Complex architecture of a closterovirus virion

In this investigation we examined the molecular architecture and functions associated with the virion components of the Beet yellows virus (BYV), family Closteroviridae. The BYV virions are filamentous particles composed of two coat proteins, the major coat protein (CP), which encapsidates the majority of the genome, and the minor coat protein (CPm) that makes a small tail-like structure at one end of the virions. The bipolar architecture of closteroviruses is unique among the plant helical viruses, and suggests that CP and CPm evolved to fulfill different functions in the life cycle of closteroviruses. Current models propose that the CPm tails are specialized virion components that participate in the vector transmission and cell-to-cell movement of the virus. In addition to CP and CPm, the cell-to-cell movement of BYV requires the contributions of three highly conserved viral genes encoding a small 6.4-kDa hydrophobic protein (p6), a HSP7O homologue (HSP7Oh) and a 64-kDa protein (p64). Using nano-liquid chromatography/tandem mass spectrometry and biochemical analyses we demonstrate that HSP7Oh and p64 are associated with virions. This conclusion is based on the co-migration of HSP7Oh and p64 with BYV virions in sucrose density gradients, and on the co-immunoprecipitation of HSP7Oh, pM and BYV capsid protein using anti-HSP7Oh or anti-p64 serum. The association of HSP7Oh and p64 with the virions is resistant to high concentrations of sodium chloride, which normally disrupt non-covalent protein interactions. Low concentrations of sodium dodecyl sulfate (SDS) or lithium chloride (LiC1), and treatment with alkaline or acidic pH resulted in the simultaneous disassembly of virions and dissociation of HSP7Oh and p64. The N-terminal domain of p64 is exposed at the virion surface and is accessible to antibodies and mild trypsin digestion. In contrast, the C-terminal domain is embedded in the virion and is inaccessible to antibodies or trypsin. The C-terminal domain of p64 is shown to be homologous to CP and CPm. Mutation of the signature motifs of capsid proteins of filamentous RNA viruses in p64 results in the formation of defective virions, which are unable to move from cell to cell. These results reveal the dual function of HSP7Oh and p64 in tail assembly and BYV motility and support the concept of the virion tail as a specialized device for BYV cell-to-cell movement

The plug-based nanovolume Microcapillary Protein Crystallization System (MPCS)

This is the published version. Copyright International Union of CrystallographyThe Microcapillary Protein Crystallization System (MPCS) embodies a new semi-automated plug-based crystallization technology which enables nanolitre-volume screening of crystallization conditions in a plasticware format that allows crystals to be easily removed for traditional cryoprotection and X-ray diffraction data collection. Protein crystals grown in these plastic devices can be directly subjected to in situ X-ray diffraction studies. The MPCS integrates the formulation of crystallization cocktails with the preparation of the crystallization experiments. Within microfluidic Teflon tubing or the microfluidic circuitry of a plastic CrystalCard, ~10-20 nl volume droplets are generated, each representing a microbatch-style crystallization experiment with a different chemical composition. The entire protein sample is utilized in crystallization experiments. Sparse-matrix screening and chemical gradient screening can be combined in one com­prehensive `hybrid' crystallization trial. The technology lends itself well to optimization by high-granularity gradient screening using optimization reagents such as precipitation agents, ligands or cryoprotectants

The plug-based nanovolume Microcapillary Protein Crystallization System (MPCS)

The Microcapillary Protein Crystallization System (MPCS) is a new protein-crystallization technology used to generate nanolitre-sized crystallization experiments for crystal screening and optimization. Using the MPCS, diffraction-ready crystals were grown in the plastic MPCS CrystalCard and were used to solve the structure of methionine-R-sulfoxide reductase

Expression of proteins in Escherichia coli as fusions with maltose-binding protein to rescue non-expressed targets in a high-throughput protein-expression and purification pipeline

The rescue of protein-expression levels by cloning genes into MBP-fusion vector is described

High-throughput protein production and purification at the Seattle Structural Genomics Center for Infectious Disease

An overview of the standard SSGCID protein-purification protocol is given and success rates and cleavage alternatives are discussed

Nanovolume optimization of protein crystal growth using the microcapillary protein crystallization system

The Microcapillary Protein Crystallization System (MPCS) is used to successfully optimize protein crystals from 28 out of 29 tested proteins. Six protein structures have been determined from diffraction-ready crystals grown inside and harvested directly from the MPCS CrystalCards, which are compatible with the recently commercialized and automated MPCS Plug Maker instrument

Solution structure of an arsenate reductase-related protein, YffB, from Brucella melitensis, the etiological agent responsible for brucellosis

B. melitensis is a NIAID Category B microorganism that is responsible for brucellosis and is a potential agent for biological warfare. Here, the solution structure of the 116-residue arsenate reductase-related protein Bm-YffB (BR0369) from this organism is reported

BrabA.11339.a: anomalous diffraction and ligand binding guide towards the elucidation of the function of a ‘putative β-lactamase-like protein’ from Brucella melitensis

The structure of a β-lactamase-like protein from B. melitensis was solved independently using two data sets with anomalous signal. Anomalous Fourier maps could confirm the identity of two metal ions in the active site. AMP-bound and GMP-bound structures provide hints to the possible function of the protein

Structure of a Nudix hydrolase (MutT) in the Mg2+-­bound state from Bartonella henselae, the bacterium responsible for cat scratch fever

B. henselae is the etiological agent responsible for cat scratch fever (bartonellosis). The crystal structure of the smaller of the two Nudix hydrolases encoded in the genome of B. henselae, Bh-MutT, was determined to 2.1 Å resolution