Automated Tools to Advance High-Resolution Imaging in Liquid.

Liquid-electron microscopy (EM), the room-temperature correlate to cryo-EM, is a rapidly growing field providing high-resolution insights of macromolecules in solution. Here, we describe how liquid-EM experiments can incorporate automated tools to propel the field to new heights. We demonstrate fresh workflows for specimen preparation, data collection, and computing processes to assess biological structures in liquid. Adeno-associated virus (AAV) and the SARS-CoV-2 nucleocapsid (N) were used as model systems to highlight the technical advances. These complexes were selected based on their major differences in size and natural symmetry. AAV is a highly symmetric, icosahedral assembly with a particle diameter of ~25 nm. At the other end of the spectrum, N protein is an asymmetric monomer or dimer with dimensions of approximately 5­7 nm, depending upon its oligomerization state. Equally important, both AAV and N protein are popular subjects in biomedical research due to their high value in vaccine development and therapeutic efforts against COVID-19. Overall, we demonstrate how automated practices in liquid-EM can be used to decode molecules of interest for human health and disease.

Liquid-EM goes viral - visualizing structure and dynamics.

Liquid-electron microscopy (EM), the room temperature correlate to cryo-EM, is an exciting new technique delivering real-time data of dynamic reactions in solution. Here, we explain how liquid-EM gained popularity in recent years by examining key experiments conducted on viral assemblies and host-pathogen interactions. We describe developing workflows for specimen preparation, data collection, and computing processes that led to the first high-resolution virus structures in a liquid environment. Equally important, we review why liquid-electron tomography may become the next big thing in biomedical research due to its ability to monitor live viruses entering cells within seconds. Taken together, we pose the idea that liquid-EM can serve as a dynamic complement to current cryo-EM methods, inspiring the "real-time revolution" in nanoscale imaging.

High-Resolution Imaging of Human Viruses in Liquid Droplets.

Liquid-phase electron microscopy (LP-EM) is an exciting new area in the materials imaging field, providing unprecedented views of molecular processes. Time-resolved insights from LP-EM studies are a strong complement to the remarkable results achievable with other high-resolution techniques. Here, the opportunities to expand LP-EM technology beyond 2D temporal assessments and into the 3D regime are described. The results show new structures and dynamic insights of human viruses contained in minute volumes of liquid while acquired in a rapid timeframe. To develop this strategy, adeno-associated virus (AAV) is used as a model system. AAV is a well-known gene therapy vehicle with current applications involving drug delivery and vaccine development for COVID-19. Improving the understanding of the physical properties of biological entities in a liquid state, as maintained in the human body, has broad societal implications for human health and disease.

Assembly of bacteriophage P2 capsids from capsid protein fused to internal scaffolding protein.

Most tailed bacteriophages with double-stranded DNA genomes code for a scaffolding protein, which is required for capsid assembly, but is removed during capsid maturation and DNA packaging. The gpO scaffolding protein of bacteriophage P2 also doubles as a maturation protease, while the scaffolding activity is confined to a 90 residue C-terminal "scaffolding" domain. Bacteriophage HK97 lacks a separate scaffolding protein; instead, an N-terminal "delta" domain in the capsid protein appears to serve an analogous role. We asked whether the C-terminal scaffolding domain of gpO could work as a delta domain when fused to the gpN capsid protein. Varying lengths of C-terminal sequences from gpO were fused to the N-terminus of gpN and expressed in E. coli. The presence of just the 41 C-terminal residues of gpO increased the fidelity of assembly and promoted the formation of closed shells, but the shells formed were predominantly small, 40 nm shells, compared to the normal, 55 nm P2 procapsid shells. Larger scaffolding domains fused to gpN caused the formation of shells of varying size and shape. The results suggest that while fusing the scaffolding protein to the capsid protein assists in shell closure, it also restricts the conformational variability of the capsid protein.

Competing scaffolding proteins determine capsid size during mobilization of Staphylococcus aureus pathogenicity islands.

Staphylococcus aureus pathogenicity islands (SaPIs), such as SaPI1, exploit specific helper bacteriophages, like 80α, for their high frequency mobilization, a process termed 'molecular piracy'. SaPI1 redirects the helper's assembly pathway to form small capsids that can only accommodate the smaller SaPI1 genome, but not a complete phage genome. SaPI1 encodes two proteins, CpmA and CpmB, that are responsible for this size redirection. We have determined the structures of the 80α and SaPI1 procapsids to near-atomic resolution by cryo-electron microscopy, and show that CpmB competes with the 80α scaffolding protein (SP) for a binding site on the capsid protein (CP), and works by altering the angle between capsomers. We probed these interactions genetically and identified second-site suppressors of lethal mutations in SP. Our structures show, for the first time, the detailed interactions between SP and CP in a bacteriophage, providing unique insights into macromolecular assembly processes.

