Biology subjects

Svoboda, T.

Publications and source records attributed to Svoboda, T..

7 recordsLinked to original sources

bioRxiv2025

Per-residue optimisation of protein structures:Rapid alternative to optimisation with constrainedalpha carbons

In recent years, the number of known protein structures has increased significantly. Predictive algorithms and experimental methods provide the positions of protein residues relative to each other with high accuracy. However, the local quality of the protein structure, including bond lengths, angles, and positions of individual atoms, often lacks the same level of precision. For this reason, protein structures are usually optimised by a force field prior to their application in further research sensitive to structural quality. Protein structure optimisation, however, is computationally challenging. In this paper, we introduce a general method Per-residue optimisation of protein structures: Rapid alternative to optimisation with constrained alpha carbons (PROPTIMUS RAPHAN). Rather than optimising the entire protein structure at once, PROPTIMUS RAPHAN divides the structure into overlapping residual substructures and optimises each substructure individually. This approach results in computational time that scales linearly with the size of the structure. Additionally, we present PROPTIMUS RAPHANGFN-FF, a reference implementation of our method employing a generic, almost QM-accurate force field, GFN-FF. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/690085v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1e9fc5borg.highwire.dtl.DTLVardef@b6d70aorg.highwire.dtl.DTLVardef@1e00427org.highwire.dtl.DTLVardef@30c66f_HPS_FORMAT_FIGEXP M_FIG C_FIG We tested PROPTIMUS RAPHANGFN-FF on 461 AlphaFold DB structures and demonstrated that our approach achieves results comparable to the optimisation of the structure with constrained alpha carbons in significantly less time. Scientific ContributionThe main contribution of this work is the PROPTI-MUS RAPHAN method and its reference parallelisable implementation PROP-TIMUS RAPHANGFN-FF. Because the time requirement increases linearly with the size of the structure, PROPTIMUS RAPHANGFN-FF optimises on average 5 000 atoms per hour and a common CPU. Therefore, prior to any research sensitive to protein structure quality, our method can be employed to obtain protein structures closer to QM-accuracy.

bioinformatics↗

bioRxiv2025

Tracing spontaneously occurring mutations in Fusarium graminearum laboratory strains resulting in reduced virulence on wheat

BackgroundFusarium graminearum is a well characterized plant pathogenic fungus which is able to infect a broad range of economically relevant crop plants. Besides yield reduction this fungus is also responsible for mycotoxin contamination of food and feed. Upon propagation under laboratory conditions, mutations may occur which would be disadvantageous for fungal fitness in nature but not in the lab where the strains are usually grown on nutrient-rich media under optimal growth conditions. In this study we characterized four phenotypically different Fusarium graminearum strains for fitness traits and compared their genomes to trace down mutations responsible for the phenotypes. ResultsThe four tested F. graminearum PH1-derived strains revealed differences in their phenotypic appearance and also in their secondary metabolite profiles expressed on different growth media. Also, two of the investigated strains (94 and 96) showed significantly reduced virulence on wheat upon point inoculation of flowering wheat. We identified one high impact mutation in each of the two strains. In strain 96 a loss of function mutation occurred in FGSG_00355 which has a high similarity to Ras GTPase activating proteins and consequently may have an impact on the cell cycle. Even though strain 96 showed enhanced DON production in vitro, the strain was no longer able to spread within the wheat ear in infection assays. In strain 94 we identified an insertion of an A rather at the end of FGSG_00052 leading to a frameshift and consequently mutation of the last three amino acids and a shift of the stop codon by seven amino acids. Even though knock-out of this putative transcription factor has been described by Son et al (2011) to have no impact on virulence, changes at the C-terminal region may result in changes of the binding affinity. ConclusionsWe tracked down the mutations which might be responsible for the changes in phenotypic appearance, secondary metabolite profile as well as virulence. Yet, a closer biological characterization is necessary to determine the impact of these mutations on the fungus. Impact statementMutations under laboratory conditions can occur spontaneously. Due to the lack of selection pressure, also potentially deleterious mutations remain unnoticed as long as all nutrients are provided. In this study we analyzed four phenotypically different Fusarium graminearum PH-1 strains among which two showed significantly reduced virulence on wheat. In one strain we identified a loss of function mutation in a Ras-GTPase activating protein resulting in enhanced growth on complete media but at the same time strongly reduced virulence. The mutation in the second strain was an insertion in the C-terminal region of a transcription factor. The exact role of the Ras-GTPase activating protein during infection and the impact C-terminal elongation of a yet uncharacterized transcription factor is yet to be investigated. This study underscores the importance of regularly checking laboratory strains on their traits that such mutations which may have an impact on your research data, do not remain unnoticed. Data summaryThe code used for web scraping is available on github (https://github.com/cicci726/webscraping/tree/main). The sequencing files are deposited at NCBI in the sequence read archive (BioProject ID: PRJNA1293145).

