Structural changes in a marine podovirus associated with
release of its genome into Prochlorococcus
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Citation
Liu, Xiangan et al. “Structural changes in a marine podovirus
associated with release of its genome into Prochlorococcus.” Nat
Struct Mol Biol 17.7 (2010): 830-836.
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http://dx.doi.org/10.1038/nsmb.1823
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Nature Publishing Group
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Thu May 26 18:26:28 EDT 2016
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http://hdl.handle.net/1721.1/61294
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Structural Changes in a Marine Podovirus Associated with Release of
its Genome into Prochlorococcus
Xiangan Liu1, Qinfen Zhang1,2, Kazuyoshi Murata1, Matthew L. Baker1, Matthew B. Sullivan3,4,
Caroline Fu1, Matthew Dougherty1, Michael F. Schmid1, Marcia S. Osburne3, Sallie W. Chisholm3
& Wah Chiu1
1
National Center for Macromolecular Imaging, Verna and Marrs McLean Department of
Biochemistry & Molecular Biology, Baylor College of Medicine, Houston, TX, 77030, USA
2
State Key lab for Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou
510275, China
3
Department of Civil and Environmental Engineering, M.I.T., Cambridge, MA 02139, USA
4
Present address: Department of Ecology and Evolutionary Biology, University of Arizona,
Tucson, AZ 85721, USA
1
Podovirus P-SSP7 infects Prochlorococcus marinus, the most abundant oceanic
photosynthetic microorganism. Single particle cryo-electron microscopy (cryo-EM) yields
icosahedral and asymmetrical structures of infectious P-SSP7 with 4.6 Å and 9 Å resolution,
respectively. The asymmetric reconstruction reveals how symmetry mismatches are
accommodated among 5 of the gene products at the portal vertex. Reconstructions of
infectious and empty particles show a conformational change of the “valve” density in the
nozzle, an orientation difference in the tail fibers, a disordering of the C-terminus of the
portal protein, and disappearance of the core proteins. In addition, cryo-electron tomography
(cryo-ET) of P-SSP7 infecting Prochlorococcus demonstrated the same tail fiber conformation
as in empty particles. Our observations suggest a mechanism whereby, upon binding to the
host cell, the tail fibers induce a cascade of structural alterations of the portal vertex complex
that triggers DNA release.
Cyanophage P-SSP7, categorized as a T7-like podovirus based on morphology and genome
sequence1, is found in Prochlorococcus, a genus of marine cyanobacteria from the Atlantic
Ocean. The Prochlorococcus group, consisting of genetically distinct ecotypes2, contributes a
significant fraction of global oceanic photosynthesis3, and T7-like phages such as P-SSP7 are
among the most abundant viral types observed in viral metagenomes from the vast, lownutrient (oligotrophic) surface ocean waters4. P-SSP7 is one of the best characterized marine
phages, as its complete genome has been sequenced 1 and the transcriptomes and proteomes
of both the virus and host cell during infection have been described 5,6. Here, we expand upon
these efforts by elucidating the structural features of the viral particle on its own using single
particle cryo-EM and during the host infection process using cryo-ET, revealing biologically
important phenomena in this system.
RESULTS
Icosahedral reconstruction of P-SSP7 capsid
Using 36,000 particle images (Fig. 1a), we obtained an icosahedral reconstruction of the P-SSP7
shell at ~4.6 Å resolution (Supplementary Fig. 1a,b and Movie 1a) using MPSA7 and EMAN8. The
density map shows the phage particle has a T=7 shell that is 25–40 Å thick and ~655 Å in
diameter from 5-fold to opposite 5-fold vertices. Using a de novo model building technique9, we
were able to build the alpha-carbon (C) backbone chain trace models for each of the 7 shell
proteins (gp10) in an asymmetric unit. Figure 1b shows one of the gp10 models (residues 1–363)
containing 5 structural features, 4 of which also appear in other phage capsid structures 9,10
(Supplementary Fig. 2 and Movie 1b): an extended amino terminus (N arm), a triangular domain
(Domain A), an elongated protrusion domain (Domain P), a long extended loop (E loop), and a
2
structurally important small loop between the N arm and E loop, near the local 2-fold axis
(which we call the F loop).
The backbones of all 7 subunits have slightly different conformations (Supplementary Fig. 1c),
but have similar structural features and topology. The whole capsid backbone model is shown
in Supplementary Fig. 1d and Movie 1c. The C backbone trace of gp10 shows its fold to be
topologically similar to the corresponding shell protein of other tailed phages such as 159,
HK9710 (Supplementary Fig. 2), P2211, T412, φ2913 and of herpes simplex type 1 virus14, even
though no sequence homology is evident. These phages appear to have different ways
(Supplementary Fig. 2) of protecting their shells against the high internal pressure (30–60 atm)
produced by tight genome packing15. P-SSP7 does not have decoration proteins such as those
used by 15 to stabilize its shell, nor does it have a covalently cross-linked chain mail like HK97
(Supplementary Fig. 2). Instead, P-SSP7 uses subunit interactions within and between its
capsomeres to stabilize its capsid shell (Fig. 1c,d) in a way that has not been seen previously in
other phage structures.
A strong density between capsomeres is found at the F loop between the charged residues,
Lys61 of one subunit and Glu120 in the domain P of another subunit (Fig. 1d). The F loop in PSSP7 is longer than in either HK97 or 15 and also interacts with the portal to accommodate
symmetry mismatch at the portal vertex, as will be seen in the asymmetric reconstruction.
Another previously unseen interaction occurs within each capsomere: the N arm (cyan-colored
loops in Fig. 1c) of one subunit wraps around the long E loop (Fig. 1c) of a neighboring subunit,
thus reducing the flexibility of the E loop without requiring  sheet formation as in HK97. All
these capsid shell features of P-SSP7 may be crucial in compensating for lack of cross-linking10
or an additional protein9 against the high internal pressure.
Asymmetric reconstruction of the P-SSP7 virion
Due to the symmetry enforcement in the reconstruction above, the density of the portal vertex
complex (defined herein as all the densities associated with the special 5-fold vertex) is lost.
Traditionally, structures of the non-icosahedral protein components were studied individually
by crystallography or NMR and fit to the low-resolution cryo-EM maps of the virion or
complexes isolated from virions16-18. It has also been possible to identify these components by
computationally isolating the tail from single particle images (e.g. T4 19 and SPP120). This divideand-conquer approach has undeniably contributed much insight into virus structure, assembly
and infection. In view of the advances in the cryo-EM method, we opted to determine the
structure of the entire mature P-SSP7 phage without any imposed symmetry, to find the
detailed structures of the portal vertex complex and the spatial relationships among protein
components of the portal vertex complex and the icosahedral shell.
