron Microscopy and X-ray Crystallography S. P. Sudhakara Rao, M. Damodara Poojaryt, Bruce W. Elliott, Jr1 Linda A. Melanson, Bruce Oriel$ and Carolyn CohenI/ Rosenstiel Basic Medical Sciences Research Center Brandeis University, Waltham, MA 022549110, U.S.A. (Received 20 October 1990; accepted 23 May
1991)
Electron microscope images of frozen-hydrated crystals of a proteolytically modified fibrinogen show excellent preservation of the structure. An electron density map of the key centric projection of the crystal at 18 A resolution has been obtained by combining the phases derived from cryo-electron microscopy with X-ray amplitudes. Simulation methods developed in earlier studies have been used to interpret the map. In contrast to the earlier images, the map allows us to visualize the coiled-coil region of the molecule and possible substructure in the /I domains. The map also shows that there is a marked difference in density in the two regions corresponding to the molecular ends where the y domains interact. A possible interpretation of this finding is provided by assuming substructure in the y domains and the breaking of molecular symmetry where these domains interact. Some additional constraints useful for the determination of the three-dimensional structure were obtained from cryo-electron micrographs of a perpendicular view at 25 A resolution. Implications of this working model for the molecular length and contacts in the filaments in both the crystal and fibrin are described. The data used here will be valuable as a star@ point for obtaining the t,hree-dimensional structure.
Keywords: fibrinogen:
cryo-electron
microscopy;
1. Introduction
t Present address: Texas A&M University? Department of Chemistry, College Station, TX 77843. 3255. U.S.A. $ Present address: Boston University, College of Basic Studies. 871 Commonwealth Ave, Boston, MA 02215, LJ.S.A. 4 Present address: Albert Einstein College of Medicine, 1396 Morris Park Ave, Bronx, NY 10461, U.S.A. 11Author to whom all correspondence should be addressed.
89 ~OS.OO/O
crystallography;
coiled coil: fibrin
were first obtained by digesting the bovine molecule with a crude protease from Pseudomonas neruginosa (Cohen & Tooney. 1974). This enzyme has now been purified and found to be specific for lysine residues (Elliott & Cohen, 1986). Previous analyses of these crystals by co-ordinated electron microscopy (using negative staining), image processing and X-ray crystallography (Weisel et al., 1981: Cohen et al., 1983) led to a low resolution (-30 A; 1 A = 0.1 nm) seven-domain model for the modified molecules. This so-called “heptad” model allows a correlation of the domains with specific regions of the amino acid sequence: in addition to a central domain formed by the three pairs of NH, termini, the COOH terminus of each chain folds independent,ly into a globular region (termed the CL /I and y domains). In the modified molecule, however, the c1 domain is removed by the protease (Wcisel et al.. 1985). The heptad model also accounts for the band pattern of negatively stained fibrin seen in the electron microscope. Despite extensive efforts, suitable isomorphous derivative crystals have not, yet been obtained, since fibrinogen is very sensitive t)o heav) metals. Previous studies on the crystal morphology,
Fibrinogen has been difficult to crystallize and its structure difficult, to solve, but detailed visualization of the molecule is essential for understanding its architecture and assembly into fibrin. Chemical studies indicate that fibrinogen is a dimer (M, 340,000): t,he molecule consists of three pairs of different polypeptide chains (termed Acr, BP and y). Native fibrinogen does not crystallize, but limited proteolytic cleavage with a variety of enzymes allows the modified molecules to form both microcrystals and crystals (Tooney & Cohen, 1977; Weisel rt nl.. 1978). Crystals suit,able for X-ray analysis
0022%2836/91/21008s-10
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1991 Academic,
Prrw
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diffracted intensity distribution and unit cell changes under different conditions (Tooney & Cohen, 1977; Weisel et al., 1978) have established aspects of the packing. The rod-shaped fibrinogen molecules of length 450 L%pack end-to-end to form filaments which run along the [3,0, l] diagonal direction, perpendicular to the crystallographic 2-fold axis. Patterson maps indicate that there are two possible positions for the molecules in the unit cell, one corresponding to a shift of about 135 A, and. the other 30 A, between symmetry-related molecules. Earlier image processing of electron micrographs of negatively stained crystal fragments viewed along the S-fold (unique) axis supported the former translation, indicating that symmetryrelated layers of molecules are staggered by about one-third the molecular length (Weisel et al., 1981; Cohen et al., 1983). The unit cell dimensions of the negatively stained crystal fragments vary greatly, however, due to dehydration and unit cell changes in preparation for electron microscopy, so that these images could not be used to phase the corresponding X-ray data from the crystals. Instead, intensities were calculated to a resolution of 30 A using various trial models derived from image analysis of the electron micrographs (Weisel et al., 1985). Recently we have taken advantage of the technique of cryoelectron microscopy, which has allowed us to obtain higher resolution images of the crystal without changes in unit cell dimensions, enabling us to phase the X-ray data directly. Here we report a description of the centric projection of the crystal at 18 L% resolution derived by using cryo-electron microscopic phases and X-ray amplitudes. Simulation methods allow us to make a preliminary interpretation of the electron density map: the results confirm certain aspects of the earlier model, reveal the or-helical coiled-coil region, possible substructure in both the fl and y domains, and the breaking of molecular symmetry at the end-to-end contacts.
2. Materials and Methods (a) Crystallization
Crystals of modified bovine fibrinogen were obtained as described (Tooney & Cohen, 1977; Weisel et al., 1978) with some modifications. (The dialysate now contained 2 IIIM-Cd&, 5 mM-Pu’aN, and was buffered with 10 mM-Mes at pH 62; KSCN was no longer used.) After dialysis, the digested fibrinogen (at a concentration of 2 to 3 mg/ml) was transferred to acid-washed vials (each containing @5 ml). Seeding has been found to be necessary when using the purified protease for digestion (Elliott C Cohen, 1986). The vials were kept at 4°C and the crystals grew in about 1 month. More recently, we have grown crystals in about 1 week at 22.5”C. Despite these
changes, it is still very difficult to obtain crystals suitable for X-ray analysis, although small crystals are readily and reproducibly obtained (see below). Diffraction from these crystals is anisotropic extending to 3.5 A in the direction of the molecular filaments and to about 6 A in perpendicular directions. The space group is P2, with a = 135 A, b = 9%1 A, c = 175 .& fl= 92”. There is one molecule per asymmetric unit in the unit cell.
(b) X-ray data collection A low resolution (20 A) and t,wo 6 A data sets have been collected. In these experiments, the crystals were cooled to 4°C. The low resolution data set was collected using precession photography on an Elliott GX6 X-ray source. Special care was taken to scale the strong reflections to the rest of the data by monitoring the incident beam with a NaI-photomultiplier detector. Data from 11 zero-level photographs taken along various crystallographic directions were scaled and merged (72 y/o complete, Rmsrg--@045). The 6 A data sets were collected both at CHESS (Cornell High Energy Synchrotron Source), by oscillation methods, and at the University of Virginia, using the multiwire area detector. The processed data sets contain about 70% of t,he total reflections with value of @09. an kg (c) Cryo-electron microscopy The P2, crystals cleave readily along planes perpendicular to the unique axis into thin sheets (Weisel et al.. 1981) suitable for the cryo-electron microscopy. Specimens were also prepared by overseeding solutions of the modified molecules (at about 4 mg/ml) at 22.5”C; small crystals grew overnight and were concentrated and fragmented by centrifugation. Crystal fragments were rapidly frozen by plunging into liquid propane cooled to liquid nitrogen temperature (Dubochet et al., 1988). Specimens were examined in a Philips EM420T transmission electron microscope equipped with a Gatan 626 cryoholder and operated at an accelerating voltage of 100 kV. Low dose images (< 10 electrons/A2) were recorded in vitreous ice at - 174°C at a magnification of 47,000 x and an underfocus of @7 pm. Micrographs were initially screened by optical diffraction (Salmon & DeRosier, 1981) to locate areas suitable for computer processing. Selected micrographs were densitometered on an Optronics Photoscan PlOOO microdensitometer (Optronits Inc., Chelmsford, MA. U.S.A.) using a sampling interval of 25 pm corresponding to 53 A on the specimen. The images were digitized on a 512 x 512 pixel array and displayed using a Grinnel graphics system with 256 grey levels (Grinnel System Corp., Santa Clara, CA, U.S.A.). Suitable areas were chosen using television screen cursors. The boxed-off areas were floated (namely, the average optical density of the perimeter of the box was subtracted from all the values within the box) and Fourier transformed. The images were corrected for lattice distortions by real-space correlation analysis (Henderson et al., 1986). The amplitudes derived from these images were corrected for the effects of the contrast transfer function (C(v)): C(v) = A(v)sinX(v)+B(v)cosX(v), x = 27r-‘(@fe2-&y4), where A and B represent the fractional contribution due to phase and amplitude contrast transfer functions, respectively and v( = e/n) is the spatial frequency in the object. The defocus, SJ was estimated from the positions of the Thon rings. C, is the spherical aberration coefficient, 1 is the electron wavelength and 0 is the scattering angle. The percentage of the amplitude contrast was determined from a comparison of the low resolution amplitudes derived from images with those obtained from X-ray diffraction; this value was about 5%, which is close to that found using pairs of defocused images (Toyoshima & Unwin, 1988). The agreement between the high resolution amplitudes in the 2 types of data was further improved with the application of a temperature factor correction based on Wilson plot analysis.
Structure of Fibrinogen
in 18 A Resolution
(bl
.
..^“-.-
.-----__~-.-”
.-,-___
I
~I--
---~ll-.~l.-^---lll--~--
.,
Figure 1. Representative electron micrographs of P2, crystals of modified bovine fibrinogen with computed Fourier transform of a selected image together with the corresponding X-ray precession photograph. (a) Electron micrograph of the [OlO] view of a thin crystal fragment of fibrinogen (protein is black). A crystal area selected for Fourier processing is shown in the inset. The images show strands running along the [3,0, l] diagonal direction of the unit cell in this projert,ion. The strands represent molecular filaments with an effective period of 450 A. (We have not measured the thickness range of the crystal fragments since our studies to date have dealt with nominally untilted views.) (b) A computed Fourier transform from an image-processed micrograph in (a) after corrections (see the text) showing Hragg reflection to 15 A resolution. (c) X-ray precession photograph of the same view of the crystal showing the low resolution reflections to about 15 A resolution. The computed transform from the cryo-electron micrograph (b) displays close correspondence with the X-ray photograph.
