Structural changes of a bacteriophage upon DNA packaging and maturation

Tailed, double-stranded DNA (dsDNA) bacteriophages, which belong to the order of Caudovirales, have a tail attached to a pentameric vertex of the icosahedral capsid shell (head) through a 12-fold portal (Johnson and Chiu, 2007). The phages package genomic dsDNA into a round procapsid using the portal in complex with an ATP-dependent terminase complex as the motor. During packaging, the procapsid shell expands to a more angular intermediate to match the size of the viral genome (Guo et al., 2014). When the phage head is full, the portal detects internal pressure and conveys a signal from the inner capsid to the exterior, which triggers a sequence of events—the terminase complex cleaving mature DNA genome from a multi-genome concatemer, the release of the terminase complex from the portal, and the attachment of the tail complex—in the completion of phage assembly (Lander et al., 2006; Johnson and Chiu, 2007). At the beginning of phage infection, the tail is responsible for receptor recognition, and the portal and tail act as a tunnel for DNA delivery into the host cytoplasm (Johnson and Chiu, 2007). These mechanisms of DNA packaging and ejection may also be conserved in many other DNA viruses, including herpesvirus (Wang et al., 2020; Yang et al., 2020). In previous studies, the structures of whole phage capsid, isolated portal, and tail components have been determined at medium to near-atomic resolutions (Fokine and Rossmann, 2014; Prevelige and Cortines, 2018). Such structural information indicates that tailed phages use the conserved structures of the capsid shell protein, as well as the DNA packaging and ejection machinery, implying strong functional similarities. However, the high-resolution in situ structures of the portal and tail are still less well understood, probably due to the intrinsic flexible assembly of these components and the overlap of different symmetrical components in the phages. The molecular mechanism through which events are detected by the portal and subsequently signaled to the exterior terminase complex to complete the phage assembly remains unclear. Escherichia coli bacteriophage T7, a member of Podoviridae, has been used as a model for understanding the DNA packaging mechanism common to tailed phages and related dsDNA viruses (Guo et al., 2013; Hu et al., 2013; Guo et al., 2014; Cuervo et al., 2019). Here, we determined structures of the DNA packaging intermediate and mature capsids of T7 using cryo-electron microscopy (cryo-EM) and symmetry-mismatch reconstruction. The structural resolutions of the portal, portal-tail complex, and cores (ejection proteins) in the two capsids have been further improved to 3.8–6.0 Å through a symmetry-mismatch and local reconstruction method that we developed. Our portal and tail structures have different conformations than recently reported recombinant portal and tail structures of T7 (Cuervo et al., 2019). The portal interacts flexibly with the capsid shell and core in both mature and intermediate capsids. Our structures reveal the conformational changes of the portal, core, and shell from the DNA packaging state to the mature state, and provide insights into the head-full packaging mechanism of the phages. The mature T7 phage was purified from cells for the cryoEM structural analysis (Fig. 1A). We obtained the mature phage icosahedral capsid shell structure at 3.5 Å resolution by using the cryo-EM and icosahedral reconstruction method (Figs. S1A and S2A–D). The icosahedral capsid shell structure is essentially identical to the previously published mature phage capsid shell structure (Guo et al., 2014). Subsequently, a 7 Å resolution asymmetric reconstruction of the mature phage with a portal-tail complex at one of the 12 icosahedral vertices (Fig. 1B) was obtained using the symmetry-mismatch reconstruction method (Liu and Cheng, 2015). The portal-tail complex structure is similar to the previously reported recombinant T7 portal-tail complex structure (Cuervo et al., 2019). The core structure is smeared in the asymmetric reconstruction. The portal and core are surrounded by DNA within the capsid, and a bulk of disordered condensed DNA is stacked on the top of the core (Fig. 1B). Such DNA organization is commonly observed in other tailed phages (Johnson and Chiu, 2007). We performed the local refinement and reconstruction focusing on the region of the portal, tail, and core. The portaltail complex was reconstructed at the same resolution of 3.8 Å (Figs. 1C, 1D and S1B), although the core structure was still smeared. Independent local reconstruction focused on the core yield the structure of the core at the resolution of 6.0 Å (Figs. S1B and S3). Another two independent local