3
Using the same data set and a newly developed reconstruction algorithm (Supplementary
Methods), we produced a ~9 Å resolution density map without assuming any symmetry (Fig. 2,
Supplementary Fig. 3a). In comparing the asymmetric density map with the C model from the
4.6 Å icosahedrally imposed map, we found that the portal vertex and its opposite 5-fold vertex
are compressed along the radius by ~3–4 Å, whereas the other 10 “normal” 5-fold vertices
appear to match with those determined from the icosahedral reconstruction. Although the
extent of such compression is smaller than the resolution of either map, small differences
between structures can be reliably revealed as shown in many structural examples21,22.
We segmented the densities at the portal vertex of the map into the following components:
nozzle, adaptor, tail fibers, portal and core proteins (Fig. 2a,b). This segmentation assignment is
based on the following considerations: (1) the connectivity of the densities, (2) the classification
of different protein parts of T7 phage23, (3) the match of the detected secondary structure
elements in both the adaptor and nozzle to the sequence based predictions of gp11 and gp12
(Supplementary Figs 4 and 5), and (4) the match of both the capsid model (Fig. 1b) and
homologs of the portal (Supplementary Fig. 6) to their corresponding densities.
The cryo-EM map, along with genomics1 and proteomics5 studies of P-SSP7 and previous T7
biochemical studies23, allowed us to annotate the segmented densities at the portal vertex
complex in terms of the corresponding gene products (Supplementary Methods). These include
the adaptor (gp11), nozzle (gp12), tail fiber (gp17), portal (gp8), and the dsDNA genome (Fig. 2b
and Supplementary Movie 2). Each of these densities (adaptor, nozzle, portal) is also crossvalidated by the expected volumes of the putative gene products. However, the nozzle may
contain slightly more volume than six copies of gp12 would occupy.
The 9 Å resolution map of P-SSP7 is sufficient to determine the copy number and symmetry for
most of the protein components in the portal vertex complex using rotational correlation
analysis. The analyses (Supplementary Fig. 3d) show 12 copies for the portal protein and 6
copies for the nozzle protein with 12 and 6 fold symmetries respectively. The 12 adaptor
proteins have weak 6-fold symmetry near the nozzle and 12-fold symmetry at the opposite end
near the portal (Supplementary Fig. 3d). The 12 copies of the adaptor protein exist in two
conformations that have alternating positions in the ring-shaped adaptor (Fig. 3c). Each of the 6
tail fibers appears to be a trimer (Fig. 2c), which splays out into three “petals” and then rejoins
at the junction with the adaptor and nozzle (Fig. 2c,d). Since the visible length and size of each
tail fiber can account for only part of its trimer, the distal portion must be very flexible and
therefore not visible in our asymmetric reconstruction. The inner core proteins are poorly
defined, and no symmetry could be identified.
Applying the appropriate symmetry to some of the portal vertex protein components, the
contrast of their structural features can be enhanced. Using SSEHunter 24 to analyze of the
symmetrized components, we were able to identify -helices (>2 turns) and -sheets (> 2
4
strands) in these densities: 2 -helices and 2 -sheets in the adaptor protein, 8  sheets in the
nozzle protein, 3  sheets in the tail fiber trimer, and 12 -helices and 3  sheets in the portal
protein (Supplementary Fig. 4). The accuracy in the identification of these secondary structure
elements was substantiated by the positional match of the helices and sheets of the capsid
protein (gp10) in this 9 Å map with its structure in the 4.6 Å icosahedral map (Fig. 1b). In
addition, we found that our assignment of the identified secondary structure elements agree
well with those predicted by bioinformatics tools for both the nozzle protein (gp12, primarily 
sheet) and the adaptor protein (gp11, a mixture of  helices and  sheets) (Supplementary Fig.
5).
Despite the absence of a crystal structure of the P-SSP7 portal subunit (gp8, 522 residues), we
matched the density and secondary structure elements of the putative portal (gp8) with models
of its structural homologs from 2925 (1IJG, 309 residues) and SPP126 (2JES, 503 residues). We
found the upper part of the density has a large conformational difference from the SPP1
portal26 structure, but the lower part density fits well with both 29 and SPP1 portal structures
(Supplementary Fig. 6a,b). This match further supports the accuracy of our secondary structure
element identification and segmentation of gp8. Based on the sequence alignment between the
29 portal and the P-SSP7 portal protein, we can deduce that the unmatched density, which
points towards the center of the capsid, belongs to the C-terminus (Supplementary Fig. 6a,b).
Furthermore, the consensus secondary structure prediction (Supplementary Fig. 6c) of the gp8
C-terminal region (residues 473–497) suggests that it is an α-helix with a glutamine (Q)-rich
motif, in agreement with our SSEHunter results (Supplementary Fig. 6d). This has not been seen
previously in other phage portal protein structures but, interestingly, sequence analysis 27,28 also
predicts -helices near the C-termini of portal proteins in other phages (T7, syn5, KIE, ε15). In PSSP7, a total of 12 Q-rich helix cluster motifs near the C terminus of the portal are surrounded
by the inner core proteins. Q-rich motifs are known to undergo reversible assembly and
disassembly in protein complexes29; the Q-rich domain here may play some role in the
disassembly and assembly of the internal core proteins, DNA translocation, and/or stabilization
of the DNA terminus. Such functional speculation requires future experimental verification.
In the asymmetric reconstruction, the putative core protein density is the most difficult region
to interpret (Fig. 2b). As suggested previously, gp15 and gp16 and possibly gp14 are core
components1,5. Because we cannot assign a copy number, observe any symmetry or resolve any
discernable secondary structure elements in the observed core density, we cannot tell if this
density is made of any or all of these three gene products. In addition, the genomics and
proteomics analyses have proposed several other gene products with completely unknown
functions and localizations (i.e. orf 23, 37, 38, 39, 41, 42, 46, 48, 50) 1,5. These unknown proteins
may have a small number of copies, be very flexible or be attached to other flexible
5
components. The resolution of our map is still too limited to determine whether these proteins
correspond to any of the observed densities.
The size of the capsid chamber can accommodate the 44,970 base pairs (bp) genome 1 which is
packed coaxially around the portal vertex axis with a spacing of ~24 Å between rings (Fig. 2b,
Supplementary Fig. 3c). The rings are packed hexagonally (Fig. 2b). The DNA near the inner
capsid surface is better resolved than the rest of the DNA, suggesting possible interactions
between the capsid and the DNA. We tentatively interpret the relatively strong density filling
the channel in the portal vertex (250 Å long, ~73bp and ~20 Å wide) to be the DNA terminus.
Density at this location was also observed in 15 phage30. This density extends as far as the
interface between the nozzle and the adaptor. A density protruding from the 6 nozzle proteins
towards the central axis appears to cap the tip of the DNA terminus, which we annotate as the
“nozzle valve” (Figs 2b–d and 3e).
