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Visualizing a Complete Siphoviridae by Single-particle Electron Microscopy: 1
The Structure of Lactococcal Phage TP901-1 2
3
Cecilia Bebeacua1,3, Livia Lai1, Christina Skovgaard Vegge2, Lone Brøndsted2, Marin van 4
Heel1, David Veesler3*§ and Christian Cambillau3* 5
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7 1 Division of Biological Sciences, Imperial College London, South Kensington Campus, 8
London SW7 2AZ, UK. 9 2 Department of Veterinary Disease Biology, University of Copenhagen, Stigbøjlen 4, DK- 10
1870 Frederiksberg C, Denmark 11 3 Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS and 12
Universités Aix-Marseille I & II, Campus de Luminy, Case 932, 13288 Marseille Cedex 09, 13
France; 14 §Present address: Department of Molecular Biology, The Scripps Research Institute, 10550, 15
N. Torrey Pines Road, La Jolla, California 92037, USA, [email protected] 16
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*Corresponding authors: [email protected]; [email protected]. 18
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Running title: A complete EM structure of Siphoviridae Phage TP901-1 20
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Key words: Lactococcus lactis, Siphoviridae, crystal structure, electron microscopy, phages 22
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Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Virol. doi:10.1128/JVI.02836-12 JVI Accepts, published online ahead of print on 7 November 2012
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SUMMARY 28
29
Tailed phages are genome delivery machines exhibiting an unequaled efficiency 30
acquired over more than 3 billion years of evolution. Siphophages from the P335 and 31
936 families infect the Gram+ bacterium Lactococcus lactis using receptor-binding 32
proteins anchored to the host-adsorption apparatus (baseplate). Crystallographic and 33
electron microscopy studies have shed light on the distinct adsorption strategies used by 34
phages of these two families suggesting that they might also rely on different infection 35
mechanisms. We report here electron microscopy reconstructions of the whole phage 36
TP901-1 (P335-species) and propose a composite EM model of this gigantic molecular 37
machine. Our results suggest conservation of structural proteins among tailed phages 38
and add to the growing body of evidence pointing to a common evolutionary origin for 39
these virions. Finally, we propose that host-adsorption apparatus architectures have 40
evolved in correlation with the nature of the receptors used during infection. 41
42
43
44
45
46
47
The abbreviations used are: SEC, size-exclusion chromatography; SLS, static Light 48
scattering; DLS, dynamic light scattering; MALS/UV/RI, on-line multi-angle laser light-49
scattering, absorbance, and refractive index; RBP, Receptor Binding Protein; MTP, Major 50
Tail Protein; TMP, Tape Measure Protein; Tal, Tail Associated Lysozyme; Dit, Distal tail 51
protein; IC, Initiation Complex; BP, Baseplate; EM, Electron microscopy; MS, Mass 52
Spectrometry. 53
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INTRODUCTION 55
Bacteriophages of the Caudovirales order are exquisitely evolved nanomachines possessing a 56
tail appendage used to recognize the host and ensure genome delivery with high specificity. 57
They are the most abundant biological entity on earth with an estimated number of 1031 tailed 58
phages in the biosphere (4). Tail morphology serves as a basis to classify Caudovirales 59
phages into three distinct families: Myoviridae, having a complex-contractile tail (e.g. T4, 60
(16)) Podoviridae, bearing a short non-contractile tail (e.g. P22; (17, 18)); Siphoviridae, 61
characterized by their long non-contractile tail (e.g. SPP1; (31)). 62
The first steps of phage infection require interactions between the phage receptor-binding 63
proteins (RBPs) and the receptors at the host cell surface. Despite the diverse infection 64
mechanisms displayed by Siphoviridae, using surface proteins and/or cell-wall saccharides as 65
receptors, their tail architecture is rather conserved. It is characterized by a long non-66
contractile tube, assembled by stacking several tens of homo-hexameric major tail protein 67