genomics↗

bioRxiv2025

Cell cycle controls pathogenic processes and mycotoxin production in Fusarium graminearum

RAS proteins control the cell cycle in all eukaryotes and lead to cancer in mammals when mutated to permanent activity. We previously isolated a spontaneous mutant of the major cereal pathogen Fusarium graminearum with a permanently active RAS allele due to a mutation in the nucleotide exchange factor GAP that is needed to inactivate RAS (Ras-GAP). In this study we evaluate the impact of a Ras-GAP deletion in F. graminearum on the phenotype and the transcriptomes of the pathogen and the host plant during infection. The mutant showed an altered secondary metabolite profile and significantly reduced virulence on wheat. The associated fungal transcriptome revealed that the mutant is unable to enter the pathogenic state and consequently, mutant cells do not switch from a saprophytic to a pathogenic program. While the wild type reprogrammed the expression of 953 genes during this switch, only six genes were significantly changed in the mutant. Genes most affected are involved in cell cycle control, response to nitrogen limitation and pathogenesis. Also, the plant responded differently and only mildly to the presence of the continuously proliferating, but avirulent fungal strain. Our data for the first time demonstrate that downregulation of the cell cycle is necessary for the production of virulence factors and pathogenicity in F. graminearum.

genetics↗

bioRxiv2025

Resistance screening of uncharacterized Lupinus angustifolius accessions towards Fusarium oxysporum f. sp. lupini

Lupins, which belong to the family of fabaceae, are plants with a high nutritional potential due to their high protein content, symbiotic nitrogen fixation and environmental adaptability. Yet, plant pathogens including Fusairum oxysporum f. sp. lupini are a considerable problem in lupin cultivation as they can cause considerable yield reduction. To find Lupinus angustifolius accessions with enhanced resistance towards Fusarium infection, we screened 20 yet uncharacterized accessions in a root infection assay. The infected roots were harvested four days post inoculation, followed by total DNA extraction. The rate of infection was estimated by calculating the relative amount of lupin and fungal DNA based on the respective qPCR cq-values. After screening of all 20 accessions, we identified L26 as the most resistant and L49 as the most susceptible accession. To verify this, infection assays of a longer time period of 15 days were performed with these two accessions. The initial results were confirmed as more severe symptoms were observed on L49. To better characterize the infection dynamics, a time series of the infection process was set up and infected roots were evaluated after one, two three and four days. These results showed that already after two days post inoculation there is a significant difference regarding infection levels between L26 and L49. Further characterizations will provide valuable information on the genes responsible for the different resistance levels.

microbiology↗

bioRxiv2024

Fusarium graminearum copper amine-oxidases redundantly increase virulence by converting tryptamine from hydrolyzed plant defense compounds into auxin

Plant pathogenic fungi have evolved different strategies to interfere with plant defense mechanisms. The well described fungal plant pathogen Fusarium graminearum is not only able to produce trichothecene toxins like deoxynivalenol, but also the plant hormone auxin. Highly elevated levels of auxin and auxin derivatives such as IAA-glucoside or IAA amino-acid conjugates were observed in wheat cultivar Apogee infected with F. graminearum. We report that F. graminearum is able to cleave tryptamine-derived hydroxycinnamic acid amides, e.g. the defense compound coumaroyl-tryptamine. In this study we investigated copper amine-oxidases, candidate genes for auxin biosynthesis converting tryptamine into the IAA precursor indole-3-acetyldehyde. After consecutive knock outs of all seven copper amine oxidases the resulting septuple knock out strain had strongly reduced ability to produce auxin. Virulence of the septuple mutant was significantly impaired while DON production in planta was comparable to the wild type. We conclude that F. graminearum, often presumed to be a simple nectrotroph, has a biotrophic phase and is able to employ plant defense compounds by converting them into defense suppressing auxin.

molecular biology↗

bioRxiv2024

Locus-specific chromatin proteomics using dCas-guided proximity labelling in Aspergillus nidulans.