There is a split of the density resulting in a separation of ~14 Å in the middle of the DNA
terminus (Fig. 2b, Supplementary Fig. 3c), which we interpret as strand separation caused by
the torsional strain on the double-stranded DNA during packaging31, and/or specific
interactions of the flipped-out bases with the portal. Each individual strand density is ~8 Å in
diameter. Thus, the size of the separated strands in our map is consistent with single-stranded
DNA rather than with double-stranded DNA. The separation occurs near the center of the
portal, whereas the proposed toroidal dsDNA structure of 29 phage32 occurs on the outside
end of its portal. The size of the 14 Å separation seems too small to accommodate a loop or
torus of double stranded DNA as found in phage 29. Therefore, these DNA features are
completely different between P-SSP7 and 29.
Symmetry mismatch at the portal vertex
Symmetry mismatch between the 5-fold capsid and the portal vertex complex has been
observed in dsDNA phages26,30,33,34. Our asymmetric map (Fig. 2a) shows how the portal (gp8)
and capsid proteins (gp10) accommodate the 5-fold and 12-fold mismatch at the molecular
level. Where the portal complex occurs instead of a penton at the portal vertex in our map,
there are 10 gp10 proteins that surround the 12 gp8 portal proteins. The interfacial density (Fig.
3a) reveals four interaction types (Fig. 3b): five portal subunits (2,4,7,9,12) contact the F loop of
cyan-colored gp10 subunits, another 5 portal subunits make contacts with one of two sites in
the domain P of purple-colored gp10 subunits (1,3,6,8 with one site and 10 with the other site).
The remaining 2 portal subunits (5,11) have no apparent density connecting to any gp10
subunit. The region of the portal density that associates with the gp10 subunits does not follow
a strict 12-fold symmetry, whereas the gp10 subunits around the portal appear to be in a
similar conformation as in the icosahedral reconstruction. Thus the symmetry mismatch at this
6
pentameric interface is accommodated by multiple types of interactions between the
structurally uniform gp10 subunits and the portal proteins.
We discovered another kind of symmetry mismatch within the portal vertex complex. The 12
copies of the adaptor constitute a ring of 6 lambda-shaped (Λ) dimers (Fig. 3c), which mediate
the mismatch between the 12-fold symmetric portal and the 6-fold symmetric nozzle. The two
types of symmetry of the adaptor proteins along their length may be used to accommodate the
transition from one symmetry type (12-fold) to another (6-fold). On the portal side, two
adjacent adaptor subunits interact with each of the 12 portal subunits (Fig. 3d), while on the
other side, four contiguous adaptor subunits interact with each of the 6 nozzle subunits (Fig.
3e). In addition, two adjacent adaptor dimers are bound together through one petal of a tail
fiber (Figs 2c,d and 3d). The fiber also binds to the nozzle near the valve and an adaptor subunit
near the nozzle side (Figs 2c,d and 3e). This intertwined organization of the portal vertex
components linked by tail fibers creates extensive interactions, which may facilitate
cooperative motions among them during phage-host infection resulting in viral genome release.
Difference between full and empty particles
Cryo-EM micrographs show a small percentage of P-SSP7 particles without DNA (Fig. 1a). An
asymmetric reconstruction of empty particles was also performed, but at lower resolution (24 Å)
due to the limited number of particles in the data set (Fig. 2e–g). The diameter of the empty
capsid was the same as that of the DNA-containing phage, whereas the portal vertex of the
empty phage was different (Fig. 2, and the portal complex difference map in Supplementary Fig.
7). Empty particles lacked the “nozzle valve” and core protein densities, the distal tip of the
nozzle was shaped differently, the fibers were extended horizontally rather than pointing
parallel to the capsid surface, the Q-rich motifs of the portal appeared disordered and the core
protein disappeared. We presume that these empty particles have completed release of their
genomes and that these structural differences reflect the changes necessary to allow the DNA
to exit the capsid. If true, then the changes in fiber orientation from inclined to horizontal
positions may trigger co-operative structural changes in the nozzle, adaptor, portal, and core.
Interestingly, signaling or structural changes from the tail fiber, which is ~150 Å away from the
Q-rich motifs, may directly influence its disordering and the disappearance of the surrounding
core proteins.
Close examination of the tail fiber in full versus empty phages at equivalent resolution (Fig. 2d,g
and Supplementary Fig. 7) shows that the three "petals" of the tail fiber do not undergo large
movement, unlike the more distal portion of the tail fiber. However, in the full phage, the
proximal end of the tail fiber (Fig. 2c) closely contacts with an adaptor subunit (yellow box in Fig.
2d), whereas in the empty phage, the density near the contact point is missing, having moved
or become disordered (yellow box in Fig. 2g). In addition, the densities near the nozzle valve
and the adaptor domains that are in contact with this tail fiber density also move or become
7
disordered in the empty phage (Fig. 2d,g). These changes result in opening the “nozzle valve”,
presumably allowing ejection of the dsDNA from the capsid. A similar mechanism has been
indirectly inferred for phages T735, T436 and SPP117,18. Our study provides a direct evidence for
this mechanism based on visualization of the valve features that are present in mature but not
in empty phages.
Tomographic reconstruction of phages during cell infection
To seek additional evidence of structural changes in the portal vertex complex that may be
necessary for infection in vivo, we used cryo-ET to examine the P-SSP7 portal vertex complex
during the actual infection process. Four different phage states can be observed: free particles
with and without DNA, and attached particles with and without DNA. Free particles with DNA
are called free phages; attached particles with DNA are referred to as infecting particles; and
empty particles are designated as post-infection phages. A slice through a representative
tomogram, (Fig. 4a, Supplementary Movie 3) shows several of these states. From 12
tomograms, 29 pre-infection, 26 infecting and 8 cell-bound post-infection phages were
extracted, aligned and averaged separately. The averaged density of the 26 infecting phages
and the central section are shown in Fig. 4b and 4c, respectively. In contrast to the pre-infection
phages, the tail fibers of the infecting phages are extended horizontally even though there was
still DNA density inside the capsid. The key observation is that the change in conformation of
the tail fibers precedes DNA release, and thus may be part of the triggering mechanism itself.
The averaged density of the cell-bound post-infection phages also shows horizontally extended
fibers. This conformation corresponds to what was seen in the single particle asymmetric
reconstruction of empty phage in Fig. 2e and 2f.