(MTP) rings, and a central core formed by a few copies of the tape measure protein (TMP) 68
extending between both tail extremities and determining its length. The proximal tail end 69
harbors the homohexameric terminator that stops tube elongation during assembly, whereas 70
the distal tail end is characterized by the presence of the tail adsorption apparatus. In phages 71
of Gram+ bacteria this structure is composed of the distal tail protein (Dit) as well as the tail 72
fiber and is termed baseplate or tip depending on the presence or absence of peripheral 73
proteins, respectively. 74
During the last few years, we have characterized the mechanisms underlying the initial steps 75
of infection by bacteriophages targeting Gram+ bacteria. Structural studies of the host 76
adsorption apparatus of the Lactococcus lactis phages p2 and TP901-1 revealed distinct 77
baseplate architectures and diverse strategies used by these two virions to initiate infection (2, 78
5, 36, 37, 39, 50). The phage p2 baseplate undergoes large conformational changes in the 79
presence of Ca2+ ions to appropriately orientate its RBPs and establish multiple interactions 80
with host saccharides at the onset of infection (36). In contrast, the TP901-1 baseplate harbors 81
RBPs already pointing in the direction of the host, suggesting that this organelle is in a 82
conformation ready for host adhesion (50). In vivo infection experiments confirmed and 83
extended these observations by demonstrating that Ca2+ ions are required for host adhesion 84
among p2-like phages (936-species) but have no influence on TP901-1-like phages (P335-85
species). Upon host recognition, a firing signal is generated and propagated along the tail up 86
to the connector to inject the dsDNA genome into the host cell, which leads to the production 87
of progeny virions (21, 31). 88
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The highly flexible nature of Siphoviridae tails makes structural characterization of such 89
phage particles difficult and explains the paucity of data reported for this organelle (31). We 90
report here the electron microscopy reconstructions of the entire TP901-1 virion using single 91
particle protocols and a methodology specially implemented to characterize its tail. Mature 92
TP901-1 virions have thin angular capsid shells filled with dsDNA, and long tails when 93
imaged by transmission electron microscopy. Based on our EM reconstructions and 94
bioinformatics analyses, we propose pseudo-atomic models for most parts of this 95
Siphoviridae virion. The conservation of canonical phage structural protein modules support 96
the evolutionary connection proposed between all tailed phages and provides insights about 97
the putative TP901-1 assembly and maturation pathway. We also put forward the idea that a 98
striking correlation exists between host adsorption device architectures and the strategies 99
employed to recognize and adsorb onto the host. 100
101
MATERIAL AND METHODS 102
103
Native Phage Production and Purification 104
TP901-1 phages were purified as previously described (Vegge et al., 2005). Briefly, phages 105
were induced with 3 µg/ml mitomycin C from lysogenic L. lactis 901-1 grown at 30 °C in 106
GM17 broth. Following cell lysis, the phage particles were precipitated and purified by 107
isopycnic centrifugation using a CsCl gradient. 108
109
Specimen preparation 110
Negative Staining. Approximately 3 µl of sample was applied onto glow-discharged carbon-111
coated grids and incubated for 1 min. The grids were blotted and 10 µl of a 2% uranyl-acetate 112
solution was added and incubated for 30 sec. Stain excess was then blotted and the grids 113
transferred to the microscope or stored. 114
Capsid cryo-EM. The sample (3µL) was applied and incubated onto glow-discharged 115
Quantifoil grids for 1 minute and subsequently blotted for 3 sec before plunging into liquid 116
ethane for vitrification using a FEI Vitrobot. 117
118
Data collection 119
Negative Stain data. Approximately 1,000 CCD images were collected using a Phillips 120
CM200 microscope with a field emission gun operated at 200 kV (CBEM, Imperial College 121
London) and a 4Kx4K TVIPS CCD camera. We used a magnification of 38,000x resulting in 122