Proximity labelling that uses promiscuous biotin ligases (BirA) fused to a bait protein is a powerful tool to identify protein interaction partners in vivo under different metabolic or developmental conditions. BirA can also be used to determine protein composition and interaction partners at specific chromatin locations when it is fused with enzymatically-disabled Cas9 (dCas9) and then guided to the location of interest by sgRNAs. We adapted this method (called CasID) for fungal cells using the nitrate assimilation gene cluster of A. nidulans as a model locus and estrogen-inducible expression of the dCas9-BirA fusion to improve condition-specific labelling. For method establishment, we first verified the presence of dCas-BirA and a known transcription factor at the nitrate locus by chromatin immunoprecipitation (ChIP). Results show that both dCas-BirA and the AreA transcription factor are present at the locus of interest under the conditions used for biotinylation. We then optimized the CasID procedure for efficient labelling and background reduction using the CasID-sgRNA strain and two control strains, one lacking the sgRNA and another one lacking the whole CasID system. Here we provide proof-of-concept for the suitability of the method by showing that biotinylated proteins are enriched in the CasID strains in comparison to the controls. After background reduction, 32 proteins remained in two independent experiments exclusively enriched in the Cas-ID-sgRNA strain. Among these proteins was NmrA, an AreA-interacting regulator, and we also found several chromatin-associated proteins. Overall, our results demonstrate that Cas-ID is suitable for locus-specific labelling and identification of chromatin-associated proteins and transcription factors in A. nidulans. However, the high background of proteins that are biotinylated out of chromatin context or unspecifically attach to the affinity purification matrix needs to be addressed by implementing a set of rigorous controls. In summary, we herewith provide a detailed protocol for application of the method that proved to be useful for the identification of novel chromatin-associated proteins and their interaction partners at a specific genomic locus in divers metabolic and developmental conditions. Author summaryThis study demonstrates that locus-specific proteomics can be carried out by dCas-BirA guided proximity labelling in Aspergillus nidulans. For establishment, we targeted the well-described bidirectional promoter region between niaD, a nitrate reductase, and niiA, a nitrite reductase. At this locus we could test by chromatin immunoprecipitation (ChIP) in combination with qPCR if both, the dCas9-BirA fusion as well as a central transcription factor are at the locus under the conditions of our Cas-ID experiment. After this first control step, we considered that unspecific labelling by dCas-BirA during the time from translation to landing at the targeted chromatin locus may be one of the most relevant drawbacks of the method. Therefore, we developed a number of control strains that would allow us to clearly discriminate between background and sgRNA-dependent specific labelling at the locus. Our protein MS results validated these estimates and only considering the results of these controls enabled us to distinguish the set of locus-specific proteins from a very high general background. Finally, enrichment of biotinylated proteins through affinity purification with streptavidin resin and subsequent LC-MS/MS analysis showed that more than 800 proteins were detected in each sample, emphasizing the high background of the purification method. After background reduction of the control samples, we were able to identify 32 proteins which were exclusively detected in the test strain in two independent measurements, including several chromatin-associated proteins and NmrA, a negative regulator of the nitrate locus transcription factor AreA.

molecular biology↗

bioRxiv2024

Rare ring conformations in PDB: Facts or wishful thinking?

Protein structural data are highly valuable for research, and many significant results have been published on their basis. A key point for their credibility and applicability is their quality. An important facet of protein structure quality is the validation of ligands. Some aspects of ligand quality have already been validated by established quality metrics. However, validation of ring conformation has yet to be comprehensively performed despite rings strongly influencing the formation of the ligands scaffold and shape. Most rings form several conformations that differ in their stability. The most stable ones occur frequently in nature and should, therefore, be found in Protein Data Bank (PDB) structures. In this article, we examined which conformations of rings occur in PDB structures. Our analysis focused on conformations of all cyclopentane, cyclohexane, and benzene rings in the PDB. Specifically, we examined 123 264 rings of 24 763 distinct ligands, which instances occur in 44 022 protein structures. In general, we found that most of the rings (98.32 %) are in energetically favourable conformations. Surprisingly, the existence of most of the energetically unfavourable ring conformations (2 067 samples, 1.68 %) is not supported by experimental data. Only 291 unfavourable ring conformations (0.24 %) are backed by experimental data that are accurate enough to distinguish the conformation, which shows that the existence of energetically unfavourable ring conformations is rarely supported by structural or experimental evidence. Our results suggest that each occurrence of untypical ring conformation in the PDB may indicate a potential error and should be carefully analysed.

bioinformatics↗