Cleavage and Structural Transitions during Maturation of Staphylococcus aureus Bacteriophage 80α and SaPI1 Capsids.

In the tailed bacteriophages, DNA is packaged into spherical procapsids, leading to expansion into angular, thin-walled mature capsids. In many cases, this maturation is accompanied by cleavage of the major capsid protein (CP) and other capsid-associated proteins, including the scaffolding protein (SP) that serves as a chaperone for the assembly process. Staphylococcus aureus bacteriophage 80α is capable of high frequency mobilization of mobile genetic elements called S. aureus pathogenicity islands (SaPIs), such as SaPI1. SaPI1 redirects the assembly pathway of 80α to form capsids that are smaller than those normally made by the phage alone. Both CP and SP of 80α are N-terminally processed by a host-encoded protease, Prp. We have analyzed phage mutants that express pre-cleaved or uncleavable versions of CP or SP, and show that the N-terminal sequence in SP is absolutely required for assembly, but does not need to be cleaved in order to produce viable capsids. Mutants with pre-cleaved or uncleavable CP display normal viability. We have used cryo-EM to solve the structures of mature capsids from an 80α mutant expressing uncleavable CP, and from wildtype SaPI1. Comparisons with structures of 80α and SaPI1 procapsids show that capsid maturation involves major conformational changes in CP, consistent with a release of the CP N-arm by SP. The hexamers reorganize during maturation to accommodate the different environments in the 80α and SaPI1 capsids.

The roles of SaPI1 proteins gp7 (CpmA) and gp6 (CpmB) in capsid size determination and helper phage interference.

SaPIs are molecular pirates that exploit helper bacteriophages for their own high frequency mobilization. One striking feature of helper exploitation by SaPIs is redirection of the phage capsid assembly pathway to produce smaller phage-like particles with T=4 icosahedral symmetry rather than T=7 bacteriophage capsids. Small capsids can accommodate the SaPI genome but not that of the helper phage, leading to interference with helper propagation. Previous studies identified two proteins encoded by the prototype element SaPI1, gp6 and gp7, in SaPI1 procapsids but not in mature SaPI1 particles. Dimers of gp6 form an internal scaffold, aiding fidelity of small capsid assembly. Here we show that both SaPI1 gp6 (CpmB) and gp7 (CpmA) are necessary and sufficient to direct small capsid formation. Surprisingly, failure to form small capsids did not restore wild-type levels of helper phage growth, suggesting an additional role for these SaPI1 proteins in phage interference.

Assembly of bacteriophage 80α capsids in a Staphylococcus aureus expression system.

80α is a temperate, double-stranded DNA bacteriophage of Staphylococcus aureus that can act as a "helper" for the mobilization of S. aureus pathogenicity islands (SaPIs), including SaPI1. When SaPI1 is mobilized by 80α, the SaPI genomes are packaged into capsids that are composed of phage proteins, but that are smaller than those normally formed by the phage. This size determination is dependent on SaPI1 proteins CpmA and CpmB. Here, we show that co-expression of the 80α capsid and scaffolding proteins in S. aureus, but not in E. coli, leads to the formation of procapsid-related structures, suggesting that a host co-factor is required for assembly. The capsid and scaffolding proteins also undergo normal N-terminal processing upon expression in S. aureus, implicating a host protease. We also find that SaPI1 proteins CpmA and CpmB promote the formation of small capsids upon co-expression with 80α capsid and scaffolding proteins in S. aureus.

A conformational switch involved in maturation of Staphylococcus aureus bacteriophage 80α capsids.

Bacteriophages are involved in many aspects of the spread and establishment of virulence factors in Staphylococcus aureus, including the mobilization of genetic elements known as S. aureus pathogenicity islands (SaPIs), which carry genes for superantigen toxins and other virulence factors. SaPIs are packaged into phage-like transducing particles using proteins supplied by the helper phage. We have used cryo-electron microscopy and icosahedral reconstruction to determine the structures of the procapsid and the mature capsid of 80α, a bacteriophage that can mobilize several different SaPIs. The 80α capsid has T=7 icosahedral symmetry with the capsid protein organized into pentameric and hexameric clusters that interact via prominent trimeric densities. The 80α capsid protein was modeled based on the capsid protein fold of bacteriophage HK97 and fitted into the 80α reconstructions. The models show that the trivalent interactions are mediated primarily by a 22-residue ß hairpin structure called the P loop that is not found in HK97. Capsid expansion is associated with a conformational switch in the spine helix that is propagated throughout the subunit, unlike the domain rotation mechanism in phage HK97 or P22.