DISCUSSION
Structure of the mature P-SSP7 phage
We used single particle cryo-EM to determine the icosahedral structure of the mature P-SSP7
phage at 4.6 Å resolution (Fig. 1). This structure is among a few examples of virus particles
whose C backbone can be traced without reference to any known crystal structure 9,37. The
topology of the P-SSP7 capsid protein bears a remarkable resemblance to HK97 and 15 phage,
whose capsid structures were previously determined at high resolution. As shown in the more
detailed comparison among these phage structures (Supplementary Fig. 2), they exhibit
different types of molecular interactions within and across capsomeres, which may be critical
for maintaining particle stability in a mature state. Our study reiterates the now commonly
observed conservation of structural motif in proteins that form a functional “nanocage” to
store and safeguard viral genome material, in spite of their high sequence diversity.
Little was known about the structure of portal vertex complex until the recent advances in cryoEM, which enabled virus structures to be solved without imposing icosahedral
8
symmetry30,32,35,38-40. The reconstruction software used in this study was designed to determine
at which of the 12 vertices the very low contrast and relatively small portal vertex complex
resides. The sub-nanometer resolution structure of the entire P-SSP7 phage, including the
portal vertex complex, gives an unambiguous view of the spatial organization of all the protein
components in the portal vertex complex. Our 9 Å resolution map (Fig. 2a) is sufficient to
reliably detect long  helices and large  sheets (Supplementary Fig. 4), as in previous cryo-EM
maps9,30,41. However, the challenge remains to delineate which densities in our map correspond
to which gene products. Our strategy uses a combination of proteomics, genomics and
structural informatics analyses to allow us to annotate the densities in the portal vertex
complex. Because of the potential overlap of densities among neighboring proteins, it is by no
means certain that the assignment of the molecular boundary of each protein is precisely
correct at the current resolution. However, we are highly confident in the following conceptual
conclusions drawn from our structural analyses: the conformationally heterogeneous dimer
encompassing the 12-copy adaptor ring; the location of the C terminus of the portal protein
with a Q-rich motif; the nozzle valve blocking the exit pathway of the DNA; a massive core
protein density next to the portal pointing toward the center of the particle; the orientations of
the tail fiber with respect to the portal vertex axis; a 250 Å long and 20 Å wide terminal end of
dsDNA with a strand separation close to the mid point of the portal protein. Even though there
have been many structural studies of phages either as a whole or in parts, most of our observed
structural features of P-SSP7 have not been seen in prior studies of phage structure.
Furthermore, the delineation of the molecular interface among the nozzle, adaptor, portal, tail
fiber and capsid protein provides a fresh and direct glimpse at the complexity of interactions
that accommodate the symmetry mismatches among these molecular components.
Interestingly, by fitting the capsid backbone models into the 10 subunit densities surrounding
the 12 copies of the portal protein (Fig. 3a,b), we find no apparent structural deformation of
the capsid protein at our current resolution level.
Structure of the empty P-SSP7 phage
Our purified sample of P-SSP7 phages contained a small percentage of the particles that
appeared to be devoid of internal DNA (Fig. 1a). We sampled those particles and obtained a 24
Å resolution map of the empty phage (Fig 2e). The low-resolution structure is due to the limited
number of empty particles available in the data pool. To compare the mature and empty phage
maps (Figs 2d,g; Supplementary Fig. 7) in a meaningful way, we blurred the resolution of the
mature phage map (Fig. 2a) equivalent to that of the empty phage (Fig. 2e). The obvious
differences in the empty phage map relative to the mature map are: the absence of DNA
density; the disappearance of the nozzle valve, the change in the fiber orientation with respect
to the portal vertex axis, and the disintegration of the C-terminus of the portal and of the core
protein. A close examination of the molecular interfacial region among the adaptor, tail fiber
9
and portal also shows unambiguous differences in density connectivity between the mature
and empty phage structures (Fig. 2d,g and Supplementary Fig. 7).
Cryo-ET Structure of P-SSP7 attached to Prochlorococcus
Cryo-ET is an emerging method for reconstructing the 3-D structure of one sample at a time42,43.
Cryo-ET is a low-resolution technique because of the radiation damage to specimens resulting
from multiple electron exposures, but it is extremely powerful for revealing structures that may
vary from one cell to another and therefore cannot be averaged as in a single particle
reconstruction. In this study, we focused on the extraction of the structures of phage particles
attached to the host cell surface. Using post-tomographic classification, aligning and averaging
of the sub-volumes of the computationally extracted phage particles attached to the cell, we
were able to find a subpopulation of phage particles in which the DNA was still present, but the
tail fiber was oriented similarly to that of the empty phage (Fig. 4b,c and Fig. 2e,f). This match
supports the hypothesis that the phage tail fiber orientation changes while attached to the host
surface in vivo before DNA release. Our study represents the first example of applying cryo-ET
to correlate the observed high-resolution structural features of a virus to its structure in the
context of a host cell.
Plausible model of viral genome release
Based on both single particle reconstructions of individual phages and tomographic
reconstructions of phages actively infecting the host cell, we propose a mechanism for DNA
release from the phage as follows. First, the most distal segments of the tail fibers explore the
host cell surface in search of a binding site to initiate the infection. As in phage T723,44, the
phage orients itself with its portal vertex complex pointing perpendicular to the cell surface so
that the tail nozzle is in contact with the cell surface and the proximal segments of the fibers
extend horizontally (Fig. 4d). This tail fiber orientation change in turn affects the interactions
among the tail fiber, adaptor and the portal causing the Q-rich motifs of the portal protein to
loosen, thereby triggering the internal core proteins to disassemble. During this process, the
DNA structure may also be changed and thus could contribute to the disassembly of the Qmotifs of the portal protein and the core proteins. Not the least among the structural change is
found in the opening of the “nozzle valve” (Fig. 2b–d and 2f,g). The cumulative conformational
changes in the portal vertex complex should then finally allow the phage DNA to eject freely.
In all, this study demonstrates the power of using cryo-EM and cryo-ET synergistically both in
vitro and in vivo, particularly for non-icosahedrally arranged protein components at a broad
range of resolutions. The structures provide a number of structural features of the P-SSP7
phage in different functional states, which have not been seen in previous studies of other
dsDNA viruses. Also, our structural interpretation sheds light on the biology of a T7-like phage
and the long-standing mystery of DNA delivery from phage to host 45,46. Given that this phage
10
type is a prominent member of the global oceanic phage community, such findings not only
impact our understanding of this ecologically important phage-host system, but also refine our
view of a biological process using a complex protein machinery to transfer the viral genome to
an infected cell.
METHODS
Methods and any associated references are available in the online version of the paper at
http://www.nature.com/nsmb/.
Accession codes: The density maps and associated models have been deposited to EBI
(accession numbers xx to be included in the galley proof).
Note: Supplementary information is available on the Nature Structural & Molecular Biology
website.
