Icosahedral reconstruction of P-SSP7 capsid
Using 36,000 particle images (), we obtained an icosahedral reconstruction of the P-SSP7 shell at ~4.6 Å resolution (Supplementary Fig. 1a,b and Movie 1a
) using MPSA7
. 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 (Cα) backbone chain trace models for each of the 7 shell proteins (gp10) in an asymmetric unit. shows one of the gp10 models (residues 1–363) containing 5 structural features, 4 of which also appear in other phage capsid structures9,10
(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 structurally important small loop between the N arm and E loop, near the local 2-fold axis (which we call the F loop).
Image and reconstruction of P-SSP7 at 4.6 Å resolution
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 Cα backbone trace of gp10 shows its fold to be topologically similar to the corresponding shell protein of other tailed phages such as ε159
(Supplementary Fig. 2
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 ε15 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 () 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 (). The F loop in P-SSP7 is longer than in either HK97 or ε15 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 ) of one subunit wraps around the long E loop () of a neighboring subunit, thus reducing the flexibility of the E loop without requiring β 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. T419
). This divide- and-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.
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 (, Supplementary Fig. 3a
). In comparing the asymmetric density map with the Cα 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
Asymmetric reconstructions of P-SSP7
We segmented the densities at the portal vertex of the map into the following components: nozzle, adaptor, tail fibers, portal and core proteins (). 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 () and homologs of the portal (Supplementary Fig. 6
) to their corresponding densities.
The cryo-EM map, along with genomics1
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 ( and Supplementary Movie 2
). Each of these densities (adaptor, nozzle, portal) is also cross-validated 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 (). Each of the 6 tail fibers appears to be a trimer (), which splays out into three “petals” and then rejoins at the junction with the adaptor and nozzle (). 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.
Symmetry mismatch at the portal vertex
Applying the appropriate symmetry to some of the portal vertex protein components, the contrast of their structural features can be enhanced. Using SSEHunter24
to analyze of the symmetrized components, we were able to identify α-helices (>2 turns) and β-sheets (> 2 strands) in these densities: 2 α-helices and 2 β-sheets in the adaptor protein, 8 β sheets in the nozzle protein, 3 β sheets in the tail fiber trimer, and 12 α-helices and 3 β 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 (). 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 β sheet) and the adaptor protein (gp11, a mixture of α helices and β 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 ϕ2925
(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 ϕ29 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 ϕ29 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 analysis27,28
also predicts α-helices near the C-termini of portal proteins in other phages (T7, syn5, KIE, ε15). In P-SSP7, 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 (). 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 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) genome1
which is packed coaxially around the portal vertex axis with a spacing of ~24 Å between rings (, Supplementary Fig. 3c
). The rings are packed hexagonally (). 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 ε15 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” ( and ).
There is a split of the density resulting in a separation of ~14 Å in the middle of the DNA terminus (, 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 ϕ29 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 ϕ29. Therefore, these DNA features are completely different between P-SSP7 and ϕ29.
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 () 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 () reveals four interaction types (): 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 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 (), 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 (), while on the other side, four contiguous adaptor subunits interact with each of the 6 nozzle subunits (). In addition, two adjacent adaptor dimers are bound together through one petal of a tail fiber ( and ). The fiber also binds to the nozzle near the valve and an adaptor subunit near the nozzle side ( and ). 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 (). 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 (). 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 (, 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 ( 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 () closely contacts with an adaptor subunit (yellow box in ), whereas in the empty phage, the density near the contact point is missing, having moved or become disordered (yellow box in ). 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 disordered in the empty phage (). 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
. 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.