3. Results and Discussion (a) Electron density map in projection Representative images of the centric [OlO] view are shown in Figure l(a). The computed transform of this view shows Bragg reflections rather uniformly up to 18 A, occasionally extending to 15 A resolution (Fig. 1(b)). After appropriate corrections (see Materials and Methods), the transforms show good correspondence with the X-ray diffraction pattern of the same view (Fig. l(c)), including unit cell parameters. (The R-factor between the X-ray amplitudes and the corrected amplitudes obtained by merging 9 images is 0.18.) The phase origins of the computed transforms of the
images selected were placed on a common 2-fold axis, and the phase for each reflection was then calculated by averaging corresponding phases from individual images, which were weighted according to the signal-to-noise ratio. Data were not used if the averaged phase deviated by more than 45” from 0 or n; in such cases, the reflections were very weak (see also the legend to Fig. 2). In this way, 93% of the X-ray amplitudes in the hOEzone were phased up to 18 A resoluti on. The quality of the data is evident from the low phase residual (143”) from merging and the tendency of the phases to follow the symmetry constraint (Fig. 2). The electron density map computed using these symmetryconstrained phases and the X-ray amplitudes is
92
S. P. S. Rao et al.
270.0
K/
I
-90.0~ 0.00
0.200 Resolution
0.400 (nm-’ )
0.600
Figure 2. Quality of phases in the merged data set derived from 9 images. The plot shows the deviation of the averaged phases from the symmetry-required values, 0 or n. Phases were constrained to 0 or K and combined with X-ray amplitudes
to compute the projection
shown in Figure 3. A map computed tional
data to 15 a showed
(b) Interpretation
very
similar
map.
using addifeatures.
of the map
The map shows features of macromolecular size running in the diagonal [s, 0, l] direction and has an overall similarity (including the relative sizes of the
molecular domains) to the lower resolution electron microscope images of this view of the crystal in negative stain. The repeating unit along the strand is a complex group of seven regions of varying size, shape and density (generally about 40 A in width). The highest density region (located at a crystallographic 2-fold axis) lies midway between two “birdshaped” regions (see the legend to Fig. 3) that are oppositely oriented on either side followed by a long region of weak density. This motif repeats every 450 A and corresponds to the projection of two symmetry-related molecular filaments in the unit cell. In order to interpret the map we applied simulation methods developed in earlier studies (Weisel et al., 1981; Cohen et al., 1983). Using a shift of 135 L% (about one-third of the molecular length) between symmetry-related molecules, the map can be interpreted by a working model that reveals new features of the molecule and its packing into filaments. For the first time, the map allows us to visualize possible substructure in the fl domain. In this projection (Fig. 3), the “bird-shaped” density corresponds to a /3 domain partly eclipsed by the central domain of the symmetry-related molecule (Fig. 4(a)). On the simplest interpretation, the a
Figure 3. Electron density map of the [OlO] projection in grey scale (protein is black) derived from the electron microscope phases (see the text) and X-ray amplitudes. A contour plot of the same map drawn with positive contours at intervals of 0.1 of the maximum density is shown in the inset. The projected unit cell (a = 135 L%,c = 180 8, B = 91”) is outlined and the 2, screw axes are indicated. The unit cell contains 2 fibrinogen molecules related by the screw symmetry along b. A schematic diagram of 2 neighboring translated filaments each with 2 “end-to-end” bonded molecules is superimposed on the map. Note that, for clarity, neither of their screw-related filaments is depicted. (In Fig. 4(a) both sets of filaments are shown.) The /?and y domains of a single molecule in a filament are labeled; note the distinctive “birdshaped” density where one of the /? domains is located (one such “bird-shaped” region in a neighboring filament is marked by an arrowhead). The shape and height of the strong density peaks reflect the size, shape and overlap of the different globular domains. The weak density regions between the domains represent the a-helical coiled-coil portion of the molecule. A small bend in the molecule near the end domains accounts in part for the slightly wavy appearance of the strands.
Structure of Fibrinogen
in 18 A Resolution
93
(a)
(b)
Figure 4. (a) Schematic diagram of the crystal packing viewed approximately along the h axis to show the arrangement of the symmetry-related molecules that overlap in the [OIO] view. For clarity the model shown is that of the symmetrical molecule (Weisel et al., 1985) but lacking the smallest “domain”. The symmetry-related molecules lying below are hatched. The location of the filaments is such that the “end-to-end” linking regions are in close proximity to a symmetry axis that results in a cluster of 4 y domains (one such region is boxed), 2 from each of the symmetry-related filaments. (1~) Simulation of the [OlO] view of the crystal by the symmetric. “short” and “long” models showing the substructures in the /I and y domains and the breakdown of the non-crystallographic symmetry at the 2 molecular ends. One of the regions where the molecules interact end-to-end to form filaments is outlined in each of’ the diagrams. As described in (a), t)he marked regions contain 4 y domains. Schematics of various types of models are superimposed on t,he simulation diagrams; these also show the nature of end-to-end linkages for these models. The precise shapes of t,he domains and their possible interactions are not yet established. (i) Electron density map of the [OIO] view as shown in Fig. 3. (ii). (iii) and (iv) Computer simulations of the symmetric (Weisel et al.. 1985), “short” and .‘long” mod&, respectively (see the text). Unlike the symmetric model, both the fi and y domains of the molecules shown in (iii) and (iv) caonsist of 2 subdomains of different sizes. As expected, the symmetric model (ii) shows equal density for all 4 interacting y domains. Tn contrast, the map in (i) shows that this region consists of 2 strong and 2 weak density features. This feat,ure is well simulated by both the models in (iii) and (iv) where the y domains interact differently at 2 molecular ends (see the text). Note that t,he positions of the larger subdomains of the y domains at the 2 molecular ends are different in the 2 structures and that in both models the molecular symmetry is broken.
domain would comprise two subdomains, the larger oriented along and the smaller at an angle to the [3,0, l] direction. The same structure would be expected for the other b domain (the highest density region), but the shape here is completely obscured by overlapping of the symmetry-related /? domain (Fig. 4(a)). The map also shows that the distance between the center of the B domain and the central “disulfide knot”) domain of the symmetry-related molecule is about 30 A (Fig. 4(a)). In the Harker section of the Patterson maps (data not shown), a strong peak at 30 A (which could have corresponded to the shift between layers) may now be accounted for most plausibly by this vector. (In images of the
negatively stained crystals, due t’o t,he lower resolution, the /l and cent,ral domains of symmetry-relat’ed molecules overlap, obscuring these features.) A striking feature of the map is the marked difference in density in the two regions corresponding to the molecular ends where t,he y domains interact (Figs 3 and 4(a)). These regions consist of two pairs of y domains: one pair from a filament in the plane and the other pair from the symmetryrelated filament. If the molecular dyad were in or near the ac plane (as shown from other findings described below) and the molecules were bonded end-to-end. the map would display equal densities for all four y domains (Fig. 4(b). panel (ii)). In
