Genome map
Supplementary Figure 1: Epsilon15 genome and identified protein products. The
genome of Epsilon15 (accession #NC_004775) is 39671 bp and contains 49 predicted open
reading frames (orfs). Six Epsilon15 virion structural proteins were matched to orfs by mass
spectrometric peptide mapping of trypsin digested phage proteins separated by SDS-PAGE,
represented by the blue boxes in the genome map. Other genes were identified by homology
to sequences of known function. The left end of the genome encodes the virion structural
proteins with a gene order typical of “lambdoid” phages (Hendrix et al, Proc Natl Acad Sci
U S A 96, 2192-7, 1999). This gene module begins with the small and large terminase
subunits, followed by the portal, scaffold, and capsid genes. The products of orfs 15 and 17
are found in virions and are likely candidates for the core and/or tail hub. The Epsilon15
tailspike gene is located further downstream, near the genes for serotype conversion. A
similar arrangement is seen in the bacteriophage P22 tailspike gene. This clustering of
tailspike is likely functional since the serotype conversion genes modify those O-antigens
which are recognized by the tailspikes during infection. At the sequence level, the structural
proteins most closely resemble the Bcep phages (NC_005887) of Burkholderia bacteria, a
human pathogen affecting cystic fibrosis patients.
stds
Epsilon15
200
116
97.4
tailspike (gp20), 115.7 kD
unknown function (gp17), 100 kD
unknown function (gp15) 91 kD
possibly product of gp16, 67.4 kD
66
portal (gp4), 61.7 kD
capsid protein (gp7**)
45
capsid protein (gp7), 36.8 kD
capsid protein (gp7*)
31
Supplementary Figure 2: Protein composition of Epsilon15 virion. Virus particles were
boiled in SDS-PAGE sample buffer for 3 minutes and sonicated to reduce viscosity of
released phage DNA. Samples were electrophoresed through 10% acrylamide gels and
protein bands visualized by Coomassie staining. Individual bands were excised and
trypsinized. The peptide mixture of these digested protein samples were separated by mass
spectrometry and their masses correlated with a conceptual digest of translated Epsilon15
reading frames. The bands and their corresponding gene products are indicated. The faint
band assigned to gp16 is tentative since the band position corresponds to larger mass than
expected. gp7* and gp7** may correspond to the degradation and cross-linked products of
gp7. gp15 and/or gp17 are likely components in the tail hub based on the genome locations
with reference to other phages15.
A
B
Supplementary Figure 3: Resolution evaluation using Fourier Shell Correlation. The
complete data set were divided evenly into two halves and 3-D reconstructions were
generated from each of the half datasets. Fourier Shell Correlation between the two
reconstructions were used to evaluate the effective resolution of the 3-D density map using
the 0.5 threshold. (A) 20 Å resolution map of Epsilon15 phage with the non-icosahedral
reconstruction method shown in Fig.1b; (B) 9.5 Å resolution map of Epsilon15 phage with
the icosahedral reconstruction method shown in Supplementary Fig. 6b.
1
2
3
4
5
6
Side
Top
*
Supplementary Figure 4: Conformation variations of the tailspikes. Shown are the side
and top views of individual tailspikes. Top view of the tailspikes exhibits 3-fold symmetry.
Structures of most tailspikes except tailspike 3 (the central knob and one of the petals close
to the label *) are very similar.
A
B
Supplementary Figure 5: The portal complex is dodecamer. (A) The azimuthal density
distribution of the portal region (purple) shown in Fig. 3d. The plot shows 12-peaks clearly.
(B) Power spectrum of (A) shows that the azimuthal density distribution is dominated by a
12-fold repeat.
A
B
500Å
Supplementary Figure 6: Structure of Epsilon15 capsid shell at 9.5 Å resolution. (A) the
300 kV images of the Epsilon15. (B) the 3-fold view of the surface rendering of the 3-D
reconstruction with icosahedral symmetry imposed. The resolution assessment is shown in
Supplementary Fig. 3b. The 7 subunits in an asymmetric unit were individually annotated
with different colors.
Epsilon15
P22
Jiang et al.
NSB 2003
HK97
Wikoff et al.
Science 2000
T4
Fokine et al.
PNAS 2005
Herpes
Baker et al.
J Virol 2005
phi29
Morais et al.
Mol. Cell 2005
Supplementary Figure 7: The shell proteins of tailed dsDNA phages share similar fold.
Shown are cryo-EM structures of Epsilon15, P22, Herpes and phi29, and crystal structures
of HK97 and T4.
A
3
5
B
α
b
3
α
b
α
b
Supplementary Figure 8: The inter-subunit interactions of tailed dsDNA phages are
similar. Shown are the local 3-fold area of HK97 capsid (A) and Epsilon15 capsid (B). A
penton and two hexons interact at this local 3-fold region. The local 3-fold symmetry was
not imposed during 3-D icosahedral reconstruction for the Epsilon15 phage.