ACKNOWLEDGMENTS
This research was supported in parts by grants from NIH (P41RR002250, R01GM079429 to
W.C.), NSF (IIS-0705644 to W.C. and M.L.B.), the Robert Welch Foundation (Q1242 to W.C.),
NSF, DOE and the Gordon and Betty Moore Foundation (to S.W.C.). We thank Maureen
Coleman, Sebastien Rodrigue, Rex Malmstrom, Jake Waldbauer and Suzanne Kern for
assistance in the infection experiments; Juan Chang, Qinglu Zeng, Simon Labrie, and Frazer
Rixon for discussions; Juan Chang, Kurt Welgehausen and Ryan Rochat for editorial assistance in
preparation of the manuscript.
AUTHOR CONTRIBUTIONS
M.B.S., M.S.O. and S.W.C provided samples. Q.Z. took single particle images. Q.Z., C.F. and K.M.
took tomographic images. X.L. introduced the methodology for single particle symmetric and
asymmetric reconstructions; computed all the maps. X.L. and W.C. interpreted the maps. M.F.S.
analyzed the difference maps. K.M. did tomographic reconstructions and performed its analysis
with M.F.S. assistance. M.L.B. and X.L. built Cα backbone models. M.T.D. and W.C. designed the
animations. X.L. and W.C. wrote the paper with contributions from all co-authors.
Figure Captions
Figure 1 Image and reconstruction of P-SSP7 at 4.6 Å resolution. (a) 300kV image of P-SSP7
embedded in vitreous ice. (b) Segmented density of the shell protein subunit (gp10) and its C
11
model from the 4.6 Å icosahedral reconstruction. (c) Subunit interactions within and across
capsomeres. Four subunits are shown; the upper two belong to one pseudo-6 fold capsomere,
the bottom two belong to another capsomere. The view is from the outside of the capsid
looking almost down the 2-fold symmetry axis. (d) A zoomed-in view, but viewed from inside
the capsid, with the corresponding density around the F loop and domain P. The densities
(annotated in yellow) near the 2-fold axis indicate interactions between Lys61 (in blue) and
Glu120 (in red) from two capsomeres.
Figure 2 Asymmetric reconstructions of P-SSP7. (a) 9.2 Å resolution map of the full phage. (b)
Central slice of the full phage. (c) Fiber trimer viewed from its proximal end. (d) A cutaway
zoomed-in view of the adaptor, nozzle and tail fibers. In (c) and (d), the full phage map was lowpass filtered to the same resolution as the empty phage map to facilitate comparison between
the structures. (e) 24 Å resolution asymmetric reconstruction of the empty phages. (f) Central
slice of the empty phage. (g) Cutaway view for the empty phage equivalent to the view in (d).
All phage protein components are annotated and colored differently. Two special features
(valve density and DNA strand separation) are observed in the slice from the full phage (b).
There are substantial conformational changes in the nozzle, tail fibers, portal and core proteins
between the full and empty particles. The yellow dashed box in (d) shows that a fiber interacts
with both nozzle and adaptor in full phage, but the links between fiber and adaptor are broken
in empty phage (g). The full phage has strong density at and around the valve (colored in
orange and red), but the density of empty phage in the corresponding location is markedly
reduced and there is no density at the valve.
Figure 3 Symmetry mismatch at the portal vertex. (a) Interface between 12 copies of portal
(greenish) and 10 copies of gp10 models (purple and cyan). The purple and cyan molecules
represent two subunits from each of the 5 surrounding hexons. The gray density segmented
from the asymmetric reconstruction is the interfacial density between capsid and portal. (b)
Schematic diagram to illustrate the 4 apparent types of interactions in our density map: F loop
interaction (2, 4, 7, 9, 12), hook of domain P interaction (1, 3, 6, 8), N terminus of long helix
interaction (10) and no interaction (5, 11). (c) 12 copies of adaptor proteins in alternating
conformations. (d) Interface between the portal (red and black footprints – black indicates one
subunit) and the adaptor. One subunit of the portal interacts with two neighboring adaptor
subunits. (e) Interface between the nozzle (red and black footprints, black for 1 subunit) and
the adaptor. One nozzle subunit interacts with 4 contiguous adaptor subunits and also interacts
with a tail fiber. The nozzle footprint outlines coverage around the center of the nozzle and
indicates the presence of the “valve”. Figure 3a, 3b and 3e are viewed from the outside along
the portal vertex complex axis, while Figure 3c and 3d are viewed from the inside of the capsid.
Figure 4 Cryo-ET of P-SSP7 infecting MED-4 cell. (a) Slice of a tomogram showing three states
of P-SSP7 in its native environment: infecting phages (all dark particles with one annotated by a
12
yellow arrow); post-infection (empty) phages still attached to the cell (white arrow); and
unattached phages. The attachment of the phages to the host cell can only be ascertained from
the visualization of the 3-D tomogram, but not in a 2-D slice as exemplified here. (b) An average
of 26 infecting phage subtomograms with the portal vertex oriented normal to the cell surface.
The phage tail fibers are extended horizontally. (c) Central slice of the averaged subtomograms
of infecting phages. (d) Schematic cartoon of P-SSP7 phage infecting the host cell (the cell
membranes are represented by the double layer at the bottom).
13
ONLINE METHODS
Preparation of P-SSP7. We prepared P-SSP7 samples based on a previous protocol1. Briefly, PSSP7 particles were propagated on Prochlorococcus MED4. The particles were precipitated by
polyethylene glycol 8000, purified on a cesium chloride step gradient (steps were ρ=1.30, 1.40,
1.50, and 1.65), spun at 104,000× g for 2 h at 4° C, and dialyzed against a buffer containing 100
mM Tris-HCl (pH 7.5), 100 mM MgSO4, and 30 mM NaCl. Just before freezing, we
ultracentrifuged the phages at 104,000× g for 1.5 h at 4° C, and re-suspended the pellet in
buffer to yield a concentration of about 1012 particles per ml for single particle cryo-EM.
Single particle cryo-EM imaging. We applied purified P-SSP7 sample to copper R1.2/1.3
Quantifoil grids (Quantifoil Micro Tools GmbH), and flash froze the grids in liquid ethane using a
Vitrobot (FEI). The sample was imaged on a JEM3200FSC electron Cryo-microscope (JEOL, Tokyo)
equipped with a field emission gun operated at 300KV and an in-column energy filter (using a
20 eV slit). The data were recorded at 50,000× nominal magnification on Kodak SO163 film with
878 and 181 micrographs from specimens imaged at specimen temperatures of 100K and 25K,
and a specimen dose of 20 to 28 e Å-2, respectively. We developed the films in full strength D19
Kodak developer for 12 minutes at 20 °C and fixed for 10 minutes in Kodak fixer. The films were
digitized at 6.35 m per pixel using a Nikon Super CoolScan 9000 ED scanner (Nikon Corp.,
Japan).