Materials and Methods
Sample preparation.
T7 phages (ATCC, BAA-1025-B2) were inoculated in 1L Escherichia coli (ATCC, BAA102) cells for 4 h at 37℃. After the cells were lysed, phages in the supernatant were precipitated with 1M NaCl and 10% polyethylene glycol 8000 overnight. The precipitated phages were resuspended in the TNM buffer (50 mM Tris-HCl, 10 mM MgCl 2 , 50 mM NaCl, pH 7.4) and then were purified on 1.56g/mL and 1.26g/mL CsCl cushions through ultracentrifugation. After centrifugation at 197,000 g for 12 h at 10℃, two virus bands were separated. The upper and lower band were collected and repeatedly ultracentrifuged on the aforementioned 1.26g/mL and 1.56g/mL CsCl cushions separately and dialyzed in TNM buffer overnight. Negative stain electron microscope observations indicated that the upper and the lower bands were empty and full particles, respectively.
The full and empty particles of T7 were imaged with an FEI Technai Arctica 200 kV electron microscope equipped with a Falcon II camera at a nominal magnification of 78,000×, corresponding to a pixel size of 1.27 Å, respectively. The full electron dose of approximately 25 e -/A 2 was fractionated into 30 movie frames, which were aligned and averaged to a single image (Li et al., 2013). The astigmatism and defocus value of each image were determined by a program that we designed. The viral particles were boxed automatically using the program ETHAN (Kivioja et al., 2000) and these boxes were then verified manually.
3D reconstructions of the unique vertices of the full and empty particles.
The icosahedral reconstructions of T7 full particles were performed using the programs (Li et al., 2017) based on the common-line algorithm (Fuller et al., 1996;Thuman-Commike and Chiu, 1997). To obtain the initial model of the asymmetric structure of the tailed phage, the location of the portal-tail complex for each particle image was determined based on the icosahedral orientation and center parameters through a search of the 12 icosahedral vertices for the unique vertex with the portal-tail complex. The correct orientation of the tailed phage for each particle image was determined using the symmetry-mismatch reconstruction method (Liu and Cheng, 2015).
(i) For each particle image, we projected the model to generate 60 projection images according to the 60 equivalent icosahedral orientations of the particle image. We searched the 60 projection images for the projection image that best matches with the particle image and then assigned the corresponding orientation to the particle image. (ii) We reconstructed the tailed phage structure using the particle images according to the newly assigned orientations. We then obtained an improved model of the portal-tail complex. (iii) We iterated steps (i) and (ii) until the orientations of all particle images were mostly stabilized and the portal-tail complex structure could not be improved further.
We further refined the orientation for each particle image by using the symmetry-mismatch reconstruction method (Liu and Cheng, 2015). (i) We segmented the portal-tail complex region from the whole phage structure determined above for use as an initial model. (ii) For each particle image, we projected the 3D tail-portal complex model to generate 5 projection images according to the 5 equivalent orientations of the unique 5-fold capsid shell vertex. We searched the 5 projection images for the projection image that best matches with the portal-tail region in the particle image and then assigned the corresponding orientation to the particle image, and this symmetry-mismatch search method is similar to that used for the bacteriophage ϕ29 reconstruction (Morais et al., 2001). (iii) We reconstructed a whole phage structure by using the particle images according to the newly assigned orientations. We then obtained an improved model of the portal-tail complex. (iv) We iterated steps (ii) and (iii) until the orientations of all particle images were mostly stabilized and the portal-tail complex structure could not be improved. A soft-mask was used for each particle image.
The orientation and center parameters of each portal-tail complex in the particle image were further refined using local refinement and reconstruction method (Wang et al., 2018;Yuan et al., 2018;Zhu et al., 2018;Wang et al., 2019). We used the portal-tail complex structure segmented from the whole phage structure as the model to refine orientation and center for the portal-tail complex region in each particle image. The reconstruction and refinement were performed iteratively to improve the resolution until the orientations and centers of the portal-tail complex regions in all images were stabilized and the portal-tail complex structure could not be improved. A soft-mask was used for the region of local refinement in each particle image.
Following the same image processing protocol, we reconstructed the whole structure of the capsid II, the portal of the capsid II, and core in the mature phage and that in capsid II.

