Supplementary Information
Interactions between fusion loops from adjacent E1* trimers.
Crystallisation of E1*HT involves contacts between the fusion peptide loops of adjacent
trimers. Because the 3-fold axis of the trimer is not crystallographic, each subunit is not in an
identical environment. All three fusion loops participate in the contacts, but the most
important ones are made between two trimers facing each other about a crystallographic dyad,
as illustrated in Figure S1 (labeled as "contact 1"). This contact involves fusion loop residues
85 to 92 of one trimer contacting fusion loop residues 93 to 96 in the adjacent trimer.
Figure S1. Diagram illustrating the contacts
between E1* trimers in the crystals. Three trimers
are shown, indicated by T1, T2 and T3. Panels A
and B give two roughly orthogonal views of the
contacts (panel B is looking down the 2-fold axis).
The main contacts are made by the fusion peptide
loops (in orange). The most important contact is
labeled 1, and takes place about a crystallographic
2-fold axis, indicated by a black line and an
ellipse. Contact 2 involves the fusion peptide loop
(cd loop) and the bc loop of the neighbouring
trimer, which is oriented almost at 90 degrees
from the plane formed by the other two. For
clarity, panel B was depth-cued by adding "fog".
The geometry is such that contact 1 can be propagated in a plane such as that of a lipid
bilayer, to make a closed ring of trimers, all interacting with the membrane and with each
other via the fusion peptide loops. This is achieved by combining the 2-fold and 3-fold
symmetry operations, i.e., by applying the 3-fold axes of the trimer to the crystallographic
dyad to generate other dyads (Figure S2). Thus, a symmetric assembly can be built by
repeating contact 1, as outlined in Figure S2. In the arrangement illustrated in Fig. S2, panel
B, trimers T2 and T3 are in absolutely equivalent environments, with the three fusion loops
engaged in identical contacts. Furthermore, this arrangement leaves a space (between trimers
T1 and T4) which can accommodate, with minor rearrangement of the contact, a 5th trimer
(T5) to make a closed assembly in which every trimer makes identical interactions with its
neighbours, exclusively via fusion loop contacts. As indicated by a curved grey arrow in
Figure S2, panel A, the product of the operations applied about the dyad and triad, oriented as
in the crystal, gives rise to an effective rotation of 78.35 deg. about an axis perpendicular to
the plane of the figure, which passes through the center of the assembly. The rearrangement
mentioned above consists in slightly adjusting the angles between triad and dyad so that that
the product of the two operations results in a rotation of 72 degrees. The observed plasticity in
this region of the molecule suggests that the contacts can easily accommodate such an
adjustment.
Figure S2. Propagation of contact 1 defined in Figure S1. If the subunits in the trimer are considered
equivalent, then it is possible to propagate contact 1 to generate trimers T3 and T4 from T1 and T2. A.
The combination of the 2-fold rotation about the dyad (indicated by a black line) and the trimer 3-fold
axis (green line) is equivalent to a rotation about an angle of 78.4 deg. about an axis perpendicular to
the plane of the Figure, to bring T1 onto T2. B. This operation can be repeated to generate trimers T3
and T4. Or alternatively, the dyad labeled 2' (drawn between T2 and T3) is generated by the 3-fold
axis of T2 applied to the dyad relating T1 and T2. Dyad 2' then generates trimers T3 and T4 from T1
and T2, respectively. C. A small adjustment of the angle between the T1 3-fold axis and the
crystallographic 2-fold (from 26.5 deg. to 20.9 deg., forcing the axis to cross at the center) results in a
combined rotation of 72 deg. about a vertical axis relating neighbouring trimers. This results in an
arrangement in which 5 trimers make a closed ring, with the fusion peptide loop from each subunit
making exactly the same contacts with its neighbours. D. Side view of the 5 trimers arrangement, with
the central 5-fold axis labeled.
The resulting assembly of 5 trimers (panels C and D) has a "volcano" appearance, with ten
fusion loops (two per trimer) forming the outer rim of a fusion "crater", about 70 Å wide, and
the remaining five forming the bottom of the crater, about 45 Å deep. Figure S3 schematizes
the general case in which a triad and a dyad intersect at a point and make a defined angle, and
gives the relations between the angles.
Figure S3. Product of rotations about 2
intersecting axes. An ellipse indicates a 2-fold
axis (d, about X) and a triangle a 3-fold axis
(t), on the XY plane, at angle  from X. The
combination of the two operations, i.e., a 180
deg. rotation about d followed by a 120 deg.
rotation about t, is equivalent to a rotation of
angle  about a. (an axis that intersects the
other two and makes angle  with X). As
shown by the equations on the right, angles 
and  are defined by the value of .
Alternatively, imposing a value for angle
results in fixed values for  and .
In order to make a closed ring of trimers, the rotation angle  about a is defined by 360/N, in
which N is the number of trimers in the ring. This defines, through the equations given in
Figure S3, the corresponding values of  and , which are given in table S1. Note that  can
not be smaller than 60 degrees (when cos = 0.5), because the right hand side of the equation
becomes bigger than 1, and there is no solution for cos. Therefore, the maximum number of
trimers in a closed ring, satisfying the 3-fold and 2-fold rotations defined above, is 6, in which
case the axes d and t are parallel, and give rise to a planar arrangement like the hexagonal
lattice observed by EM (see Figure 2). The other angles reported in table S1 correspond to the
angles between symmetry axes within the platonic solids, as listed in the last row. The table
shows that the observed angle between 2-fold and 3-fold in the crystal is in between the angle
corresponding to a cube and a dodecahedron, being closest to the latter.
Table S1. Possible rings of N trimers, with angle  = 360/N.
N
2
3
4
OBS.
5
6