Single particle 3D reconstruction. We used ETHAN47 to automatically extract raw particle
images from 1,059 micrographs, and manually screened ~48,000 particles using the EMAN8
Boxer program. Most of the micrographs had a defocus range of 0.5–2.0µm.
We used Multi-Path Simulated Annealing (MPSA)7 algorithm based on cross common lines48 to
determine the icosahedral orientation of each particle. An initial model was built by assigning
random orientations for a small number of particles. The EMAN8 make3d program was used to
reconstruct the 3D map. We iteratively refined the map to 4.6 Å resolution with updated
models in each cycle. The final icosahedral reconstruction used about 36,000 particles selected
by the consistency criterion in MPSA7 procedure. The resolution of the map was assessed by
the 0.5 FSC criterion of the Fourier shell correlation from two split data sets.
The previous asymmetric reconstruction method30,49 for 15 phage did not work for P-SSP7. We
introduced a different approach (Supplementary Methods and Supplementary Fig. 8) to
reconstruct P-SSP7 virion structure with no imposed symmetry. In this process, we started with
the same data set of 36,000 particle images as in the icosahedral reconstruction. We used a
total of 15,000 particle images for the asymmetric reconstruction, due to the removal of
particles with inconsistent orientations in the last few iterations. The final asymmetric map of
14
the mature phage was reconstructed at ~9 Å. We used the same procedures for the empty
particle reconstruction with 1,200 particles at ~24 Å resolution.
Structure analysis of the maps. The segmentation of each of the molecular components in the
reconstructions was done manually with Amira software. The methods for segmentation and
gene annotation are detailed in Supplementary Methods. All visualizations were done with
Chimera50. The 4.6 Å icosahedral map had clear boundaries between the gp10 subunits. We
traced the Cα backbones of the seven gp10 proteins in an asymmetric unit of the 4.6 Å map as
described previously9,51.
Preparation of phage infection for cryo-ET imaging. The Prochlorococcus MED4 host cells were
grown in a modified, lighted Percival incubator using a "sunbox light pattern" (0-110 E m-2 s-1
light, that slowly ramps 5 hours at sunrise/sunset with 4 hours stable in the middle, and 10
hours darkness) at a temperature of 22–24° C depending upon the time of day, yielding a
growth rate of about mu = 0.65. In late exponential phase, we concentrated these cells to a
concentration of ~109 cells per ml. Then, 100 l of cells (~109 cells per ml) were mixed with 400
l of phage (~1010 phage per ml as counted by fluorescence counts), to achieve a multiplicity of
infection of ~40 viruses per cell. 5 µl aliquots of the cell and phage mixture were taken at 3
minute intervals from 4 to 90 minutes post-infection, added to 1 µl of 150 Å colloidal gold
(EMS), applied to R3.5/1 copper Quantifoil grids (Quantifoil Micro Tools GmbH), and rapidly
frozen with the Leica plunger (EM-CPC, Leica Microsystems). Grids were kept in liquid nitrogen
and transferred into JEM3200FSC electron cryomicroscope (JEOL, Tokyo) for collecting the tilt
series on a Gatan 4k×4k CCD camera (Model 895) using Serial EM software52.
Subtomogram averaging. The tilt series from the frozen grids after 60 minutes post-infection
were aligned using gold fiducials, and tomograms were reconstructed by weighted back
projection using IMOD software53. We extracted three different types of phages (named free,
infecting, and empty) from 12 tomograms. We used the following three steps to align the
extracted particles. First, the 20 Å low-pass filtered subtomograms, in which the particle tails
were excluded by a tight spherical mask, were aligned to the single particle icosahedral map.
Second, all the icosahedrally aligned particles, including their tails, were further rotated so that
each portal vertex complex was oriented along the Z axis. Third, to find the unique azimuthal
orientation of the portal vertex complex out of the five orientation choices, we masked the
portal vertex complex, applied 6-fold symmetry, and compared the symmetrized map with the
single particle asymmetric map in 72° (5-fold) steps. The resulting aligned subtomograms of the
three different types were separately averaged in Fourier space by weighting the missing
wedge to reduce the orientation bias54.
15
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19
a
c
F loop
E loop
E loop
F loop
700 Å
N arm
Lys61
N terminus
b
Glu120
Val363
N arm
Domain P
d
Domain A
Met1
Glu120
E loop
Lys61
F loop
C terminus
a
c
b
Contacts the
upper part
of adaptor
dsDNA
Core
proteins
Capsid
DNA strand
separation
d
Portal
Fiber
Valve
Nozzle
e
Contacts the
adaptor
and nozzle
Adaptor
f
g
Capsid
Portal
Fiber
Nozzle
Adaptor
I
a
12
11
V
II
1
10
7
6
5
d
1
2
10
3
9
4
7
5
6
II
1
2
3
7
III
c
12
8
4
IV
8
11
10
9
3
8
12
V
2
9
11
I
b
6
IV
5
4
III
e
a
b
500 nm
c
d
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GD4GE4GI4GK4HC=%1'*%1&',(*+&,!&, %,-*( 7$!%!,'-*'&$-+!'&'&$1
,' , '+ &+!,!+ & & (*'-,+ , , & +-(('*, 1 !&:(, &$1+!+ !&$-!&
+,*-,-*$ %, + /!, #&'/& ! :*+'$-,!'& +,*-,-*+ &9'* +)-&:+ +'&*1
+,*-,-* (*!,!'&7 + *+-$,4 / &&',, , '$$'/!& &+!,1 ,-*+ ,' , *+(,!.
& (*'-,+6 &'22$ (*',!& <(DE=4 ,!$ !*+ <(DJ=4 (,'* <(DD= & ('*,$ (*',!& <(K=7
'* &+!,1 %1 '&,!& (DH & (DI & ('++!$1 (DG7 %'& , + ++!&%&,+4 !, !+
$+' ('++!$ , , ', * 1, -&++!&&(*'-,<+=%1'&,*!-,,'+'%', '+*.
&+!,!+, ,&&',!+*&!, *-,'+%$$+!24+%$$&-%*''(!+'*$0!!$!,17
SUPPLEMENTARY FIGURES
b
a
1
0.9
FSC
0.8
0.7
0.6
4.6Å
0.5
0.4
0.3
0.2
0.1
0
1/84.2
1/23.4
1/13.6
1/9.6
1/7.4
1/6.0
1/5.1
1/4.4
1/3.9
1/3.5
1/3.1
1/2.8
Spatial frequency (1/Å)
c
d
Supplementary Figure 1 Capsid icosahedral reconstruction at 4.6 Å resolution. (a) 4.6 Å resolution
density map reconstructed from 36,000 particles with icosahedral symmetry enforced. (b) Fourier
shell correlation (FSC) curve. The map has 4.6 Å resolution using 0.5 FSC criterion. (c) Two different
views of the seven gp10 models comprising the asymmetric unit, illustrating the conformation
variability among the seven gp10 monomers. (d) Gp10 models for the whole capsid.
a
c
b
363/375AA
d
335/335AA
e
P-SSP7
282/385AA
f
Epsilon15
HK97
Supplementary Figure 2 Different modes of capsid folding and intercapsomere stabilization.