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a pixel size of 2.32 Å/pixel (4.64 Å/pixel coarsened by 2) over a range of nominal defocus 123
values comprised between 0.5 and 1.5 µm. 124
Capsid cryo-EM. We collected 200 CCD images using the same setup as for stain data but 125
with a magnification of 50,000x (resulting in a pixel size of 1.765 Å) and nominal defocus 126
values ranging from −1.5 μm to −3 μm. 127
128
Image Processing. 129
All processing was carried out using the IMAGIC software (44). Defocus estimation and 130
correction for the microscope CTF was carried out using IMAGIC CTF2D_FIND and 131
CTF2D_FLIP programs. Particles were selected using the program PICK_M_ALL and 132
filtered, normalized and masked before further processing. The number of particles used in 133
each reconstruction is presented in table 1. 134
Full phage. In order to evaluate the overall dimension of the tail and the number of MTP 135
rings, 1000 particles were manually selected from the images where the phages were observed 136
in isolation and with a relatively straight tail. The full-phage particles were extracted into 137
boxes of 1200x1200 pixels, coarsened by 2 to speed-up processing and submitted to single-138
particle analysis imposing c6 symmetry. Particles were then pre-treated as described above 139
and submitted to five rounds of alignment by classification (7), and subsequently MSA 140
classified with 10 images per class (43). An initial model was generated by back-projecting a 141
selected class average with C6-fold symmetry. The initial model was re-projected and the re-142
projections were used for the initial angular assignment of the aligned particles by projection 143
matching (12, 43). As previously described (36), particles were positioned in a side view 144
orientation with the symmetry axis perpendicular to the projection direction. Therefore, maps 145
were re-projected along the equator (IMAGIC Euler angle β equal to 90°) with a difference of 146
20°. Subsequent cycles of refinement over the entire dataset including alignment, projection 147
matching, and model calculations were iterated until stabilized. 148
Fragments processing. The EM map generated for the full phage was cut into 7 continuous 149
segments of 72x72x72 pixels corresponding to: 1) the connector, 2-6) the tail, and 7) the 150
baseplate. The aligned particles resulting from the full-phage refinement described above 151
were cut in the same way and re-masked to generate 7 subsets. The 1st (connector) and 7th 152
(baseplate) subsets were further refined by projection matching using the corresponding 153
segment of the map obtained from cropping the full-phage initial map. Refinement was 154
carried out for several rounds over 2° imposing c12 symmetry for the connector and c6 155
symmetry for the baseplate. For further analysis and interpretation, however, we used the 156
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baseplate that we previously obtained (2). Fragments 2 to 6 (corresponding to the tail) were 157
combined (~4000 particles) and submitted to helical processing. 158
Tail helical processing. The 4000 particles combined as described above were submitted to 159
helical processing (Table 1). The helical map was produced using the package IHRSR++ (8). 160
The rotational symmetry used was c6 and, as the particles were already aligned, the maximum 161
allowed in-plane rotational angle was set to 10°. The initial helical parameters were 162
determined using the Brandeis Helical Package (27) to calculate the Bessel orders of the basic 163
layer lines (6 and -6) (Fig. 1A,B) and the Ruby-Helix package to estimate a repeat distance of 164
110 Å (23). These were later refined by IHRSR to a helical rise of 38 Å and a rotation 165
between subunits of 22.4°. 166
Reconstruction of the capsid. 1500 particles were manually selected and extracted into boxes 167
of 256x256 pixels and submitted to MSA classification (Table 1). An initial model was 168
created by back-projecting a single class average with icosahedral symmetry. The initial 169
model was refined by projection matching over the entire dataset and with an angular 170
sampling rate of 1° for several rounds imposing icosahedral symmetry until stabilized. 171
