Resolution and Probabilistic Models of Components in CryoEM Maps of Mature P22 Bacteriophage

CryoEM continues to produce density maps of larger and more complex assemblies with multiple protein components of mixed symmetries. Resolution is not always uniform throughout a cryoEM map, and it can be useful to estimate the resolution in specific molecular components of a large assembly. In this study, we present procedures to 1) estimate the resolution in subcomponents by gold-standard Fourier shell correlation (FSC); 2) validate modeling procedures, particularly at medium resolutions, which can include loop modeling and flexible fitting; and 3) build probabilistic models that combine high-accuracy priors (such as crystallographic structures) with medium-resolution cryoEM densities. As an example, we apply these methods to new cryoEM maps of the mature bacteriophage P22, reconstructed without imposing icosahedral symmetry. Resolution estimates based on gold-standard FSC show the highest resolution in the coat region (7.6 Å), whereas other components are at slightly lower resolutions: portal (9.2 Å), hub (8.5 Å), tailspike (10.9 Å), and needle (10.5 Å). These differences are indicative of inherent structural heterogeneity and/or reconstruction accuracy in different subcomponents of the map. Probabilistic models for these subcomponents provide new insights, to our knowledge, and structural information when taking into account uncertainty given the limitations of the observed density.     

Antimicrobial and anti-pathogenic activity of some thioureides derivatives against Erwinia amylovora phytopathogenic strains

A series of N-(1-methyl-1 Hpyrazole-4-carbonyl)-thiourea derivatives were assessed for their in vitro antimicrobial and anti-pathogenic activity against twenty-two strains of Erwinia amylovora isolated from different regions in Romania. The compounds were solubilised in dimethylsulfoxide and screened for their in vitro antimicrobial activity. The qualitative screening of the susceptibility spectra of various strains to the compounds was performed by adapted diffusion techniques (distribution of the tested compound solution directly on the solid medium previously seeded with the bacterial inoculums). The quantitative assay of the minimal inhibitory concentration (MIC, microg/mL) was based on liquid medium two-fold microdilutions. The subinhibitory concentrations of the tested substances were investigated for their influence on biofilm development on inert substrata. The present study showed that six new thiourea compounds exhibited a low antibacterial activity (MIC values > 500 microg/ml), but the subinhibitory concentrations inhibited the biofilm development on inert substrata. Thus, these results could suggest the usefulness of the tested compounds as control agents for preventing the first stage (colonization) of the infection with the fire blight pathogen.     

Hepatitis B, C and D coinfection in HIV-infected patients: prevalence and progress

The HAART therapy has improved life expectancy enabling long latency conditions caused by the hepatitis viruses that became the leading cause of death in HIV infected patients. In this study a group of 300 patients aged from 18 to 63 years were selected in order to assess the prevalence and consequences of HIV and the hepatitis B (HBV), C (HCV) and D (HDV) viruses coinfections. Study groups were designed for each coinfection. These groups were in turn divided in case groups formed of coinfected participants and control groups consisting of mono-infected participants. This classification was obtained by testing the participants for the presence of specific infection markers using the ELISA technique. As a result, in regard to the HIV/HBV coinfection the study group consisted of 16 coinfected participants and 114 HBV-infected participants resulting in a prevalence of the coinfection of 14%. In the case of the HIV/HDV coinfection the study group consisted of 5 coinfected participants and 45 HDV-infected participants. The prevalence of the HIV/HCV coinfection was 25% out of the 170 HCV-infected participants. The effect of the coinfections on the expression and levels of the infection markers was analyzed in constrast to those encountered in the case of the mono-infection. The observed changes in the expression of the specific hepatitis markers indicate the impact of the coinfection with HIV on the progression of the hepatitis infections. In addition, the inadequate immune response towards the hepatitis viruses in the case of the coinfected participants leads to the development of cirrhosis and end stage liver disease.     

A “yellow” laccase with “blue” spectroscopic features,from Sclerotinia sclerotiorum

Reported here are the production, purification and characterization of a laccase from the phytophathogenic fungus Sclerotinia sclerotiorum. This laccase is identified by mass spectrometry with a sequence coverage of 74.9% (458/577 AA) revealing that the protein is identical or highly homologous to a predicted oxidoreductase from this species (A7EM18 in the Uniprot database); the closest homologous protein previously isolated from a fungus is the Melanocarpus albomyces, with only 35% identity. The UV–vis spectral features of this laccase classify it as a “yellow” one. The EPR spectrum nevertheless demonstrates resemblance to blue laccases – including the type 1 center not detectable in UV–vis spectra. The presence of type 3 coppers was proven by fluorescence spectrum and by 330 nm band in UV–vis. The purified laccase has an apparent molecular mass of 70 kDa and appears as a monomer. The values of K_M and k_cat were determined for ABTS, 2,6-dimethoxyphenol, p-phenylenediamine and guaicol and are typical of a laccase. The optimal pH value is around 4 except for ABTS, for which activity is linearly increasing with acidity. The high laccase activity in liquid culture makes S. sclerotiorum a useful source of laccase for practical applications.     

The 3.5-Å CryoEM Structure of Nanodisc-Reconstituted Yeast Vacuolar ATPase Vo Proton Channel

1. Download high-res image (505KB)
2. Download full-size image