The top panels (a-c) show the folding of the capsid protein in P-SSP7, ε15 (3C5B) and HK97 (
1OHG) respectively. The numbers under corresponding folding figures show the resolved amino
acids and the total number of amino acids. The bottom panels (d-f ) show interactions among 3
neighboring hexons of each phage. (d) P-SSP7 has strong interactions (Lys61 and Glu120)
around local 2-fold axes (shown thicker and in color). In contrast, the capsomeres of ε15 are held
together through capsid decoration proteins (gp10, brown shading) near its local 2-fold axes.
HK97, on the other hand, has cross-links around each local 3-fold axis, with the color of chains
indicating each interaction pair.
a
1
0.9
0.8
FSC
0.7
0.6
9.2Å
0.5
0.4
0.3
0.2
0.1
0
1/84.2
1/24.1
1/14.0
1/9.9
1/7.7
1/6.4
1/5.3
Spatial frequency (1/Å)
d
Cross correlation coefficient
b
c
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1
Portal
Top of adaptor
Bottom of adaptor
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
−0.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
−0.1
−180 −150 −120
Fibers
Nozzle
−90
−60
−30
0
30
60
90
120
150
180
Rotation angle (degree)
Supplementary Figure 3 Asymmetric reconstruction at 9 Å resolution. (a) Fourier shell correlation
(FSC) curve; the asymmetrically reconstructed P-SSP7 map has ~9 Å resolution using 0.5 FSC
criterion. (b) The portal vertex complex of P-SSP7 includes the nozzle (slate blue), adaptor (pink), tail
fibers (blue) and portal (yellow). (c) dsDNA is packed hexagonally with a spacing of ~24 Å between
rings. (d) Rotational correlation curves for each protein component of the portal vertex complex (b).
The curve is generated by taking a given density slice, rotating it, and then correlating it with the
original density slice. Due to the interpolation averaging in rotated density slices, the peak at zero
degrees has the highest correlation value.
Capsid
(3 α , 3 β )
Portal
(12 α, 3 β)
Adaptor dimer
(4 α, 4 β)
Nozzle
(0 α, 8 β)
Fiber trimer
(0 α, 3 β)
Supplementary Figure 4 Segmented P-SSP7 capsid and portal vertex complex components
from the 9 Å asymmetric reconstruction. The secondary structures (helix: green cylinder, sheet:
density map. The capsid subunit density is segmented from the asymmetric map. The nozzle,
12-fold, respectively). The adaptor has 6 dimeric subunits. The dimer’s two upper domains are
similar and contain 2 β sheets each. The lower domains are not similar, but each contains 2 α
helices. Each fiber is a trimeric complex.
gp11
Pred:
AA: MATTTIQPDTELSAVNSILGSIGQSPLTTLNYNNPETAFV YNLLVEANKDVQGEGWHFNTEDHVLVTPDATTKYINVPSN
50
60
70
80
10
20
30
40
Pred:
AA: YLRYDLHSGHVDKSMDLVKRNGRLYDKVGHTDQFDDDLYLDIVTLYPFEDVPPIFQRYIISKAAVRAATQLVANRELVAL
130
140
150
160
90
100
110
120
Pred:
AA: LQVQEQSARANVLEYECNQGDHSFMGWPHESSYRPYQPYK ALQR
170
180
190
200
gp12
Pred:
AA: MASVTQTIPTLTGGLSQQPDELKIPGQVSVANNVIPDVTHGLLKRPGGKLVASISDNGTAALNSQTNGKWFSYYRDETES
50
60
70
80
10
20
30
40
Adaptor (dimer)
Pred:
AA: YIGQVSRSGDINMWRCSDGQAMTVNYDSGTATALTTYLTHTNDEDIQTLTLNDYTFLTNRTKTVAMSSTVEPVRPPEVFI
130
140
150
160
90
100
110
120
Pred:
AA: DLKATAYARQYAVNLFDNTTTTAVSTVTRIDVELIKSSNNYCDSNGAMVARTSRPSNSTRCDDSAGDGRDAYAPNVGTKV
170
180
190
200
210
220
230
240
Pred:
AA: FNVTDGASLTDEANSGSYTYTIDVKDSSNNSVNRGVNLYFRIRTVGQSVPFTTGSGSSATTTYQARYTTTFDLLYGGTGW
250
260
270
280
290
300
310
320
Pred:
AA: QEGDYFYVWMKDGYYKITVEAISTANVQANLGLIRPNPTPFDTETAVTAESIIGDIRTAIIATGNFTSANVQQIGTGLYV
370
380
390
400
330
340
350
360
Pred:
AA: TRPSGTFNVTAPSSDLLRVMSGEVANVDDLPSQCKHGYVVKVANSEADADDYYVKFFGHNNRDGDGVWEECAKPSRNIEF
450
460
470
480
410
420
430
440
Pred:
AA: DKGTMPIQLVRQANGTFTVSQATWQNAEVGDELTNPNPSFVGKTINQLVFFRNRLVFLSDENVIMSRPGEFFNFWSKTAT
530
540
550
560
490
500
510
520
Pred:
AA: TFTPQDVIDLSCSSTYPAIVYDGIQVNAGLLLFTKNQQFMLTTDSDILSPETAKINAVSSYNFNEKTHPVSLGTTVAFID
570
580
590
600
610
620
630
640
Pred:
AA: NANQFTRFFEMSNVVRQGEPDVVDQSKVISRLLDKNISLVSVSRENSVVFFSQKDTDKIYCFRYFTSGEKRLLQAWTTWT
650
660
670
680
690
700
710
720
Pred:
AA: ITGNIQYHCMLDDALYVVTRNNNKDQIVKYSLKLDDAGHFVTDTQGTTSTDDDSIYRVHLDHSSSVTAASNTYNTTTIKT
770
780
790
800
730
740
750
760
Pred:
AA: TIPKPNGYESTKQLVAYDTDAGNDLGRYALVTVSGSNLEIPGNWSNNSFIIGYLYEMDVQLPTLYVTQQVGDKYRSDAKS
810
820
830
840
850
860
870
880
Pred:
Nozzle
AA: SLIVHRIKFSFGPLGVYSTTIQRDGKPDFTETKELGLAGVVGASRLPIVPEVIETVPCYERNTNLKVNVKSEHPAPATLY
930
940
950
960
890
900
910
920
Pred:
AA: SLAWEGDFTNRFYKRV
970
Supplementary Figure 5 Justification of gene products assignment of the adaptor and nozzle. The
left panel show the predicted (PSIPRED) secondary structures of gp11 and gp12 of P-SSP7. The right
panel shows the secondary structure elements identified by SSEHunter are superimposed on the
segmented densities. The observed secondary structure elements in both adaptor and nozzle
subunits agree with the sequence based predictions for gp11 and gp12, respectively.