Resolution. The resolution of the reconstructions was estimated by Fourier shell correlation 172
and the 1⁄2 bit threshold correlation criterion (45) (Fig. 1C). 173
174
Fitting and analyses were carried out using the UCSF Chimera package (29) (Resource for 175
Biocomputing, Visualization, and Informatics at the University of California, San Francisco, 176
with support from the National Institutes of Health). 177
178
Sequence alignments were performed using the profile-profile alignment and fold recognition 179
algorithm FFAS03 (Fold and Function Assignment System) as well as HHpred. Typically, 180
predictions with FFAS03 scores lower than −9.5 contain <3% of false positives. 181
182
Accession codes. The capsid, connector and tail reconstructions have been deposited at the 183
EMDB with accession codes EMD- 2133, EMD- 2227 and EMD- 2228, respectively. 184
185
RESULTS 186
187
The capsid 188
Bacteriophage capsids are robust containers designed to carry and protect the viral genome 189
that is packaged at liquid crystalline density within its interior (48). We computed a 190
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reconstruction of the TP901-1 capsid at 15 Å resolution using ~1500 particle images and 191
applying icosahedral symmetry (Fig. 2A). The mature capsid is approximately 660 Å wide 192
along its 5-fold axes and is made of 60 hexamers and 11 pentamers of the ORF36 major 193
capsid protein (MCP), organized with a T=7 symmetry, as well as a dodecamer of the portal 194
protein occupying a unique vertex. Due to the 60-fold averaging applied during the 195
reconstruction process, the portal density is averaged out and its structure has been 196
independently investigated by reconstructing the connector region only. The capsid interior is 197
filled with the dsDNA genome organized as concentric layers regularly spaced by ~25 Å (Fig. 198
2B), as typically observed in other Caudovirales phages (48). 199
The plethora of MCP structures reported to date demonstrates the conservation of the so-200
called HK97 “Johnson fold” among tailed phages, herpesviruses and some archaeal 201
counterparts (46, 48, 53). Hence, we expect the TP901-1 MCP to exhibit a similar fold and 202
this is further supported by the detection of weak sequence similarity (but with high 203
confidence) with the T7 and HK97 MCP sequences using the FFAS03 server and HHpred 204
(Table 2) (13). A pseudo-atomic model of the TP901-1 MCP shell was thus produced by 205
rigid-body fitting of the icosahedral asymmetric unit of the mature HK97 capsid (PDB 206
1OHG) within the EM reconstruction (Fig. 2C). The seven subunits of the icosahedral 207
asymmetric unit are well accommodated in the capsid density and form a 32 Å thick shell 208
surrounding the viral genome. 209
210
The head-to-tail connecting region 211
The connector ensures the cohesion of the phage capsid with its tail and is often made of three 212
different components organized as successive rings: the portal protein and two head 213
completion proteins. It is located at a unique capsid vertex where it replaces a penton motif 214
(Fig. 3A-C). We achieved a reconstruction of the connector using ~1000 particles and 215
applying 12-fold symmetry along the connector channel axis. 216
The portal is a dodecameric protein, disclosing a conserved fold in tailed phages and 217
herpesviruses (46, 48), which is involved in DNA packaging during assembly and allowing 218
release at the onset of infection. The TP901-1 portal (ORF32) has an overall length 219
comparable to the equivalent protein in phage SPP1 (452 vs 503 residues) and both sequences 220
share 26% identity and 45% similarity. We used a SPP1 dodecameric portal model (19) to fit 221
into the proximal region of the connector reconstruction revealing a good agreement between 222
the atomic model and the EM map at this resolution (Fig. 3D-E) and confirming the structural 223
similarity between TP901-1 and SPP1 portal proteins suggested by FFAS03 and HHpred 224
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(Table 2). Sequence analysis of the TP901-1 portal indicates that no P22-like coiled-coil 225
structure is present at its C-terminal region suggesting that this phage only relies on the 226
turbine region of the dodecamer to trigger packaging termination when the genome reaches 227