The Staphylococcus aureus pathogenicity island 1 protein gp6 functions as an internal scaffold during capsid size determination.

Staphylococcus aureus pathogenicity island 1 (SaPI1) is a mobile genetic element that carries genes for several superantigen toxins. SaPI1 is normally stably integrated into the host genome but can become mobilized by "helper" bacteriophage 80α, leading to the packaging of SaPI1 genomes into phage-like transducing particles that are composed of structural proteins supplied by the helper phage but having smaller capsids. We show that the SaPI1-encoded protein gp6 is necessary for efficient formation of small capsids. The NMR structure of gp6 reveals a dimeric protein with a helix-loop-helix motif similar to that of bacteriophage scaffolding proteins. The gp6 dimer matches internal densities that bridge capsid subunits in cryo-electron microscopy reconstructions of SaPI1 procapsids, suggesting that gp6 acts as an internal scaffolding protein in capsid size determination.

Functional domains of the bacteriophage P2 scaffolding protein: identification of residues involved in assembly and protease activity.

Bacteriophage P2 encodes a scaffolding protein, gpO, which is required for correct assembly of P2 procapsids from the gpN major capsid protein. The 284 residue gpO protein also acts as a protease, cleaving itself into an N-terminal fragment, O, that remains in the capsid following maturation. In addition, gpO is presumed to act as the maturation protease for gpN, which is N-terminally processed to N, accompanied by DNA packaging and capsid expansion. The protease activity of gpO resides in the N-terminal half of the protein. We show that gpO is a classical serine protease, with a catalytic triad comprised of Asp 19, His 48 and Ser 107. The C-terminal 90 amino acids of gpO are required and sufficient for capsid assembly. This fragment contains a predicted alpha-helical segment between residues 197 and 257 and exists as a multimer in solution, suggesting that oligomerization is required for scaffolding activity. Correct assembly requires the C-terminal cysteine residue, which is most likely involved in transient gpN interactions. Our results suggest a model for gpO scaffolding action in which the N-terminal half of gpO binds strongly to gpN, while oligomerization of the C-terminal alpha-helical domain of gpO and transient interactions between Cys 284 and gpN lead to capsid assembly.

Cryo-electron tomography of porcine reproductive and respiratory syndrome virus: organization of the nucleocapsid.

Porcine reproductive and respiratory virus (PRRSV) is an enveloped positive-sense RNA virus of the family Arteriviridae that causes severe and persistent disease in pigs worldwide. The PRRSV virion consists of a lipid envelope that contains several envelope proteins surrounding a nucleocapsid core that encapsidates the RNA genome. To provide a better understanding of the structure and assembly of PRRSV, we have carried out cryo-electron microscopy and tomographic reconstruction of virions grown in MARC-145 cells. The virions are pleomorphic, round to egg-shaped particles with an average diameter of 58 nm. The particles display a smooth outer surface with only a few protruding features, presumably corresponding to the envelope protein complexes. The virions contain a double-layered, hollow core with an average diameter of 39 nm, which is separated from the envelope by a 2-3 nm gap. Analysis of the three-dimensional structure suggests that the core is composed of a double-layered chain of nucleocapsid proteins bundled into a hollow ball.

Capsid size determination by Staphylococcus aureus pathogenicity island SaPI1 involves specific incorporation of SaPI1 proteins into procapsids.

The Staphylococcus aureus pathogenicity island SaPI1 carries the gene for the toxic shock syndrome toxin (TSST-1) and can be mobilized by infection with S. aureus helper phage 80alpha. SaPI1 depends on the helper phage for excision, replication and genome packaging. The SaPI1-transducing particles comprise proteins encoded by the helper phage, but have a smaller capsid commensurate with the smaller size of the SaPI1 genome. Previous studies identified only 80alpha-encoded proteins in mature SaPI1 virions, implying that the presumptive SaPI1 capsid size determination function(s) must act transiently during capsid assembly or maturation. In this study, 80alpha and SaPI1 procapsids were produced by induction of phage mutants lacking functional 80alpha or SaPI1 small terminase subunits. By cryo-electron microscopy, these procapsids were found to have a round shape and an internal scaffolding core. Mass spectrometry was used to identify all 80alpha-encoded structural proteins in 80alpha and SaPI1 procapsids, including several that had not previously been found in the mature capsids. In addition, SaPI1 procapsids contained at least one SaPI1-encoded protein that has been implicated genetically in capsid size determination. Mass spectrometry on full-length phage proteins showed that the major capsid protein and the scaffolding protein are N-terminally processed in both 80alpha and SaPI1 procapsids.