Atomic model building and refinement.
Our models of the two gp8 (in the mature phage and capsid II), gp11, gp12, and gp17 N-terminus were built based on our cryo-EM density maps using the COOT software (Emsley et al., 2010). The models were refined using real-space refinement as implemented in Phenix (Adams et al., 2010). We built the model of gp8 in capsid II with the reference to the model of gp8 in mature phage. Refinement and validation statistics are presented in Table S1.

Supplementary text
Definition of reference orientation coordinate system of reconstruction.
The Euler angles of our portal and tail reconstruction are defined as follows. First, we place three mutually perpendicular 2-fold axes of the icosahedron coincident to the Cartesian x, y, and z axes, according to the icosahedral reconstruction (Baker et al., 1999;Thuman-Commike and Chiu, 2000). Then, the reference orientation coordinate system used in the symmetry-mismatch reconstruction is defined by the rotation θ=31.72° so that the axis of the tailed 5-fold vertex coincides with the z axis ( Fig. S10). As such, ϕ defines the angle by which the portal-tail complex rotates around the 5-fold vertex axis of the capsid shell.
Flexible interaction between the portal and shell.
The local reconstruction method allows us to determine the orientation and center distributions of the portal with respect to the shell in the mature and capsid II particles through the use of Euler angles (Fig. S10) and 2-D translation. The Euler angles and centers ( Fig. S11) show the difference between the asymmetric refinement result and the subsequent local refinement result. The rotation angles θ and ω and translations x and y of the portal position with respect to the shell in the mature particle were observed to be four narrow Gaussian distributions of particle numbers for each angles θ and ω and translations x and y ranging from -2° to 2°and from 0 to 3 Å, respectively ( Fig. S11A and S11C). These distributions can be regarded as random errors. However, the distribution of the ϕ, which represents the rotation angle of the portal around the 5-fold axis with respect to the shell, was observed to be an overlap of two Gaussian distributions with the means at -3° and 3° (a span of 6°) respectively. (Fig. S11A). It suggests that many of the particles have a wrong ϕ angle assigned to the portal (as ϕ, ϕ+72°, ϕ+144°, ϕ+216° and ϕ+288° are degenerate), requiring a change of up to 3° in either direction during local refinement.
We also determined the orientation and center distributions of the portal in the capsid II particle ( Fig. S11B and S11D). All these distributions were Gaussian curves, which reflect the flexible interactions between the portal and shell and the lower resolution of this reconstruction. The ranges of the distributions of the capsid II portal were clearly wider than those of the mature phage portal. These results could not prove that the portal rotates axially, as suggested previously (Hendrix, 1978;Simpson et al., 2000).

Structural comparisons with recombinant portal and tail structures of T7.
We compared our in situ portal and tail structures with recently reported recombinant portal-tail (gp8-gp11-gp12) and portal (gp8)  A superposition of the recombinant portal-tail structure (6R21) with our portal-tail structure showed that the portal in the two structures are mostly identical, with the exception of the conformation of the kinked helix (α10) and the tunnel loop. This N-terminal part of α10 in our portal structures points almost perpendicularly toward the channel axis, whereas the counterpart in their recombinant portal-tail structure tilts upward, resulting in a wider channel. In addition, the α-helices in the two crowns vary slightly. The hexameric gp12 nozzles in the two structures are identical, with the exception of a loop (residues 736-744), which was flexible in the recombinant portal-tail structure, was resolved in our structure. The gp11 proteins in the two structure have an identical main body but they have different N-terminal loops. In our structure, two adjacent gp11 N-terminal loops approach each other, resulting in a 6-fold symmetric arrangement of the gp11 dimer ( Fig. S6B and S6C). These structural variations occur in the regions interacting with the gp17 trimers, which were absent from their structures.
The portal structure 6QX5 is similar to our portal structure in the mature phage, although most of the secondary structure does not fit well. The portal structure 6QXM is similar to our portal structure in the capsid II, however, the crown is not present in their portal, and the N-terminal part of α10 in their portal structure tilts upward (in contrast to the rough perpendicularity toward the channel axis in our structure).