the headful density (18, 26, 40). 228
The remaining region of the connector reconstruction reported here was assumed to account 229
for the two rings of head-completion proteins and the tail terminator. Sequence analyses using 230
the FFAS03 and HHpred servers allowed us to link the TP901-1 ORF38 and ORF39 to the 231
SPP1 head-completion proteins gp15 and gp16, respectively (Table 2). These latter two 232
proteins form dodecameric rings of known structures in vivo (21). We docked the SPP1 gp15 233
(PDB 2KBZ) and gp16 (PDB 2KCA) rings directly underneath the portal dodecamer in the 234
connector EM map. The SPP1 gp15 head-completion protein ring matches reasonably well 235
the dimensions of the corresponding TP901-1 connector moiety (Fig 3D, F) while the SPP1 236
gp16 dodecamer is only moderately accounted for by the density (data not shown). Due to the 237
symmetry mismatch between the connector and the tail, we have not attempted to fit any tail 238
terminator model in the connector reconstruction and this region is analyzed in the next 239
section. 240
241
The tail 242
Bacteriophage tails ensure genome delivery to the target cell with an unequaled efficacy in the 243
viral world. To investigate the tail structure, we first produced a low-resolution, 6-fold 244
averaged, reconstruction of the whole TP901-1 phage from selected virions exhibiting a 245
straight (unbent) tail. We used this reconstruction to assess that the tail tube is made of 34 246
stacks comprising a tail terminator hexamer, located at the interface with the connector, and 247
33 MTP hexamers forming the rest of the tube. We then boxed small tail segments (each 248
including seven complete MTP stacks) from the tails and combined in one dataset 249
subsequently processed with the appropriate helical symmetry. 250
The TP901-1 tail extends over 1180 Å (Fig. 4A), between the connector and the baseplate, 251
and its diameter varies between 110 Å (at the level of the MTP rings) and 90 Å (at the 252
intersections between rings) (Fig. 4B). The MTP hexameric stacks are rotated by 22.4° 253
between each other from the distal to the proximal tail extremity, and the interhexamer 254
distance is 38 Å (Fig. 4B). The tail tube delineates a 42 Å wide central channel that is 255
continuous with the connector and baseplate ones to form the genome ejection pathway (10, 256
19, 21, 31, 49, 50) (Fig. 4C,D). We attributed the 28 Å wide elongated density filling the tail 257
interior to the Tape Measure protein (TMP), the molecular ruler controlling the tail length 258
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during assembly (28) (Fig. 4C,D). The TMP is believed to be oligomeric and to form a long 259
helical region extending through the tail tube and anchored by one globular domain at each 260
extremity. Consistent with what has been observed in phage SPP1, no contacts are observed 261
between the TMP and the surrounding MTP rings, either due to their nonexistence or because 262
of a symmetry discrepancy between the two structures. Weak interactions between the TMP 263
and the MTP hexamers probably facilitate ejection of the former before DNA ejection 264
through the tail channel. Although of lower resolution, the overall dimensions of the TP901-1 265
tail components are in good agreement with the equivalent ones in the SPP1 tail, supporting 266
the validity of our reconstruction. 267
268
The host-adsorption device 269
The baseplate is the control center for infectivity and is in charge of host recognition, 270
attachment and initiation of infection. Combining our recently reported TP901-1 baseplate 271
crystal structure with the EM reconstruction (2) shows the detailed organization of this 280 Å 272
wide and 150 Å high organelle exhibiting an overall 6-fold symmetry (Fig. 5A-B). From the 273
proximal to distal end, it is formed by 18 copies of BppU (ORF48) arranged around a central 274
Dit hexamer (ORF46) and holding 54 RBPs (ORF49) organized as eighteen trimers (50) (Fig. 275
5A-C). The RBPs orientate their 54 receptor-binding sites toward the distal extremity in a 276
way suitable for establishing interactions with the pellicle layer of the host without requiring 277
conformational changes (6, 50). The Tail-associated lysine (Tal, ORF47) forms a 150 Å long 278