94
S. P. S. Rae et al.
contrast, the map appears to show that the electron density of the y domain is significantly lower at one end of the molecule than at the other end (Figs 3 and 4(b), panel (i)), indicating that the molecule cannot possess perfect 2-fold symmetry in the ac plane. This feature has also been observed in images of negatively stained crystals. In principle, asymmetric cleavage of the y domains during proteolytic modification could account for the difference in density. Studies by SDS/polyacrylamide gel electrophoresis, however, show that the y-chains are essentially intact: they have lost a mass equivalent to about 15 residues and retain the ability to be crosslinked by factor XIII (Elliott et al., 1987). A plausible explanation of this feature of the map is that there is a breakdown of symmetry at the two molecular ends, possibly due to packing constraints in the crystal lattice. On this view, the map can be simulated by assuming that the y domain consists of two subdomains of unequal size (the distal one being larger) which are oriented slightly differently at the two ends of the molecule. Such a division is supported by recent scanning microcalorimetric studies by Medved’ et al. (1986). It is consistent as well with the strong homology of the amino acid sequences of the /? and y chains, and the projected appearance of the b domain described above. (Note, however, that the subdomains of the /I and y chains appear to be arranged differently (see below).) By considering the packing constraints in the crystal, together with the positions of the density peaks in the map, we have found only two types of molecular model. In one (termed “short”), the distal y subdomains are laterally displaced from the molecular axis in the same direction, but to different extents, and are displaced from the ac plane in opposite directions (Fig. 4(b), panel iii)). In this case, the molecules have a length of 450 A when projected along the molecular axis (but a contour length of 480 A). In the other (termed “long”), the distal y subdomain at each molecular end lies nearly in the ac plane, with one of them tilted by about 15” from the molecular axis (Fig. 4(b), panel (iv)). Consequently, the joint between the molecules in the filament is not “end-to-end” but involves a lateral overlap of a distal and proximal subdomain. The length of the molecule would then be about 480 A, although the effective repeat in the filament is still 450 A. (c) Support for the “long” model from a perpendicular view A choice between the two models can be made from cryo-images of other views of the lattice. In this connection, the [OOl] view (Fig. 5(a)), which is perpendicular to the [OIO] projection, is particularly informative. Although the best images of this view obtained thus far have a resolution of only about 25 A, they provide support for the “long” model. The image (Fig. 5(b), panel (i)) is very distinctive: it consists of a strong density striation spaced at 135 A
(the a cell dimension of the crystal) separated by weak density regions. Simulations were carried out as described to compare the symmetric, “long” and “short” models. Only the latter two models accounted satisfactorily for the strong densities because of the similar disposition of the central and fi domains. The lower density peaks which correspond to the projection of the y domains, however, were well simulated by the “long” model (Fig. 5(b), panel (iv)), but by neither of the others (Fig. 5(b), panels (ii) and (iii)). This result has been checked by simulating images of negatively stained crystals which have unit cell parameters similar to the hydrated crystals in this view. These findings indicate that, to a first approximation, the y domains are disposed nearly along the length of the molecule as in the “long” model. (d) Visualization
of the coiled coil
The region of weak density in the [OlO] projection (Fig. 3) between the central and /I domains corresponds to part of the u-helical coiled coil portion of the molecule. This region appears rather diffuse in the map and is generally not visible in images of negatively stained crystals. Although the overall width of a three-stranded a-helical coiled coil is about 23 A, this dimension cannot be estimated accurately from the map because of the limited resolution and the overlap of symmetry-related molecules in projection. Within the coiled coil, there are two very weak peaks, centered at about 100 A from the midpoint of the central domain (Fig. 3). In studies from this laboratory, evidence for the presence of a small “domain” near the center of the coiled coil was obtained from a specific stainexcluding region in images of negatively stained bovine fibrin and certain microcrystal forms of fibrinogen (Weisel et al., 1981, 1985; Cohen et al.: 1983). This small domain was incorporated into the heptad model as the presumed site of plasmin cleavage (Weisel et al.? 1983). In human fibrin however, this stain exclusion is not pronounced. The intensity of this band in images of negatively stained bovine fibrin is considerably reduced after limited proteolysis; strong density would therefore not be expected for this region of the modified molecule in the electron density map. In attempts to account for this density we have re-examined available amino acid sequences of a number of fibrinogens (Chung et al., 1981; Crabtree & Kent, 1982; Henschen et aE., 1983; Crabtree et aE., 1985; Brown et al., 1989) and do not find any major interruption in the heptad pattern of residues in the a-helical coiled coil which could correspond to a non-helical domain (see also Cohen & Parry, 1990). Moreover, the breaks in the heptad repeat appear to vary from one chain to another (Conway & Parry, 1991). The two very weak peaks seen in this region of the map (and represented in Figs 3 to 6) should therefore probably not be identified with a specific domain; nor is it clear that they correspond to regions of plasmin attack. The origin of the additional density in
Xtructure of Fibrinogen
in 18 d Resolution
95
(a)
Figure 5. (a) Schematic diagram of the crystal packing viewed approximately along the c axis. As in Fig. 4(a), the symmet)rical molecule is shown, but here the length of the b axis is doubled for clarity. The filaments form sheets in the UC planes which stack along the b axis as shown in the Figure. The symmetry-related layer is hatched. (b) Simulation of the [OOl] projection of the crystal by the symmetric, “short” and “long” models. The marked regions in these diagrams consist of the 2 y domains, one from each of the end-to-end interacting molecules in a filament. Schematic models are superimposed on each of the simulation diagrams. Note that in this view the projected filament repeat is about 405 A. (i) Electron density map of the [OOI] projection derived from the electron microscope phases and X-ray amplitudes. This view is much rarer than the [OlO] projection (Figs 1, 3 and 4), although it is frequently seen in negative stain. The image was processed as described, but no symmetry has been imposed on the phases; nevertheless the screw symmetry along the b axis is clearlz.visible. The projected unit cell (a = 135 A, b = 98 A, y = 90”) and the location of the symmetry axes are marked. (ii), (m) and (iv) Computer simulations of the symmetric (Weisel et al., 1985) “short” and “long” models, respectively. Neither the strong density striations (corresponding to the overlapping of central and /? domains) nor the weak density regions (corresponding to the interacting y domains) in (i) are well simulated by the symmetric model (see (ii)). Both the “short” and “long” models generate the strong density striations equally well. Note, however, that the pattern of t,he weak density regions (boxed) is correctly simulated only by the “long” model.
images of negatively to be established.
stained bovine
fibrin
remains
(e) Inferences on crystal packing and molecular symmetry The chemistry and morphology of the fibrinogen dimer indicate that there is a 2-fold axis perpendicular to the long axis of the molecule.
This dyad is not
expressed in the P2, crystal, nor in the other crystal forms of modified fibrinogen (Hewat et al., 1983; Gollwitzer & Rode, 1986), which all show one dimeric molecule in the asymmetric unit. Information about the orientation of the molecular Z-fold axis in the P2, crystal, as well as its location, may, however, be obtained by analysis of the X-ray amplitudes. Our results from R-factor and correlation coefficient searches, as well as Patterson maps computed using 20 A data, show that the major part of the molecule contains a 2-fold axis near the
ac plane; this dyad is located at about 65 A from the origin (our unpublished results). Moreover, by assuming that this elongated molecule consists mainly of discrete spherical domains, a Patterson self-rotation function search was carried out (using both 20 and 6 A data), which also showed that the molecular dyad is oriented about 5” to the ac plane. Our interpretation of the [OlO] projection above was based on these results, but we required as well the assumptions of two subdomains in the y domain and the breaking of symmetry at the two molecular ends. In both models described only the distal y subdomains appear to depart from the molecular 2-fold symmetry. Although we lack detailed three-dimensional information on the packing in the lattice, the molecules in neighboring filaments appear to interact closely at the region of the y domains in the CLC plane. Moreover, whatever the model, the contacts made at the two ends of the molecule are different
96
S. P. S. ho
(see Fig. 4). In both models, the distal y subdomain at one end appears to make close contact with a region of the coiled-coil portion of the neighboring translated filament. The resulting bend in this region produces the “waviness” of the filaments. Such a contact is not possible for the distal subdomain at the other end of the molecule which appears to interact with a coiled-coil region of the molecule from the symmetry-related filament. Moreover, the tendency of the crystals to cleave readily in a direction parallel to the ac plane demonstrates that the interactions of the filaments in this plane are considerably stronger than those between the symmetry-related layers. It is therefore likely that these packing contacts in the crystal, together with some flexibility of the molecule, lead to the departure of the two ends from exact 2-fold symmetry. (f) Implications
et al.
(b)
(cl
for $brin assembZy
Despite the preliminary nature of our current interpretation, it is worthwhile to consider the implications for fibrin assembly of the novel features that we have inferred for the molecule and its packing in the filaments. In all of our previous studies we have taken fibrin as but one of the polymorphic forms of fibrinogen (Cohen et al., 1983). In other words, the crystalline arrays produced by modified fibrinogen, aa well as fibrin, may be accounted for on the basis of the same molecular filaments, packed in various ways, with minor differences due to different packing environments. In the P2, crystals we have shown that both classes of filaments (symmetry-related as well as translated along a) are shifted by about one-third of the molecular length with respect to one another; in fibrin, the shift is one-half the molecular length. We have therefore examined the possible packing in fibrin of the filaments derived from the “long” model. Fibrin assembly appears to involve the formation of half-staggered dimers to which additional molecules are added to yield a two-stranded protofibril. In these aggregates each y domain is involved in at least two kinds of linkages: one to the complementary binding sites in the central domain exposed by the loss of the fibrinopeptides; and the other to the y domain on the neighboring molecule in the filament. Because of the half-staggered arrangement, an essential feature of the fibrin packing would be equivalent linkages of the neighboring y domains in a filament with a central domain. We can build a model for fibrin, using filaments closely similar to those in the crystal lattice, by slightly flexing the molecules at the ends to restore 2-fold symmetry. A half-staggered assembly of filaments generated in this manner from the “long” model accounts well for the 225 A repeat and the band pattern in the images of negatively stained fibrin (Fig. 6). In this assembly, the central domain fits into the pocket formed by the two interacting y domains (Weisel et al., 1983), giving a “closed” protofibril (Fig. 6(a)). It
Figure 6. A model for fibrin assembly. Tn order to construct a packing model for fibrin, the “long” model is made symmetrical by slightly displacing the distal y sub domains. The length of the molecule is still about 480 A. (a) A half-staggered assembly of filaments (viewed as in Fig. 4(b), panel (iv)) generating a “closed” protofibril (Weisel, 1987). The bonding of neighboring y domains in the filaments in fibrin is virtually the same as that in the filaments in the crystal, but symmetrical linkages with the central domain of the staggered filament can now be made. An enlarged portion of a filament (boxed) showing this overlapping region is seen in the inset (top right). The protofibrils aggregate laterally in a complex way (but still in a half-staggered fashion) to form fibrin which is shown schematically in (b). (c) An electron micrograph of negatively stained fibrin showing how the domain sizes and positions in the model account for the stain-excluding bands. As noted in Results, section (d), however, the origin of the extra density in the region where we have shown a small “domain” is not yet accounted for. Notes that because of the overlapping of the adjacent, end domains (in (a) and (b)), the effective repeat in the filament is 450 A and that in the protofibril is 225 A.
is also of interest that the lateral overlapping of the y domains in the filaments now provides a simple structural explanation for the finding of an ant’iparallel arrangement of y chain crosslinks (Doolittle. 1981). (We may note that a similar study for the filaments derived from the “short” model gives a less satisfactory packing for fibrin.) On the basis of this analysis of fibrin assembly, as well as the simulation
results
of the two-dimensional
data discussed
above, we consider that the “long” model at present. gives the best 6t to the available data. (g) Additional
implications
We summarize our current picture of the model with a schematic diagram of the organization of the polypeptide chains in the molecule (Fig. 7). Such a picture is, of course, heuristic, since it is based on
Structure of Fibrinogen
97
in 18 A Resolution
other proteins having domains with this motif will also be discovered (Baker et al., 1990). We are extending our structural analysis of fibrinogen to three dimensions microscopy, and have recently
-. P Figure 7. A schematic representation of the organization of the polypeptide chains in the native fibrinogen molecule (based on the “long” model) showing the correlation of the structural features with specific regions of the chains. The diagram depicts the sequence of human fibrinogen, but the available sequences of bovine fibrinogen are closely homologous to those of human fibrinogen. The view is as shown in Fig. 4(b), panel (iii), where the molecular symmetry axis is in the plane of the paper. The NH, termini of the 3 pairs of non-identical chains (Acc, BP and y) form the central domain. The portions of the chains between 2 sets of disulfide rings (1) are mainly a-helical forming an irregular 3-stranded coiled coil. (In the rod region as drawn, the a-chain is at the bottom and the /I chain at the top: AM, predicted to be a-helical coiled coil with breaks indicated by vertical lines; p, possible nonhelical segments (Conway & Parry, 1991).) The COOH termini of the Bfl and y chains fold independently into globular domains with substructure: about 2/3 of each chain (after the outer disulfide ring) forms a larger subdomain and the rest a smaller subdomain. This region of the /I chain is shown in bold. (Not illustrated are the disulfide bonds common to both chains, and an additional disulfide bond unique to the /l chain.) The flexible COOH termini of the Aa chains fold back to form an additional central domain which is, however, absent from the molecules aft)er proteolytic rleavage (see also Weisel et aZ., 1986).
low resolution
two-dimensional
data.