a
b
d
Q-rich
motif
c
MKARERYNQLTTARQMFLDKAVECSELTLPYLIDDDISSRPNHKSLTVPWQSVGAKCCVTLAAKLMLAVLPPQ
TSFFKLQVRDDKLGEELDPQIRSELDLSFSKMERMIMDYIAASNDRVAVHQALKHLIVGGNALIFMGKDGLKT
FPLTRYVINRDGDGNVLEIVTKELISRKVLDIELPEPKPNTGIDESSTTNDDVTIYTYVKLDKSSGRWVWHQE
AFDKIIPDSRSTAPKNASPWLPLRFNTVDGEDYGRGRVEEFLGDLKSLDGLSQSLIEGAAAASKVVFLVSPSS
TTKPATIAKAGNGAIVQGRPEDVAVIQVGKTADFSTAANMATAIEKRLLEAFLVMNVRNAERVTAEEVRLTQL
ELEQQLGGIFSLLVIEFLIPYLNRTLLVLQRSNQIPKLPKDIVRPTIVAGVNALGRGQDRESLTAFVGTIAQT
LGPEALMQYLNPLEAIKRLAAAQGIDVLNLVKTEQQLAEEQQAAQQQAAQQSLVDQAGQMTGSPLMDPTKNPQ
LMDEEQPPMEE
--HHHHHHHHHH---HHHHHHHHHHHHHH-----------------------HHHHHHHHHHHHHHH-------EEEEE-----------HHHHHHHHHHHHHHHHHHHHHHHH---HHHHHHHHHHHHHH---EEEE-----EE
E---EEEEEE-----EEEEEEEEEEHHHHHHHHHHHH-HHHHHH---------EEEEEEEE------EEEEEE
-----EEE-----------EEEEEEEE------------H-HHHHHHHHHHHHHHHHHHHH------------------------EEE--------EEE-------HHHHHHHHHHHHHHHHHHHH---------HHHHHHHHHH
HHHHH---HHHHHHHHHHHHHHHHHHHHHHH---------------EEE--HHHHHHHHHHHHHHHHHHHHHH
----H------HHHHHHHHHHH-----------HHHHHHHHHHHHHHHHHH-----HHH------------HH
HHHH-------
Supplementary Figure 6 P-SSP7 Portal structure. (a) φ29 portal X-ray structure (1IJG, 309AA)
is superimposed on one subunit’s portal density. The φ29 portal X-ray structure fits the lower
density well. (b) SPP1 portal X-ray structure (2JES, 503AA) is superimposed on the portal
density. The C-terminus of SPP1 structure (in red) does not fit well in the upper density. The
unfitted density should belong to the C-terminal region of the portal subunit of P-SSP7. (c)
P-SSP7 portal sequence (522AA) and secondary structure prediction (JPRED). A Q-rich motif (in
blue) was detected and predicted to be a helix. The Q-rich motif has been assigned to the long
helix near the C-terminus (see d). (d) The secondary structure elements identified by SSEHunter
are superimposed on the portal density.
Q-rich
motif
region
Portal
Fibers
Valve and
Adaptor
region
Nozzle
Supplementary Figure 7 Portal vertex complex difference map subtracting the empty phage
map from the full one. The full phage portal vertex map was low-pass filtered to the same
resolution as the empty phage map and normalized. In blue are the positive difference
densities and in red are the negative densities. Prominent positive difference densities include
1, the distal portion of the full phage tail fibers; 2, the nozzle valve and lower portion of the
adaptor; 3, the lower portion of the nozzle. Both lower parts of the adaptor and nozzle appear
to be disordered in empty phage. Prominent negative difference densities include 4, the distal
portion of the empty phage tail fibers and 5, the portal protein, which appears to expand
radially in the empty phage map.
Icosahedral Reconstruction
(very low threshold)
Final 9Å
Asymmetric Map
Initial 3D portal
complex
Initial
3D mask
3D portal
complex
3D mask
5
4
4
5
5
2
3
n
3
5
4
361
3
2
1
1
Raw particle images with
known icosahedal
orientations
2D masks
projected from
the 3D mask in
all possible
orientations
400
2D references
projected from the
3D portal complex
in random
orientations
1
particle 4
All resolved
raw particle
images
4
3
n
2
1
particle 2
particle 3
resolved
asymmetric
orientation
particle
1
res ved
resol
ve
resolved
sol
ssol
resolved
asymmetric
asym
asymm
symm
ymmetric
mmetric
e
etri
mm
m
m
orientation
orie
orien
e tatio
tat
a nasymmetric
at
with resolved
orientation
orien
or
rient
asymmetric
orientation
60
3
2
60
Raw particle image has 60
possible asymmetric
orientations
Trial
Orientation
1
3
2
1
Masking the predicted portal
complex location based on a
given trial orientation
1. Calculate phase residuals of
the cross common lines
between each of the 60 trial
portal complexes and all the
400 2D references
2. Output the orientation with
the best phase residual
Removing the
unmasked part
Supplementary Figure 8 Flow chart diagram of the asymmetric reconstruction. See
supplementary methods for description.
D7
E7
F7
G7
H7
!&47,$7,*-,-*'(+!$'&DH,*!'( *.$+&'%'*&!2,!'&&
(#!&9!&",!'&((*,-+7-,14IDE:I<ECCI=7
&474!$474!&474 !-47?!&47*1':+1%%,*!*'&+,*-,!'&'
,*!'( EE*.$+'*&!2,!'&'!,+(#!&&!&,!&% !&*17
+-4DCJF:KE<ECCI=7
!-474!&474#&47? !-47.*!&,&+,' -&*+'!'+ *$(*,!$
!%+,'*+'$.(*',!&+'&*1+,*-,-*$%&,+-+!&-$,!:, !%-$,
&&$!&'(,!%!2,!'&$'*!, %7
+/*4DD:EJ<ECCJ=7
#*4774-47? !-47
&,!!,!'&'+'&*1+,*-,-*$%&,+!&
!&,*%!,:*+'$-,!'&&+!,1%(+7+.4J:DL<ECCJ=7
!&$$47,$7&'%:/!0(*++!'&1&%!+'%*!&.!*-+& '+,*.$
,-*+'':.'$-,!'&7--14KF:I<ECCJ=7