trimeric tail fiber appended to the Dit ring and extending beyond the baseplate core at its 279
distal extremity. We modeled the tail fiber N-terminal domains using the closed p2 ORF16 280
trimer, which is expected to share a virtually identical fold and to undergo a similar 281
conformational change to open the DNA ejection conduit during infection (10, 36, 46, 49, 282
50). While no structure is available to model the tail fiber central region, the last ~150 283
residues of each monomer form a domain belonging to the peptidase M23 family that is 284
probably involved in peptidoglycan digestion at the onset of infection to allow the virion 285
accessing the cytoplasmic membrane (Table 2) (15, 51). 286
287
DISCUSSION 288
289
The overall structure of TP901-1 phage 290
The structure determination of the TP901-1 phage capsid, tail, connector and baseplate makes 291
it possible to have an intermediate resolution view of this large viral molecular machine (Fig. 292
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6). The TP901-1 capsid is 660 Å wide along its 5-fold axes and is virtually identical to the 293
phage HK97 one (53). Indeed, the TP901-1 MCP seems to harbor the so-called HK97 294
“Johnson fold” that is conserved among Caudovirales phages and in some viruses infecting 295
Eukaryotes and Archaea (46, 48, 53). Analysis of the TP901-1 MCP sequence reveals that the 296
protein does not harbor a scaffolding domain fused at its N-terminus, in contrast to the HK97 297
situation. Instead, the virion genome exhibits an upstream ORF encoding a protein product of 298
~200 residues predicted to possess a high helical content and likely acting as a scaffolding 299
protein. Based on these observations, we propose that the TP901-1 capsid assembly and 300
maturation pathway is reminiscent of that of phage P22, which expels the intact scaffolding 301
proteins upon initiation of dsDNA packaging rather than via proteolysis (11). 302
The TP901-1 connector structure is globally similar to that of SPP1: the SPP1 portal 303
dodecamer as well as the most proximal dodecamer of head-completion protein (SPP1gp15) 304
are reasonably well accommodated in the corresponding regions of the TP901-1 305
reconstruction. The most distal ring of head-completion protein should logically correspond 306
to the SPP1 gp16 dodecamer (the “stopper”) according to sequence comparisons and to the 307
observed density occluding the DNA exit channel in the reconstruction. However, the EM 308
density appears to only moderately account for it probably due to the limited quality and 309
resolution of the map in this region. Interestingly, an additional ring-like structure surrounds 310
the connector at the level of the gp15 dodecamer and we propose that this additional region of 311
density might result from binding of additional proteins forming the collar/whiskers observed 312
in micrographs of TP901-1 and that have been averaged out during the reconstruction (14, 313
51). This additional ring might also be due to large conformational changes occuring upon tail 314
attachment 315
Considerable efforts have been made to understand how the dsDNA genome is driven from 316
the phage capsid up to the host cytoplasm during infection. In the case of phage SPP1, it has 317
been demonstrated that the high pressure with which the genome is packaged into the head is 318
not enough to power its complete entry into the target cell (34). Other proteinaceous factors 319
have been shown to participate in this phenomenon in bacteriophages T5 and T7, by pulling 320
DNA into the host cell (20, 24). All the proteins building the central channel that allows DNA 321
transit from the capsid to the host cell form a ~40Å wide central channel with conserved 322
negative electrostatic properties. No structure of the hexameric (biologically relevant) form of 323
the MTP has been reported so far. However, considering the low pI observed for most MTPs 324
(e.g. pI~4.8 in TP901-1 or pI~4.7 in SPP1), it is likely that this protein contributes to the 325
overall negative potential of the central ejection tunnel. The fact that the genome transit 326
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pathway is negatively charged has an obvious functional implication: as the dsDNA backbone 327