Note
that
in
t)his drawing (as well as in the models used for the simulations) the /I and y domains are arranged differently. In keeping with the high homology of the sequences, however, the tertiary structures of the two domains are depicted as very similar. Such a picture presents a problem, but this disposition could be produced by a simple rotation near the out,er disulfide ring that might be related to the additional disulfide bridge in the /I chain. At this stage of structure determination, any such details of domain structure are purely speculative. These findings are relevant also to the structure and interactions of the extracellular matrix glycoprotein cytotactin (also called tenascin (see Erickson B Rourdon, 1989)). The COOH-terminal end of each arm of this “hexabrachion” structure appears as a ‘~knob” in electron micrographs of rotary-shadowed molecules (Erickson & Iglesias, I9S4).
This
knob
contains
207 amino
acid residues
are highly homologous to the sequence of the b and y domains of human fibrinogen, including the position of an intrachain disulfide bridge (Jones et al., 1989). We would. t,herefore, expect that the that
(‘OOH-terminal
domain
of
cytotactin
has
a
substructure similar to that of the fibrinogen /I?and 7 domains. The lateral overlapping of the y domains currently envisaged for the filaments of fibrinogen may thus display features of some of cytotactin’s various int.eractions. Tt is likely that a number of
using cryo-electron obtained three non-
centric views of the crystals at about 20 A resohtion. We are now attempting to collect sufficient views to phase a major portion of the X-ray data. This paper provides a starting point’ for obtaining a three-dimensional map by density modification techniques, utilizing phase constraints from both centric and non-centric views. As the structure of the molecular filaments is extended t’o three dimensions and to higher resolution, we should be able t’o clarify the physical basis for the apparent molecular asymmetry as well as delineate feat,ures of domain substructure with more reliability. We thank J. W. Weisel for many helpful discussions, I). Stokes for contributions to the analysis of the model as well as for valuable suggestions, J. I’. Griffith for carrying out the rotation function search and for his encouragcment in our use of cryo-electron microscopy. R. Kretsinger. S. Sobottka and R. J. Chandross of the University of Virginia for their generous help in the use of the area detector, C. V. Stauffacher and W. C. Phillips for help with data collect,ion, R. Stryker and K. Giese for crystallization studies. J. Black for photography, ,J. Khorana for drawings, P. Vibert and P. Walian for help with the manuscript, and L. Seidel and B. Finkelstein for typing. Supported by grant AR17346 from NIH to c‘.C. Funds to purchase and maintain the computer system were obtained from a shared instrumentation grant l-SlORR04671-01 awarded to David $1. DeRosier by the National Institutes of Health. Funds to purchase and maintain the Philips EM420T transmission electron microscope were obtained from a shared inst’rument,ation grant l-SlO-RR02464-01 awarded t#o(‘.(‘. by t,he National Institutes of Health.
References Baker, N. E., Mlodzik, M. t Rubin, G. M. (1990). Spacing differentiation in the developing Drosophila eye: a fibrinogen-related lateral inhibitor encoded by scabrous. Science, 250, 1370-1377. Brown, W. M., Dziegielewska, K. M., Foreman. R. (1. &
Saunders, N. R. (1989). Nucleotide and deduced amino acid sequence of a y subunit of bovine fibrinogen. Nucl. Acids Res. 17, 6397. Chung, D. C., Rixon, M. W., MacGillivray, R. T. A. & Davie, E. W. (1981). Characterization of a cDNA clone coding for the B chain of bovine fibrinogen. PTOC. Nat. Acad. Sci., IT2.A. 78. 1466-1470. Cohen, C. & Parry, D. A. D. (1990). a-Helical coiled coils and bundles: how to design an a-heliral protein. Proteins: Struct. Fun& Genet. 7, l-15. Cohen, C. & Tooney, N. M. (1974). Crystallization of a modified fibrinogen. Nature (London), 251, 659-660. Cohen, C., Weisel, J. W., Phillips, G. N., Jr. Stauffacher, C. V., Fillers, ,J. P. & Daub, E. (1983). The structure of fibrinogen and fibrin: I. Electron microscopy and X-ray crystallography of fibrinogen. In Molecular Biology of Fibrinogen and Fibrin (Mosesson. N. W. & Doolittle. R. F., eds), vol. 408. pp. 194&213, Annals of the New York Academy of Sciences. Nrw York.
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Conway, J. F. & Parry, D. A. D. (1991). Three-stranded a-fibrous proteins: the heptad repeat and its implications for structure. Zntl. J. Biol. Macromol. 13, 14-16. Crabtree, G. R. & Kant, J. A. (1982). Organization of the rat y-fibrinogen gene: alternative mRNA splice patterns produce the yA and yB(y’) chains of fibrinogen. Cell, 31, 159-166. Crabtree, G. R., Comeau, C. M., Fowlkes, D. M., Fornace, A. J., Jr, Malley, J. D. & Kant, J. A. (1985). Evolution and structure of the fibrinogen genes. Random insertion of introns or selective loss? J. Mol. Biol. 185, 1-19. Doolittle, R. F. (1981). Fibrinogen and fibrin. Sci. Amer. 245, 126-135. Dubochet, *J., Adrian, M., Chang, J. J., Homo, J. C.. Lepault, J., McDowell, A. W. 6 Schultz, P. (1988). Cryo-electron microscopy of vitrified specimens. Quart. Rev. Biophys. 21, 129-228. Elliott, B. W., Jr & Cohen, C. (1986). Isolation and characterization of a lysine-specific protease from Pseudomw aeruginosa. J. Biol. Chem. 261, 11259-l 1265. Elliott, B. W., Jr, Stryker, R. & Cohen, C. (1987). Biochemical characterization of modified molecules of bovine fibrinogen that form crystals. Fed. Proc. Fed. Amer. Sot. Exp. Biol. 46, 2243. Erickson, H. P. & Bourdon, M. A. (1989). Tenascin: an extracellular matrix protein prominent in specialized embryonic tissues and tumors. Annu. Rev. Cell Biol. 5, 71-92. Erickson, H. P. & Iglesias, J. L. (1984). A six-armed oligomer isolated from cell surface fibronectin preparations. Nature (London), 3 11, 267-269. Gollwitzer, R. & Bode, W. (1986). X-ray crystallographic and biochemical characterization of single crystals formed by proteolytically modified human fibrinogen. Eur. J. Biochem. 154, 437-443. Henderson, R., Baldwin, J. M., Downing, K. H., Lepault, J. & Zemlin, F. (1986). Structure of purple membrane from Halobacterium halobium: recording, measurement and evaluation of electron micrographs at 3.5 A resolution. tJltramicroscopy, 19, 147-178. Edited
Henschen, A., Lottspeich, F., Kehl, M. & Southan, C. (1983). Covalent structure of fibrinogen. In Molecular Biology of Fibrinogen and Fibrin (Mosesson, N. W. & Doolittle, R. F.. eds), vol. 408, pp. 28-43, Annals of the New York Academy of Sciences, New York. Hewat, E. A., Tranqui, L. & Wade, R. H. (1983). Electron microscope structural study of modified fibrin and a related modified fibrinogen aggregate. J. Mol. Biol. 170, 203-222. Jones, F. S., Hoffman, S., Cunningham, B. A. & Edelman, G. M. (1989). A detailed structural model of cytotactin: protein homologies, alternative RNA splicing and binding regions. Proc. Nat. Acad. 9ci., U.S.A. 86, 1905-1909. Medved’, L. V., Litvinovich, S. V. & Privolov, I’. L. ( 1986). Domain organization of the terminal parts in the fibrinogen molecule. FEBS Letters, 202, 298-302. Salmon, E. D. & DeRosier, D. J. (1981). A surveying optical diffractometer. J. Microsc. 123, 239-247. Tooney, N. M. & Cohen, C. (1977). Crystalline states of a modified fibrinogen. J. Mol. Biol. 110, 363-385. Toyoshima, C. & Unwin, N. (1988). Contrast transfer for frozen-hydrated specimens: determination from pairs of defocused images. Ultramicroscopy, 25, 279-292. Weisel, J. W. (1987). Molecular symmetry and binding sites in fibrin assembly. Thromb. Res. 48, 615-617. Weisel, J. W.. Warren, S. C. t Cohen, C. (1978). Crystals of modified fibrinogen. Size, shape and packing of molecules. J. Mol. Biol. 126, 159-183. Weisel, J. W.? Phillips, G. N., Jr & Cohen C. (1981). A model from electron microscopy for the molecular structure of fibrinogen and fibrin. Nature (London), 289, 263-267. Weisel, J. W., Phillips, G. N.,
Note added in proof: Recent improvements in image collection and processing have the [OlO] view to beyond 10 A resolution. A projection map from these data is lower resolution map reported here and shows additional features of the molecular a resolution of about 10 A have also been obtained for certain near-perpendicular goal is a 3-dimensional map to about this resolution.
enabled us to phase consistent with the filaments. Phases to views. Our current
Fibrinogen
Structure in Projection
at 18 A Resolution
Electron Density by Co-ordinated Cryo-electron Microscopy and X-ray Crystallography S. P. Sudhakara Rao, M. Damodara Poojaryt, Bruce W. Elliott, Jr1 Linda A. Melanson, Bruce Oriel$ and Carolyn CohenI/ Rosenstiel Basic Medical Sciences Research Center Brandeis University, Waltham, MA 022549110, U.S.A. (Received 20 October 1990; accepted 23 May
1991)
Electron microscope images of frozen-hydrated crystals of a proteolytically modified fibrinogen show excellent preservation of the structure. An electron density map of the key centric projection of the crystal at 18 A resolution has been obtained by combining the phases derived from cryo-electron microscopy with X-ray amplitudes. Simulation methods developed in earlier studies have been used to interpret the map. In contrast to the earlier images, the map allows us to visualize the coiled-coil region of the molecule and possible substructure in the /I domains. The map also shows that there is a marked difference in density in the two regions corresponding to the molecular ends where the y domains interact. A possible interpretation of this finding is provided by assuming substructure in the y domains and the breaking of molecular symmetry where these domains interact. Some additional constraints useful for the determination of the three-dimensional structure were obtained from cryo-electron micrographs of a perpendicular view at 25 A resolution. Implications of this working model for the molecular length and contacts in the filaments in both the crystal and fibrin are described. The data used here will be valuable as a star@ point for obtaining the t,hree-dimensional structure.
