Filamentous phage are elongated semiflexible ssDNA viruses that infect bacteria. The M13 phage, belonging to the family inoviridae, has a length of ∼1 μm and a diameter of ∼7 nm. Here we present a structural model for the capsid of intact M13 bacteriophage using Rosetta model building guided by structure restraints obtained from magic-angle spinning solid-state NMR experimental data. The C5 subunit symmetry observed in fiber diffraction studies was enforced during model building. The structure consists of stacked pentamers with largely alpha helical subunits containing an N-terminal type II β-turn; there is a rise of 16.6-16.7 Å and a tilt of 36.1-36.6°between consecutive pentamers. The packing of the subunits is stabilized by a repeating hydrophobic stacking pocket; each subunit participates in four pockets by contributing different hydrophobic residues, which are spread along the subunit sequence. Our study provides, to our knowledge, the first magicangle spinning NMR structure of an intact filamentous virus capsid and further demonstrates the strength of this technique as a method of choice to study noncrystalline, high-molecular-weight molecular assemblies.solid-state NMR | magic-angle spinning | filamentous bacteriophage | structure determination | Rosetta modeling F ilamentous bacteriophage are long, thin, and semiflexible rod viruses that infect bacteria (1, 2). These large assemblies (∼15-35 MDa) contain a circular single-stranded (ss) DNA genome encapsulated in a protein shell. All filamentous phage have a similar life cycle and virion structure despite the relatively high number of strains, with DNA sequence homology varying from almost complete to very little. The unique phage properties make them ideal for a large range of applications such as phage display (3), DNA cloning and sequencing (4, 5), nanomaterial fabrication (6-8), and as drug-carrying nanomachines (9). In addition, filamentous viruses form a variety of liquid crystals driving the development of both theory and practice of softmatter physics (10, 11). Filamentous viruses are also associated with various diseases, e.g., CTXϕ phage in cholera toxin (12) and Pf4 phage in cystic fibrosis (13).Phage belonging to the Ff family (M13, fd, f1) are F-pilusspecific viruses that share almost identical genomes and very similar structures. M13 is a 16-MDa virus having a diameter of ∼7 nm and a length of ∼1 μm. The capsid is composed of several thousand identical copies of a major coat protein subunit arranged in a helical array surrounding a core of a circular ssDNA. The major coat proteins constitute ∼85% of the total virion mass, the ssDNA ∼12%, and all other minor proteins (gp3, gp6, gp7, gp9) that are specific for infection and assembly constitute about 3% of the total virion mass (1, 14).Previous structural models for a small number of phages have been obtained by means of X-ray fiber diffraction (15-19), static solid-state NMR (20, 21), and cryo-EM (22). Structural models for the Ff family have been proposed based on the three methods; however, ...

The filamentous bacteriophage viruses of the Ff family, fd and M13, slightly differ in their genome, and their 50-residue-long major capsid proteins have a single site difference: the uncharged asparagine-12 in M13 is replaced with a negatively charged aspartate in fd. We have used magic-angle spinning solid-state NMR spectroscopy to site-specifically assign the resonances belonging to the capsid protein of M13. Assignment of several mobile residues was facilitated by using J-based spectroscopy, which in addition provided sugar-base contacts in the M13-DNA stemming from two-bond scalar couplings. A comparison between M13 and fd bacteriophages reveals that the two virions have a very conserved and stable structure, manifested in negligibly small chemical shift differences and similar dynamic properties for nearly all resonances. The principal difference between the two phages involves residues in the vicinity of residue 12. We suggest that the elimination of the single charge at position 12 throughout the entire assembly affects the electrostatic and hydrogen-bonding interaction network governing inter- and intraresidue contacts, mainly by the rearrangement of the positively charged lysine residue at position 8.

The fd bacteriophage is a filamentous virus consisting of a circular single-stranded DNA (ssDNA) wrapped by thousands of copies of a major coat protein subunit (the capsid). The coat protein subunits are mostly α-helical and curved, and are arranged in the capsid in consecutive pentamers related by a translation along the main viral axis and a rotation of ~36° (C5S2 symmetry). The DNA is right-handed and helical, but information on its structure and on its interface with the capsid is incomplete. We present here an approach for assigning the DNA nucleotides and studying its interactions with the capsid by magic-angle spinning solid-state NMR. Capsid contacts with the ssDNA are obtained using a two-dimensional (13)C-(13)C correlation experiment and a proton-mediated (31)P-(13)C polarization transfer experiment, both acquired on an aromatic-unlabeled phage sample. Our results allow us to map the residues that face the interior of the capsid and to show that the ssDNA-capsid interactions are sustained mainly by electrostatic interactions between the positively charged lysine side chains and the phosphate backbone. The use of natural abundance aromatic amino acids in the growth media facilitated the complete assignment of the four nucleotides and the observation of internucleotide contacts. Using chemical shift analysis, our study shows that structural features of the deoxyribose carbons reporting on the sugar pucker are strikingly similar to those observed recently for the Pf1 phage. However, the ssDNA-protein interface is different, and chemical shift markers of base pairing are different. This experimental approach can be utilized in other filamentous and icosahedral bacteriophages, and also in other biomolecular complexes involving structurally and functionally important DNA-protein interactions.

The fd bacteriophage is a filamentous virus that is widely used for bio- and nanotechnology applications ranging from phage display to battery materials. The possibility of obtaining a detailed description of its structural properties regardless of its state is therefore essential not only for understanding its physical arrangement and its bacterial infection process but also for many other applications. Here we present a study of the fd phage by magic-angle spinning solid-state NMR. While current structures rely on a Y21M mutant, experiments performed on a strain bearing a wild-type capsid report on high symmetry of the phage and lack of explicit subunit polymorphism. Chemical shift analysis confirmed that the coat protein mostly consists of a rigid right-handed curved α-helix (residues 6-47 of 50), preceded by a flexible loop-structured N-terminus. We were able to qualitatively assign the resonances belonging to the DNA, including the deoxyribose sugars and the thymine bases. These chemical shifts are consistent with base stacking and a C2'-endo/C3'-exo sugar pucker.

Filamentous bacteriophage (phage) are single-stranded DNA viruses that infect bacteria. Single-site mutants of fd phage have been studied by magic-angle spinning nuclear magnetic resonance and by small-angle X-ray scattering. Detailed analysis has been performed that provides insight into structural variations on three length scales. The results, analyzed in conjunction with existing literature data, suggest that a single charge mutation on the capsid surface affects direct interviral interactions but not the structure of individual particles or the macroscale organization. On the other hand, a single hydrophobic mutation located at the hydrophobic interface that stabilizes capsid assembly alters the atomic structure of the phage, mainly affecting intersubunit interactions, affects its macroscale organization, that is, the pitch of the cholesteric liquid crystal formed by the particles, but skips the nanoscale hence does not affect direct interparticle interactions. An X-ray scattering under osmotic pressure assay provides the effective linear charge density of the phage and we obtain values of 0.6 Å and 0.4 Å for fd and M13 phage, respectively. These values agree with a simple consideration of a single cylinder with protein and DNA charges spread according to the most recent atomic-resolution models of the phage.