is negatively charged too due to the presence of phosphate groups, a strong repulsion occurs 328
with the corresponding phage regions to which it is exposed favoring genome transit. 329
Besides ejection, this property might have an impact on phage assembly. A survey of the pI of 330
various TMP proteins revealed that these tail components are strongly basic (e.g. pI~8.8 in 331
TP901-1 or pI~10 in SPP1). Therefore, association of the central TMP oligomer with the 332
surrounding tail tube (formed of stacked MTP hexamers) is likely to rely, at least partially, on 333
strong complementary electrostatic interactions. 334
335
The distal tail architecture is governed by the host adsorption strategy 336
All bacteriophages belonging to the Siphoviridae family share a canonical tail organization 337
but differ mainly at their distal tail part. Dairy phages belonging to the P335 and 936 species 338
harbor complex baseplates in comparison to other phages of the same family and this seems 339
to correlate with the different host adsorption strategy. Indeed, they interact only with host 340
cell-wall saccharides, putatively the phosphosaccharides present in the pellicle of Gram+ 341
bacteria, for specific recognition and attachment (6, 32, 36, 38, 39, 41). Besides, many other 342
siphophages, such as SPP1 and the lactococcal phages belonging to the c2-species, bind 343
reversibly to saccharidic receptors in a first step before interacting irreversibly with a 344
membrane protein that initiate infection (1, 9, 25, 31, 33, 35, 42, 52). As the affinity between 345
phage antireceptors and their saccharidic partners is generally moderate (in the low 346
micromolar range), several RBPs are involved in binding to ensure strong interactions based 347
on avidity (22, 47). In contrast, the interaction between antireceptors and their proteinaceous 348
receptors is strong, as illustrated by the tight binding reported between the T5 pb5 protein and 349
the outer membrane transporter FhuA (30) or the λ gpJ and LamB (3). Siphoviridae members 350
can therefore be dichotomized in two categories based on the observation of their distal tail 351
architecture. On one hand, some phages harbor a large baseplate accommodating up to several 352
tens of RBPs to interact only with the saccharidic part of the host cell wall. On the other hand, 353
bacteriophages devoid of any baseplate and possessing a simplified tail-tip rely on an 354
irreversible binding with a transmembrane protein present in the target cell to ensure their 355
commitment. 356
357
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ACKNOWLEDGMENTS 358
This work was supported, in part, by grants from the Marseille-Nice Génopole, the CNRS and 359
the Agence Nationale de la Recherche (grants ANR-07-BLAN-0095, "Siphophages" and 360
ANR-11-BSV8-004-01 “Lactophages”). A PhD grant from the "ministère Français de 361
l'enseignement supérieur et de la recherche" no. 22976-2006" was attributed to D.V. MvH 362
acknowledges financial support from the EU/NOE (NOE - PE0748), from the Dutch ministry 363
of economic affairs (Cyttron Project: BIBCR_PX0948) and from the BBSRC (Grant: 364
BB/G015236/1). Molecular graphics images were produced using the UCSF Chimera 365
package from the Resource for Biocomputing, Visualization, and Informatics at the 366
University of California, San Francisco (supported by NIH P41 RR-01081). 367
368
369
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532
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Figure legends 534
535
Figure 1: EM parameters of TP901-1 structure. (A, B) Determination of the helical 536
parameters of the TP901 tail. An average of aligned tail tube segments (A) was used to 537
generate the Fourier transform (B). The meridional line is marked by a dotted line. The layer 538
lines are marked by arrows that also indicate their Bessel orders (6, 6). This indexation 539
showed the six-fold rotational symmetry of the TP901 helical tail. (C) Fourier Shell 540
Correlation (FSC) curves of the final 3D reconstructions obtained by correlation of two 541
different 3Ds created by splitting the particles set into two subsets. The resolution was 542
estimated by the ½-bit cutoff threshold criterion as 15Å for the capsid, 21Å for the connector, 543