Keywords: fibrinogen:
cryo-electron
microscopy;
1. Introduction
t Present address: Texas A&M University? Department of Chemistry, College Station, TX 77843. 3255. U.S.A. $ Present address: Boston University, College of Basic Studies. 871 Commonwealth Ave, Boston, MA 02215, LJ.S.A. 4 Present address: Albert Einstein College of Medicine, 1396 Morris Park Ave, Bronx, NY 10461, U.S.A. 11Author to whom all correspondence should be addressed.
89 ~OS.OO/O
crystallography;
coiled coil: fibrin
were first obtained by digesting the bovine molecule with a crude protease from Pseudomonas neruginosa (Cohen & Tooney. 1974). This enzyme has now been purified and found to be specific for lysine residues (Elliott & Cohen, 1986). Previous analyses of these crystals by co-ordinated electron microscopy (using negative staining), image processing and X-ray crystallography (Weisel et al., 1981: Cohen et al., 1983) led to a low resolution (-30 A; 1 A = 0.1 nm) seven-domain model for the modified molecules. This so-called “heptad” model allows a correlation of the domains with specific regions of the amino acid sequence: in addition to a central domain formed by the three pairs of NH, termini, the COOH terminus of each chain folds independent,ly into a globular region (termed the CL /I and y domains). In the modified molecule, however, the c1 domain is removed by the protease (Wcisel et al.. 1985). The heptad model also accounts for the band pattern of negatively stained fibrin seen in the electron microscope. Despite extensive efforts, suitable isomorphous derivative crystals have not, yet been obtained, since fibrinogen is very sensitive t)o heav) metals. Previous studies on the crystal morphology,
Fibrinogen has been difficult to crystallize and its structure difficult, to solve, but detailed visualization of the molecule is essential for understanding its architecture and assembly into fibrin. Chemical studies indicate that fibrinogen is a dimer (M, 340,000): t,he molecule consists of three pairs of different polypeptide chains (termed Acr, BP and y). Native fibrinogen does not crystallize, but limited proteolytic cleavage with a variety of enzymes allows the modified molecules to form both microcrystals and crystals (Tooney & Cohen, 1977; Weisel rt nl.. 1978). Crystals suit,able for X-ray analysis
0022%2836/91/21008s-10
X-ray
0
1991 Academic,
Prrw
Limited
90
S. P. S. Rao et al.
diffracted intensity distribution and unit cell changes under different conditions (Tooney & Cohen, 1977; Weisel et al., 1978) have established aspects of the packing. The rod-shaped fibrinogen molecules of length 450 L%pack end-to-end to form filaments which run along the [3,0, l] diagonal direction, perpendicular to the crystallographic 2-fold axis. Patterson maps indicate that there are two possible positions for the molecules in the unit cell, one corresponding to a shift of about 135 A, and. the other 30 A, between symmetry-related molecules. Earlier image processing of electron micrographs of negatively stained crystal fragments viewed along the S-fold (unique) axis supported the former translation, indicating that symmetryrelated layers of molecules are staggered by about one-third the molecular length (Weisel et al., 1981; Cohen et al., 1983). The unit cell dimensions of the negatively stained crystal fragments vary greatly, however, due to dehydration and unit cell changes in preparation for electron microscopy, so that these images could not be used to phase the corresponding X-ray data from the crystals. Instead, intensities were calculated to a resolution of 30 A using various trial models derived from image analysis of the electron micrographs (Weisel et al., 1985). Recently we have taken advantage of the technique of cryoelectron microscopy, which has allowed us to obtain higher resolution images of the crystal without changes in unit cell dimensions, enabling us to phase the X-ray data directly. Here we report a description of the centric projection of the crystal at 18 L% resolution derived by using cryo-electron microscopic phases and X-ray amplitudes. Simulation methods allow us to make a preliminary interpretation of the electron density map: the results confirm certain aspects of the earlier model, reveal the or-helical coiled-coil region, possible substructure in both the fl and y domains, and the breaking of molecular symmetry at the end-to-end contacts.
2. Materials and Methods (a) Crystallization
Crystals of modified bovine fibrinogen were obtained as described (Tooney & Cohen, 1977; Weisel et al., 1978) with some modifications. (The dialysate now contained 2 IIIM-Cd&, 5 mM-Pu’aN, and was buffered with 10 mM-Mes at pH 62; KSCN was no longer used.) After dialysis, the digested fibrinogen (at a concentration of 2 to 3 mg/ml) was transferred to acid-washed vials (each containing @5 ml). Seeding has been found to be necessary when using the purified protease for digestion (Elliott C Cohen, 1986). The vials were kept at 4°C and the crystals grew in about 1 month. More recently, we have grown crystals in about 1 week at 22.5”C. Despite these
changes, it is still very difficult to obtain crystals suitable for X-ray analysis, although small crystals are readily and reproducibly obtained (see below). Diffraction from these crystals is anisotropic extending to 3.5 A in the direction of the molecular filaments and to about 6 A in perpendicular directions. The space group is P2, with a = 135 A, b = 9%1 A, c = 175 .& fl= 92”. There is one molecule per asymmetric unit in the unit cell.
(b) X-ray data collection A low resolution (20 A) and t,wo 6 A data sets have been collected. In these experiments, the crystals were cooled to 4°C. The low resolution data set was collected using precession photography on an Elliott GX6 X-ray source. Special care was taken to scale the strong reflections to the rest of the data by monitoring the incident beam with a NaI-photomultiplier detector. Data from 11 zero-level photographs taken along various crystallographic directions were scaled and merged (72 y/o complete, Rmsrg--@045). The 6 A data sets were collected both at CHESS (Cornell High Energy Synchrotron Source), by oscillation methods, and at the University of Virginia, using the multiwire area detector. The processed data sets contain about 70% of t,he total reflections with value of @09. an kg (c) Cryo-electron microscopy The P2, crystals cleave readily along planes perpendicular to the unique axis into thin sheets (Weisel et al.. 1981) suitable for the cryo-electron microscopy. Specimens were also prepared by overseeding solutions of the modified molecules (at about 4 mg/ml) at 22.5”C; small crystals grew overnight and were concentrated and fragmented by centrifugation. Crystal fragments were rapidly frozen by plunging into liquid propane cooled to liquid nitrogen temperature (Dubochet et al., 1988). Specimens were examined in a Philips EM420T transmission electron microscope equipped with a Gatan 626 cryoholder and operated at an accelerating voltage of 100 kV. Low dose images (< 10 electrons/A2) were recorded in vitreous ice at - 174°C at a magnification of 47,000 x and an underfocus of @7 pm. Micrographs were initially screened by optical diffraction (Salmon & DeRosier, 1981) to locate areas suitable for computer processing. Selected micrographs were densitometered on an Optronics Photoscan PlOOO microdensitometer (Optronits Inc., Chelmsford, MA. U.S.A.) using a sampling interval of 25 pm corresponding to 53 A on the specimen. The images were digitized on a 512 x 512 pixel array and displayed using a Grinnel graphics system with 256 grey levels (Grinnel System Corp., Santa Clara, CA, U.S.A.). Suitable areas were chosen using television screen cursors. The boxed-off areas were floated (namely, the average optical density of the perimeter of the box was subtracted from all the values within the box) and Fourier transformed. The images were corrected for lattice distortions by real-space correlation analysis (Henderson et al., 1986). The amplitudes derived from these images were corrected for the effects of the contrast transfer function (C(v)): C(v) = A(v)sinX(v)+B(v)cosX(v), x = 27r-‘(@fe2-&y4), where A and B represent the fractional contribution due to phase and amplitude contrast transfer functions, respectively and v( = e/n) is the spatial frequency in the object. The defocus, SJ was estimated from the positions of the Thon rings. C, is the spherical aberration coefficient, 1 is the electron wavelength and 0 is the scattering angle. The percentage of the amplitude contrast was determined from a comparison of the low resolution amplitudes derived from images with those obtained from X-ray diffraction; this value was about 5%, which is close to that found using pairs of defocused images (Toyoshima & Unwin, 1988). The agreement between the high resolution amplitudes in the 2 types of data was further improved with the application of a temperature factor correction based on Wilson plot analysis.
Structure of Fibrinogen
in 18 A Resolution
(bl
.