180
120
90
78.4
72
60

90
54.7
35.3
26.5
20.9
0

90
54.7
45
37.7
31.7
0
triangular
prism
tetrahedron
cube

dodecahedron
Figure S4. Possible arrangement of 3, 4, 5 and 6 E1* trimers in closed rings, generated by 2fold related contacts via the fusion peptide. The trimers were rotated about the contact area on
the dyad such that the angle between 2-fold and 3-fold matches the angle given in Table 1S.
Figure S4 shows the resulting possible rings of E1* trimers. The heads of the trimers are
bulkier than the fusion loop end, and therefore in the parallel arrangement of trimers in the 6fold ring the tips of domain II are forced to splay apart in order to make contact. The head-to-
head contacts observed are identical to those obtained by fitting the trimers into the EM
reconstruction. The "legs" of the tripod-like trimers were not visible at the resolution of the
reconstruction, and only the heads were visualized, and the high B-factor of the tips of domain
II suggests that they might indeed follow the fusion loop contacts by splaying apart. The
rings of five are very similar to those observed in the E1* trimer "rosettes" shown in Figure 3
of the main text.
Figure S5. Generation of a dodecahedral body (“rosette”) by propagating the 2-fold related
contacts between trimers, imposing an angle of 20.9 deg. between 2-fold and 3-fold axes
(from Table S1) to generate 20 trimers, each interacting with the others via the fusion peptide
loop. A: surface representation. B. Ribbons representation. C. The top part in B was removed
to show the type of curvature that such interaction would impose on a membrane.
Figure S5 shows a rosette generated from the crystal structure by introducing 3 identical
contacts to each of the 20 trimers present in a dodecahedron. This construction involves using
the "free", outward facing side of each trimer in the rings of 5, so that each ring becomes
surrounded by 5 other rings to make a closed body. During the trimer’s interactions with
membranes, however, the geometry of the lipid bilayer is likely to restrict the assembly and
allow only the formation of one pentagonal face of the dodecahedron, since the other faces
would be out of the plane of the membrane. In contrast, the rosettes, which are obtained by
dialysing away the detergent, are free to form a closed body with the fusion loop regions on
the interior, buried from solvent.