21Å resolution for the helical tail, and 25Å resolution for the baseplate. 544
545
Figure 2. The 15 Å resolution CryoEM reconstruction of the TP901-1 mature capsid. (A) 546
Surface rendering of the icosahedral reconstruction low-pass filtered at 15 Å and viewed along 547
an icosahedral 2-fold axis. The capsid measures 660 Å along its 5-fold axis. (B) Cross-section 548
of the capsid reconstruction showing the layers of the dsDNA genome organized as concentric 549
shells. (C) Pseudo-atomic model of the TP901-1 mature capsid fitted in the reconstruction. 550
551
Figure 3. The 20 Å resolution reconstruction of the TP901-1 connector. (A-C) The 552
connector occupies a unique capsid vertex. (A) Side view. (B) View from the distal extremity 553
along the tail tube. (C) Cross section of the capsid showing the portal protruding into it. (D) 554
The SPP1 portal and the first head completion protein dodecamer (SPP1 gp15) are fitted into 555
the connector reconstruction. Note the additional density surrounding the gp15 ring that likely 556
corresponds to the collar/whiskers. The stopper (equivalent to SPP1 gp16) and the tail-557
terminator are postulated to account for the remaining density. (E) Side view showing the 558
fitting of the SPP1 portal dodecamer into the corresponding TP901-1 EM density along the 559
tail axis (the region corresponding to the capsid density has been computationally removed for 560
clarity). (F) Side view showing the fitting of the SPP1 first head completion protein into the 561
corresponding TP901-1 EM density along the tail axis 562
563
Figure 4. The 20 Å resolution reconstruction of the TP901-1 tail. (A) 6-fold averaged, 564
reconstruction of the TP901-1 phage tail from a few selected virions exhibiting a almost 565
straight tail, making it possible to obtain the number of MTPs. (B) Detailed view of the 566
reconstruction of a segment of the tail (7 MTP rings) using helical symmetry. The helical 567
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parameters of the tail are shown. (C) Cross-section of the tail segment (blue) along its long 568
axis revealing the internal TMP (violet). (D) Cross-section of the tail segment orthogonal to 569
its long axis (the coloring scheme is the same as in (B)). 570
571
Figure 5. The 20 Å resolution reconstruction of the TP901-1 distal tail region (the 572
baseplate). The TP901-1 baseplate crystal structure was rigid-body fitted in the 573
reconstruction. The color-coding is as follows: Dit (green), BppU (red) and BppL (light blue). 574
The MTP hexamers were modeled using the Hcp type VI secretion system protein (dark blue). 575
The N-terminal region of the tail fiber was modeled using the phage p2 ORF16 in closed 576
conformation (pink). (A) Side view. (B) View along the tail axis from the distal extremity 577
toward the capsid. (C) Cross-section of the baseplate EM map. The assignment of the EM 578
density to the different ORFs has been performed using the Xray structure. 579
580
Figure 6. The assembled complete structure of TP901-1 phage. The complete phage 581
assembled by fitting the individually refined reconstructions into the map obtained for the full 582
phage. 583
584
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Table 1: Summary of the data processing strategies employed for the various TP901-1 reconstructions. Method Symmetry resolution Nr. of particles Capsid Cryo-EM Icosahedral 15 Å 1500 Connector Negative-staining EM c12 21 Å 1000 Tail Negative-staining EM Helical 20 Å 4000 Baseplate Negative-staining EM c6 25 Å 10000
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Table 2 : Sequence analyses of TP901-1 structural proteins. FFAS03 scores lower than -9.5 are considered significant.
TP901-1 ORFs FFAS03 score
FFAS03 %identity
HHpred e-value
To (sequence)
ORF36 – Major capsid protein -62.0 -38.6
17 16
3.7x10-27
5.1x10-28 T7 MCP-10A HK97 gp5
ORF32 - Portal -89.5 19* 3.4x10-60 SPP1 gp6 ORF38 – Head completion protein
-33.2 14 1.8x10-19 SPP1 gp15
ORF39 – Head completion protein, stopper
-12.8 10 0.0034 SPP1 gp16
ORF42 – Major tail protein -22.3 11 8.5x10-10 Lambda gpV ORF47 – Tail associated lysin -53.3 31 4.6x10-26 S. aureus glycyl-glycine
endopeptidase LytM
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