..^“-.-
.-----__~-.-”
.-,-___
I
~I--
---~ll-.~l.-^---lll--~--
.,
Figure 1. Representative electron micrographs of P2, crystals of modified bovine fibrinogen with computed Fourier transform of a selected image together with the corresponding X-ray precession photograph. (a) Electron micrograph of the [OlO] view of a thin crystal fragment of fibrinogen (protein is black). A crystal area selected for Fourier processing is shown in the inset. The images show strands running along the [3,0, l] diagonal direction of the unit cell in this projert,ion. The strands represent molecular filaments with an effective period of 450 A. (We have not measured the thickness range of the crystal fragments since our studies to date have dealt with nominally untilted views.) (b) A computed Fourier transform from an image-processed micrograph in (a) after corrections (see the text) showing Hragg reflection to 15 A resolution. (c) X-ray precession photograph of the same view of the crystal showing the low resolution reflections to about 15 A resolution. The computed transform from the cryo-electron micrograph (b) displays close correspondence with the X-ray photograph.
3. Results and Discussion (a) Electron density map in projection Representative images of the centric [OlO] view are shown in Figure l(a). The computed transform of this view shows Bragg reflections rather uniformly up to 18 A, occasionally extending to 15 A resolution (Fig. 1(b)). After appropriate corrections (see Materials and Methods), the transforms show good correspondence with the X-ray diffraction pattern of the same view (Fig. l(c)), including unit cell parameters. (The R-factor between the X-ray amplitudes and the corrected amplitudes obtained by merging 9 images is 0.18.) The phase origins of the computed transforms of the
images selected were placed on a common 2-fold axis, and the phase for each reflection was then calculated by averaging corresponding phases from individual images, which were weighted according to the signal-to-noise ratio. Data were not used if the averaged phase deviated by more than 45” from 0 or n; in such cases, the reflections were very weak (see also the legend to Fig. 2). In this way, 93% of the X-ray amplitudes in the hOEzone were phased up to 18 A resoluti on. The quality of the data is evident from the low phase residual (143”) from merging and the tendency of the phases to follow the symmetry constraint (Fig. 2). The electron density map computed using these symmetryconstrained phases and the X-ray amplitudes is
92
S. P. S. Rao et al.
270.0
K/
I
-90.0~ 0.00
0.200 Resolution
0.400 (nm-’ )
0.600
Figure 2. Quality of phases in the merged data set derived from 9 images. The plot shows the deviation of the averaged phases from the symmetry-required values, 0 or n. Phases were constrained to 0 or K and combined with X-ray amplitudes
to compute the projection
shown in Figure 3. A map computed tional
data to 15 a showed
(b) Interpretation
very
similar
map.
using addifeatures.
of the map
The map shows features of macromolecular size running in the diagonal [s, 0, l] direction and has an overall similarity (including the relative sizes of the
molecular domains) to the lower resolution electron microscope images of this view of the crystal in negative stain. The repeating unit along the strand is a complex group of seven regions of varying size, shape and density (generally about 40 A in width). The highest density region (located at a crystallographic 2-fold axis) lies midway between two “birdshaped” regions (see the legend to Fig. 3) that are oppositely oriented on either side followed by a long region of weak density. This motif repeats every 450 A and corresponds to the projection of two symmetry-related molecular filaments in the unit cell. In order to interpret the map we applied simulation methods developed in earlier studies (Weisel et al., 1981; Cohen et al., 1983). Using a shift of 135 L% (about one-third of the molecular length) between symmetry-related molecules, the map can be interpreted by a working model that reveals new features of the molecule and its packing into filaments. For the first time, the map allows us to visualize possible substructure in the fl domain. In this projection (Fig. 3), the “bird-shaped” density corresponds to a /3 domain partly eclipsed by the central domain of the symmetry-related molecule (Fig. 4(a)). On the simplest interpretation, the a
Figure 3. Electron density map of the [OlO] projection in grey scale (protein is black) derived from the electron microscope phases (see the text) and X-ray amplitudes. A contour plot of the same map drawn with positive contours at intervals of 0.1 of the maximum density is shown in the inset. The projected unit cell (a = 135 L%,c = 180 8, B = 91”) is outlined and the 2, screw axes are indicated. The unit cell contains 2 fibrinogen molecules related by the screw symmetry along b. A schematic diagram of 2 neighboring translated filaments each with 2 “end-to-end” bonded molecules is superimposed on the map. Note that, for clarity, neither of their screw-related filaments is depicted. (In Fig. 4(a) both sets of filaments are shown.) The /?and y domains of a single molecule in a filament are labeled; note the distinctive “birdshaped” density where one of the /? domains is located (one such “bird-shaped” region in a neighboring filament is marked by an arrowhead). The shape and height of the strong density peaks reflect the size, shape and overlap of the different globular domains. The weak density regions between the domains represent the a-helical coiled-coil portion of the molecule. A small bend in the molecule near the end domains accounts in part for the slightly wavy appearance of the strands.
Structure of Fibrinogen
in 18 A Resolution
93
(a)
(b)
Figure 4. (a) Schematic diagram of the crystal packing viewed approximately along the h axis to show the arrangement of the symmetry-related molecules that overlap in the [OIO] view. For clarity the model shown is that of the symmetrical molecule (Weisel et al., 1985) but lacking the smallest “domain”. The symmetry-related molecules lying below are hatched. The location of the filaments is such that the “end-to-end” linking regions are in close proximity to a symmetry axis that results in a cluster of 4 y domains (one such region is boxed), 2 from each of the symmetry-related filaments. (1~) Simulation of the [OlO] view of the crystal by the symmetric. “short” and “long” models showing the substructures in the /I and y domains and the breakdown of the non-crystallographic symmetry at the 2 molecular ends. One of the regions where the molecules interact end-to-end to form filaments is outlined in each of’ the diagrams. As described in (a), t)he marked regions contain 4 y domains. Schematics of various types of models are superimposed on t,he simulation diagrams; these also show the nature of end-to-end linkages for these models. The precise shapes of t,he domains and their possible interactions are not yet established. (i) Electron density map of the [OIO] view as shown in Fig. 3. (ii). (iii) and (iv) Computer simulations of the symmetric (Weisel et al.. 1985), “short” and .‘long” mod&, respectively (see the text). Unlike the symmetric model, both the fi and y domains of the molecules shown in (iii) and (iv) caonsist of 2 subdomains of different sizes. As expected, the symmetric model (ii) shows equal density for all 4 interacting y domains. Tn contrast, the map in (i) shows that this region consists of 2 strong and 2 weak density features. This feat,ure is well simulated by both the models in (iii) and (iv) where the y domains interact differently at 2 molecular ends (see the text). Note that t,he positions of the larger subdomains of the y domains at the 2 molecular ends are different in the 2 structures and that in both models the molecular symmetry is broken.
domain would comprise two subdomains, the larger oriented along and the smaller at an angle to the [3,0, l] direction. The same structure would be expected for the other b domain (the highest density region), but the shape here is completely obscured by overlapping of the symmetry-related /? domain (Fig. 4(a)). The map also shows that the distance between the center of the B domain and the central “disulfide knot”) domain of the symmetry-related molecule is about 30 A (Fig. 4(a)). In the Harker section of the Patterson maps (data not shown), a strong peak at 30 A (which could have corresponded to the shift between layers) may now be accounted for most plausibly by this vector. (In images of the
negatively stained crystals, due t’o t,he lower resolution, the /l and cent,ral domains of symmetry-relat’ed molecules overlap, obscuring these features.) A striking feature of the map is the marked difference in density in the two regions corresponding to the molecular ends where t,he y domains interact (Figs 3 and 4(a)). These regions consist of two pairs of y domains: one pair from a filament in the plane and the other pair from the symmetryrelated filament. If the molecular dyad were in or near the ac plane (as shown from other findings described below) and the molecules were bonded end-to-end. the map would display equal densities for all four y domains (Fig. 4(b). panel (ii)). In
94
S. P. S. Rae et al.
contrast, the map appears to show that the electron density of the y domain is significantly lower at one end of the molecule than at the other end (Figs 3 and 4(b), panel (i)), indicating that the molecule cannot possess perfect 2-fold symmetry in the ac plane. This feature has also been observed in images of negatively stained crystals. In principle, asymmetric cleavage of the y domains during proteolytic modification could account for the difference in density. Studies by SDS/polyacrylamide gel electrophoresis, however, show that the y-chains are essentially intact: they have lost a mass equivalent to about 15 residues and retain the ability to be crosslinked by factor XIII (Elliott et al., 1987). A plausible explanation of this feature of the map is that there is a breakdown of symmetry at the two molecular ends, possibly due to packing constraints in the crystal lattice. On this view, the map can be simulated by assuming that the y domain consists of two subdomains of unequal size (the distal one being larger) which are oriented slightly differently at the two ends of the molecule. Such a division is supported by recent scanning microcalorimetric studies by Medved’ et al. (1986). It is consistent as well with the strong homology of the amino acid sequences of the /? and y chains, and the projected appearance of the b domain described above. (Note, however, that the subdomains of the /I and y chains appear to be arranged differently (see below).) By considering the packing constraints in the crystal, together with the positions of the density peaks in the map, we have found only two types of molecular model. In one (termed “short”), the distal y subdomains are laterally displaced from the molecular axis in the same direction, but to different extents, and are displaced from the ac plane in opposite directions (Fig. 4(b), panel iii)). In this case, the molecules have a length of 450 A when projected along the molecular axis (but a contour length of 480 A). In the other (termed “long”), the distal y subdomain at each molecular end lies nearly in the ac plane, with one of them tilted by about 15” from the molecular axis (Fig. 4(b), panel (iv)). Consequently, the joint between the molecules in the filament is not “end-to-end” but involves a lateral overlap of a distal and proximal subdomain. The length of the molecule would then be about 480 A, although the effective repeat in the filament is still 450 A. (c) Support for the “long” model from a perpendicular view A choice between the two models can be made from cryo-images of other views of the lattice. In this connection, the [OOl] view (Fig. 5(a)), which is perpendicular to the [OIO] projection, is particularly informative. Although the best images of this view obtained thus far have a resolution of only about 25 A, they provide support for the “long” model. The image (Fig. 5(b), panel (i)) is very distinctive: it consists of a strong density striation spaced at 135 A
(the a cell dimension of the crystal) separated by weak density regions. Simulations were carried out as described to compare the symmetric, “long” and “short” models. Only the latter two models accounted satisfactorily for the strong densities because of the similar disposition of the central and fi domains. The lower density peaks which correspond to the projection of the y domains, however, were well simulated by the “long” model (Fig. 5(b), panel (iv)), but by neither of the others (Fig. 5(b), panels (ii) and (iii)). This result has been checked by simulating images of negatively stained crystals which have unit cell parameters similar to the hydrated crystals in this view. These findings indicate that, to a first approximation, the y domains are disposed nearly along the length of the molecule as in the “long” model. (d) Visualization
of the coiled coil
The region of weak density in the [OlO] projection (Fig. 3) between the central and /I domains corresponds to part of the u-helical coiled coil portion of the molecule. This region appears rather diffuse in the map and is generally not visible in images of negatively stained crystals. Although the overall width of a three-stranded a-helical coiled coil is about 23 A, this dimension cannot be estimated accurately from the map because of the limited resolution and the overlap of symmetry-related molecules in projection. Within the coiled coil, there are two very weak peaks, centered at about 100 A from the midpoint of the central domain (Fig. 3). In studies from this laboratory, evidence for the presence of a small “domain” near the center of the coiled coil was obtained from a specific stainexcluding region in images of negatively stained bovine fibrin and certain microcrystal forms of fibrinogen (Weisel et al., 1981, 1985; Cohen et al.: 1983). This small domain was incorporated into the heptad model as the presumed site of plasmin cleavage (Weisel et al.? 1983). In human fibrin however, this stain exclusion is not pronounced. The intensity of this band in images of negatively stained bovine fibrin is considerably reduced after limited proteolysis; strong density would therefore not be expected for this region of the modified molecule in the electron density map. In attempts to account for this density we have re-examined available amino acid sequences of a number of fibrinogens (Chung et al., 1981; Crabtree & Kent, 1982; Henschen et aE., 1983; Crabtree et aE., 1985; Brown et al., 1989) and do not find any major interruption in the heptad pattern of residues in the a-helical coiled coil which could correspond to a non-helical domain (see also Cohen & Parry, 1990). Moreover, the breaks in the heptad repeat appear to vary from one chain to another (Conway & Parry, 1991). The two very weak peaks seen in this region of the map (and represented in Figs 3 to 6) should therefore probably not be identified with a specific domain; nor is it clear that they correspond to regions of plasmin attack. The origin of the additional density in
Xtructure of Fibrinogen
in 18 d Resolution
95
(a)
Figure 5. (a) Schematic diagram of the crystal packing viewed approximately along the c axis. As in Fig. 4(a), the symmet)rical molecule is shown, but here the length of the b axis is doubled for clarity. The filaments form sheets in the UC planes which stack along the b axis as shown in the Figure. The symmetry-related layer is hatched. (b) Simulation of the [OOl] projection of the crystal by the symmetric, “short” and “long” models. The marked regions in these diagrams consist of the 2 y domains, one from each of the end-to-end interacting molecules in a filament. Schematic models are superimposed on each of the simulation diagrams. Note that in this view the projected filament repeat is about 405 A. (i) Electron density map of the [OOI] projection derived from the electron microscope phases and X-ray amplitudes. This view is much rarer than the [OlO] projection (Figs 1, 3 and 4), although it is frequently seen in negative stain. The image was processed as described, but no symmetry has been imposed on the phases; nevertheless the screw symmetry along the b axis is clearlz.visible. The projected unit cell (a = 135 A, b = 98 A, y = 90”) and the location of the symmetry axes are marked. (ii), (m) and (iv) Computer simulations of the symmetric (Weisel et al., 1985) “short” and “long” models, respectively. Neither the strong density striations (corresponding to the overlapping of central and /? domains) nor the weak density regions (corresponding to the interacting y domains) in (i) are well simulated by the symmetric model (see (ii)). Both the “short” and “long” models generate the strong density striations equally well. Note, however, that the pattern of t,he weak density regions (boxed) is correctly simulated only by the “long” model.
images of negatively to be established.
stained bovine
fibrin
remains
(e) Inferences on crystal packing and molecular symmetry The chemistry and morphology of the fibrinogen dimer indicate that there is a 2-fold axis perpendicular to the long axis of the molecule.
This dyad is not
expressed in the P2, crystal, nor in the other crystal forms of modified fibrinogen (Hewat et al., 1983; Gollwitzer & Rode, 1986), which all show one dimeric molecule in the asymmetric unit. Information about the orientation of the molecular Z-fold axis in the P2, crystal, as well as its location, may, however, be obtained by analysis of the X-ray amplitudes. Our results from R-factor and correlation coefficient searches, as well as Patterson maps computed using 20 A data, show that the major part of the molecule contains a 2-fold axis near the
ac plane; this dyad is located at about 65 A from the origin (our unpublished results). Moreover, by assuming that this elongated molecule consists mainly of discrete spherical domains, a Patterson self-rotation function search was carried out (using both 20 and 6 A data), which also showed that the molecular dyad is oriented about 5” to the ac plane. Our interpretation of the [OlO] projection above was based on these results, but we required as well the assumptions of two subdomains in the y domain and the breaking of symmetry at the two molecular ends. In both models described only the distal y subdomains appear to depart from the molecular 2-fold symmetry. Although we lack detailed three-dimensional information on the packing in the lattice, the molecules in neighboring filaments appear to interact closely at the region of the y domains in the CLC plane. Moreover, whatever the model, the contacts made at the two ends of the molecule are different
96
S. P. S. ho
(see Fig. 4). In both models, the distal y subdomain at one end appears to make close contact with a region of the coiled-coil portion of the neighboring translated filament. The resulting bend in this region produces the “waviness” of the filaments. Such a contact is not possible for the distal subdomain at the other end of the molecule which appears to interact with a coiled-coil region of the molecule from the symmetry-related filament. Moreover, the tendency of the crystals to cleave readily in a direction parallel to the ac plane demonstrates that the interactions of the filaments in this plane are considerably stronger than those between the symmetry-related layers. It is therefore likely that these packing contacts in the crystal, together with some flexibility of the molecule, lead to the departure of the two ends from exact 2-fold symmetry. (f) Implications
et al.
(b)
(cl
for $brin assembZy
Despite the preliminary nature of our current interpretation, it is worthwhile to consider the implications for fibrin assembly of the novel features that we have inferred for the molecule and its packing in the filaments. In all of our previous studies we have taken fibrin as but one of the polymorphic forms of fibrinogen (Cohen et al., 1983). In other words, the crystalline arrays produced by modified fibrinogen, aa well as fibrin, may be accounted for on the basis of the same molecular filaments, packed in various ways, with minor differences due to different packing environments. In the P2, crystals we have shown that both classes of filaments (symmetry-related as well as translated along a) are shifted by about one-third of the molecular length with respect to one another; in fibrin, the shift is one-half the molecular length. We have therefore examined the possible packing in fibrin of the filaments derived from the “long” model. Fibrin assembly appears to involve the formation of half-staggered dimers to which additional molecules are added to yield a two-stranded protofibril. In these aggregates each y domain is involved in at least two kinds of linkages: one to the complementary binding sites in the central domain exposed by the loss of the fibrinopeptides; and the other to the y domain on the neighboring molecule in the filament. Because of the half-staggered arrangement, an essential feature of the fibrin packing would be equivalent linkages of the neighboring y domains in a filament with a central domain. We can build a model for fibrin, using filaments closely similar to those in the crystal lattice, by slightly flexing the molecules at the ends to restore 2-fold symmetry. A half-staggered assembly of filaments generated in this manner from the “long” model accounts well for the 225 A repeat and the band pattern in the images of negatively stained fibrin (Fig. 6). In this assembly, the central domain fits into the pocket formed by the two interacting y domains (Weisel et al., 1983), giving a “closed” protofibril (Fig. 6(a)). It
Figure 6. A model for fibrin assembly. Tn order to construct a packing model for fibrin, the “long” model is made symmetrical by slightly displacing the distal y sub domains. The length of the molecule is still about 480 A. (a) A half-staggered assembly of filaments (viewed as in Fig. 4(b), panel (iv)) generating a “closed” protofibril (Weisel, 1987). The bonding of neighboring y domains in the filaments in fibrin is virtually the same as that in the filaments in the crystal, but symmetrical linkages with the central domain of the staggered filament can now be made. An enlarged portion of a filament (boxed) showing this overlapping region is seen in the inset (top right). The protofibrils aggregate laterally in a complex way (but still in a half-staggered fashion) to form fibrin which is shown schematically in (b). (c) An electron micrograph of negatively stained fibrin showing how the domain sizes and positions in the model account for the stain-excluding bands. As noted in Results, section (d), however, the origin of the extra density in the region where we have shown a small “domain” is not yet accounted for. Notes that because of the overlapping of the adjacent, end domains (in (a) and (b)), the effective repeat in the filament is 450 A and that in the protofibril is 225 A.
is also of interest that the lateral overlapping of the y domains in the filaments now provides a simple structural explanation for the finding of an ant’iparallel arrangement of y chain crosslinks (Doolittle. 1981). (We may note that a similar study for the filaments derived from the “short” model gives a less satisfactory packing for fibrin.) On the basis of this analysis of fibrin assembly, as well as the simulation
results
of the two-dimensional
data discussed
above, we consider that the “long” model at present. gives the best 6t to the available data. (g) Additional
implications
We summarize our current picture of the model with a schematic diagram of the organization of the polypeptide chains in the molecule (Fig. 7). Such a picture is, of course, heuristic, since it is based on
Structure of Fibrinogen
97
in 18 A Resolution
other proteins having domains with this motif will also be discovered (Baker et al., 1990). We are extending our structural analysis of fibrinogen to three dimensions microscopy, and have recently
-. P Figure 7. A schematic representation of the organization of the polypeptide chains in the native fibrinogen molecule (based on the “long” model) showing the correlation of the structural features with specific regions of the chains. The diagram depicts the sequence of human fibrinogen, but the available sequences of bovine fibrinogen are closely homologous to those of human fibrinogen. The view is as shown in Fig. 4(b), panel (iii), where the molecular symmetry axis is in the plane of the paper. The NH, termini of the 3 pairs of non-identical chains (Acc, BP and y) form the central domain. The portions of the chains between 2 sets of disulfide rings (1) are mainly a-helical forming an irregular 3-stranded coiled coil. (In the rod region as drawn, the a-chain is at the bottom and the /I chain at the top: AM, predicted to be a-helical coiled coil with breaks indicated by vertical lines; p, possible nonhelical segments (Conway & Parry, 1991).) The COOH termini of the Bfl and y chains fold independently into globular domains with substructure: about 2/3 of each chain (after the outer disulfide ring) forms a larger subdomain and the rest a smaller subdomain. This region of the /I chain is shown in bold. (Not illustrated are the disulfide bonds common to both chains, and an additional disulfide bond unique to the /l chain.) The flexible COOH termini of the Aa chains fold back to form an additional central domain which is, however, absent from the molecules aft)er proteolytic rleavage (see also Weisel et aZ., 1986).
low resolution
two-dimensional
data.
Note
that
in
t)his drawing (as well as in the models used for the simulations) the /I and y domains are arranged differently. In keeping with the high homology of the sequences, however, the tertiary structures of the two domains are depicted as very similar. Such a picture presents a problem, but this disposition could be produced by a simple rotation near the out,er disulfide ring that might be related to the additional disulfide bridge in the /I chain. At this stage of structure determination, any such details of domain structure are purely speculative. These findings are relevant also to the structure and interactions of the extracellular matrix glycoprotein cytotactin (also called tenascin (see Erickson B Rourdon, 1989)). The COOH-terminal end of each arm of this “hexabrachion” structure appears as a ‘~knob” in electron micrographs of rotary-shadowed molecules (Erickson & Iglesias, I9S4).
This
knob
contains
207 amino
acid residues
are highly homologous to the sequence of the b and y domains of human fibrinogen, including the position of an intrachain disulfide bridge (Jones et al., 1989). We would. t,herefore, expect that the that
(‘OOH-terminal
domain
of
cytotactin
has
a
substructure similar to that of the fibrinogen /I?and 7 domains. The lateral overlapping of the y domains currently envisaged for the filaments of fibrinogen may thus display features of some of cytotactin’s various int.eractions. Tt is likely that a number of
using cryo-electron obtained three non-
centric views of the crystals at about 20 A resohtion. We are now attempting to collect sufficient views to phase a major portion of the X-ray data. This paper provides a starting point’ for obtaining a three-dimensional map by density modification techniques, utilizing phase constraints from both centric and non-centric views. As the structure of the molecular filaments is extended t’o three dimensions and to higher resolution, we should be able t’o clarify the physical basis for the apparent molecular asymmetry as well as delineate feat,ures of domain substructure with more reliability. We thank J. W. Weisel for many helpful discussions, I). Stokes for contributions to the analysis of the model as well as for valuable suggestions, J. I’. Griffith for carrying out the rotation function search and for his encouragcment in our use of cryo-electron microscopy. R. Kretsinger. S. Sobottka and R. J. Chandross of the University of Virginia for their generous help in the use of the area detector, C. V. Stauffacher and W. C. Phillips for help with data collect,ion, R. Stryker and K. Giese for crystallization studies. J. Black for photography, ,J. Khorana for drawings, P. Vibert and P. Walian for help with the manuscript, and L. Seidel and B. Finkelstein for typing. Supported by grant AR17346 from NIH to c‘.C. Funds to purchase and maintain the computer system were obtained from a shared instrumentation grant l-SlORR04671-01 awarded to David $1. DeRosier by the National Institutes of Health. Funds to purchase and maintain the Philips EM420T transmission electron microscope were obtained from a shared inst’rument,ation grant l-SlO-RR02464-01 awarded t#o(‘.(‘. by t,he National Institutes of Health.
References Baker, N. E., Mlodzik, M. t Rubin, G. M. (1990). Spacing differentiation in the developing Drosophila eye: a fibrinogen-related lateral inhibitor encoded by scabrous. Science, 250, 1370-1377. Brown, W. M., Dziegielewska, K. M., Foreman. R. (1. &
Saunders, N. R. (1989). Nucleotide and deduced amino acid sequence of a y subunit of bovine fibrinogen. Nucl. Acids Res. 17, 6397. Chung, D. C., Rixon, M. W., MacGillivray, R. T. A. & Davie, E. W. (1981). Characterization of a cDNA clone coding for the B chain of bovine fibrinogen. PTOC. Nat. Acad. Sci., IT2.A. 78. 1466-1470. Cohen, C. & Parry, D. A. D. (1990). a-Helical coiled coils and bundles: how to design an a-heliral protein. Proteins: Struct. Fun& Genet. 7, l-15. Cohen, C. & Tooney, N. M. (1974). Crystallization of a modified fibrinogen. Nature (London), 251, 659-660. Cohen, C., Weisel, J. W., Phillips, G. N., Jr. Stauffacher, C. V., Fillers, ,J. P. & Daub, E. (1983). The structure of fibrinogen and fibrin: I. Electron microscopy and X-ray crystallography of fibrinogen. In Molecular Biology of Fibrinogen and Fibrin (Mosesson. N. W. & Doolittle. R. F., eds), vol. 408. pp. 194&213, Annals of the New York Academy of Sciences. Nrw York.
98
S. P. S. Rae et al.
Conway, J. F. & Parry, D. A. D. (1991). Three-stranded a-fibrous proteins: the heptad repeat and its implications for structure. Zntl. J. Biol. Macromol. 13, 14-16. Crabtree, G. R. & Kant, J. A. (1982). Organization of the rat y-fibrinogen gene: alternative mRNA splice patterns produce the yA and yB(y’) chains of fibrinogen. Cell, 31, 159-166. Crabtree, G. R., Comeau, C. M., Fowlkes, D. M., Fornace, A. J., Jr, Malley, J. D. & Kant, J. A. (1985). Evolution and structure of the fibrinogen genes. Random insertion of introns or selective loss? J. Mol. Biol. 185, 1-19. Doolittle, R. F. (1981). Fibrinogen and fibrin. Sci. Amer. 245, 126-135. Dubochet, *J., Adrian, M., Chang, J. J., Homo, J. C.. Lepault, J., McDowell, A. W. 6 Schultz, P. (1988). Cryo-electron microscopy of vitrified specimens. Quart. Rev. Biophys. 21, 129-228. Elliott, B. W., Jr & Cohen, C. (1986). Isolation and characterization of a lysine-specific protease from Pseudomw aeruginosa. J. Biol. Chem. 261, 11259-l 1265. Elliott, B. W., Jr, Stryker, R. & Cohen, C. (1987). Biochemical characterization of modified molecules of bovine fibrinogen that form crystals. Fed. Proc. Fed. Amer. Sot. Exp. Biol. 46, 2243. Erickson, H. P. & Bourdon, M. A. (1989). Tenascin: an extracellular matrix protein prominent in specialized embryonic tissues and tumors. Annu. Rev. Cell Biol. 5, 71-92. Erickson, H. P. & Iglesias, J. L. (1984). A six-armed oligomer isolated from cell surface fibronectin preparations. Nature (London), 3 11, 267-269. Gollwitzer, R. & Bode, W. (1986). X-ray crystallographic and biochemical characterization of single crystals formed by proteolytically modified human fibrinogen. Eur. J. Biochem. 154, 437-443. Henderson, R., Baldwin, J. M., Downing, K. H., Lepault, J. & Zemlin, F. (1986). Structure of purple membrane from Halobacterium halobium: recording, measurement and evaluation of electron micrographs at 3.5 A resolution. tJltramicroscopy, 19, 147-178. Edited
Henschen, A., Lottspeich, F., Kehl, M. & Southan, C. (1983). Covalent structure of fibrinogen. In Molecular Biology of Fibrinogen and Fibrin (Mosesson, N. W. & Doolittle, R. F.. eds), vol. 408, pp. 28-43, Annals of the New York Academy of Sciences, New York. Hewat, E. A., Tranqui, L. & Wade, R. H. (1983). Electron microscope structural study of modified fibrin and a related modified fibrinogen aggregate. J. Mol. Biol. 170, 203-222. Jones, F. S., Hoffman, S., Cunningham, B. A. & Edelman, G. M. (1989). A detailed structural model of cytotactin: protein homologies, alternative RNA splicing and binding regions. Proc. Nat. Acad. 9ci., U.S.A. 86, 1905-1909. Medved’, L. V., Litvinovich, S. V. & Privolov, I’. L. ( 1986). Domain organization of the terminal parts in the fibrinogen molecule. FEBS Letters, 202, 298-302. Salmon, E. D. & DeRosier, D. J. (1981). A surveying optical diffractometer. J. Microsc. 123, 239-247. Tooney, N. M. & Cohen, C. (1977). Crystalline states of a modified fibrinogen. J. Mol. Biol. 110, 363-385. Toyoshima, C. & Unwin, N. (1988). Contrast transfer for frozen-hydrated specimens: determination from pairs of defocused images. Ultramicroscopy, 25, 279-292. Weisel, J. W. (1987). Molecular symmetry and binding sites in fibrin assembly. Thromb. Res. 48, 615-617. Weisel, J. W.. Warren, S. C. t Cohen, C. (1978). Crystals of modified fibrinogen. Size, shape and packing of molecules. J. Mol. Biol. 126, 159-183. Weisel, J. W.? Phillips, G. N., Jr & Cohen C. (1981). A model from electron microscopy for the molecular structure of fibrinogen and fibrin. Nature (London), 289, 263-267. Weisel, J. W., Phillips, G. N.,
Note added in proof: Recent improvements in image collection and processing have the [OlO] view to beyond 10 A resolution. A projection map from these data is lower resolution map reported here and shows additional features of the molecular a resolution of about 10 A have also been obtained for certain near-perpendicular goal is a 3-dimensional map to about this resolution.
enabled us to phase consistent with the filaments. Phases to views. Our current