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As structure prediction methods are generating millions of publicly available protein structures, searching these databases is becoming a bottleneck. Foldseek aligns the structure of a query protein against a database by describing tertiary amino acid interactions within proteins as sequences over a structural alphabet. Foldseek decreases computation times by four to five orders of magnitude with 86%, 88% and 133% of the sensitivities of Dali, TM-align and CE, respectively. Foldseek speeds up protein structural search by four to five orders of magnitude.

Proteins are key to all cellular processes and their structure is important in understanding their function and evolution. Sequence-based predictions of protein structures have increased in accuracy 1 , and over 214 million predicted structures are available in the AlphaFold database 2 . However, studying protein structures at this scale requires highly efficient methods. Here, we developed a structural-alignment-based clustering algorithm—Foldseek cluster—that can cluster hundreds of millions of structures. Using this method, we have clustered all of the structures in the AlphaFold database, identifying 2.30 million non-singleton structural clusters, of which 31% lack annotations representing probable previously undescribed structures. Clusters without annotation tend to have few representatives covering only 4% of all proteins in the AlphaFold database. Evolutionary analysis suggests that most clusters are ancient in origin but 4% seem to be species specific, representing lower-quality predictions or examples of de novo gene birth. We also show how structural comparisons can be used to predict domain families and their relationships, identifying examples of remote structural similarity. On the basis of these analyses, we identify several examples of human immune-related proteins with putative remote homology in prokaryotic species, illustrating the value of this resource for studying protein function and evolution across the tree of life. The novel Foldseek clustering algorithm defines 2.30 million clusters of AlphaFold structures, identifying remote structural similarity of human immune-related proteins in prokaryotic species.

Chemical investigation of an undescribed Australian fungus, Aspergillus nanangensis , led to the identification of the nanangenines – a family of seven new and three previously reported drimane sesquiterpenoids. The structures of the nanangenines were elucidated by detailed spectroscopic analysis supported by single crystal X-ray diffraction studies. The compounds were assayed for in vitro activity against bacteria, fungi, mammalian cells and plants. Bioinformatics analysis, including comparative analysis with other acyl drimenol-producing Aspergilli, led to the identification of a putative nanangenine biosynthetic gene cluster that corresponds to the proposed biosynthetic pathway for nanangenines.

Background Fungi are prolific producers of secondary metabolites (SMs), which are bioactive small molecules with important applications in medicine, agriculture and other industries. The backbones of a large proportion of fungal SMs are generated through the action of large, multi-domain megasynth(et)ases such as polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). The structure of these backbones is determined by the domain architecture of the corresponding megasynth(et)ase, and thus accurate annotation and classification of these architectures is an important step in linking SMs to their biosynthetic origins in the genome. Results Here we report synthaser, a Python package leveraging the NCBI’s conserved domain search tool for remote prediction and classification of fungal megasynth(et)ase domain architectures. Synthaser is capable of batch sequence analysis, and produces rich textual output and interactive visualisations which allow for quick assessment of the megasynth(et)ase diversity of a fungal genome. Synthaser uses a hierarchical rule-based classification system, which can be extensively customised by the user through a web application ( http://gamcil.github.io/synthaser ). We show that synthaser provides more accurate domain architecture predictions than comparable tools which rely on curated profile hidden Markov model (pHMM)-based approaches; the utilisation of the NCBI conserved domain database also allows for significantly greater flexibility compared to pHMM approaches. In addition, we demonstrate how synthaser can be applied to large scale genome mining pipelines through the construction of an Aspergillus PKS similarity network. Conclusions Synthaser is an easy to use tool that represents a significant upgrade to previous domain architecture analysis tools. It is freely available under a MIT license from PyPI ( https://pypi.org/project/synthaser ) and GitHub ( https://github.com/gamcil/synthaser ).

Novel and rare fungal species continue to serve as an invaluable source of new natural products. In this study, we conducted a comprehensive metabologenomic assessment of the endemic Australian fungus Aspergillus luteorubrus MST-FP2246. Genome sequencing revealed that A. luteorubrus harbours a rich biosynthetic potential, including 42 biosynthetic gene clusters (BGCs) involved in secondary metabolite biosynthesis. Extensive chemical profiling of A. luteorubrus led to the identification of ten metabolites, including one novel compound, luteolactone A (1), and nine previously reported metabolites, dimethoxyphthalide (2), marilone B (3), (+)-dihydrocanadensolide (4), ascosteroside C (5), ascosteroside D (6), viridicatumtoxin A (7), aszonalenin (8), 6-hydroxyaszonalenin (9), and the recently described polyketide glycoside, luteodienoside A (10). By integrating prior knowledge with BGC analysis, eight of these metabolites were mapped to their corresponding BGCs, while the remaining BGCs represent an opportunity for novel metabolite discovery. Together, these findings underscore the rich biosynthetic capacity of A. luteorubrus and establish it as a promising target for future genome mining and novel secondary metabolites discovery.

Advances in computational structure prediction will vastly augment the hundreds of thousands of currently available protein complex structures. Translating these into discoveries requires aligning them, which is computationally prohibitive. Foldseek-Multimer computes complex alignments from compatible chain-to-chain alignments, identified by efficiently clustering their superposition vectors. Foldseek-Multimer is 3–4 orders of magnitudes faster than the gold standard, while producing comparable alignments; this allows it to compare billions of complex pairs in 11 h. Foldseek-Multimer is open-source software available at GitHub via https://github.com/steineggerlab/foldseek/ , https://search.foldseek.com/search/ and the BFMD database. Foldseek-Multimer offers a fast strategy for complex-to-complex alignment to quickly identify compatible sets of chain-to-chain alignments by their superpositions. It can compare billions of complex pairs in 11 h.

The necrotrophic fungal pathogen Cochliobolus victoriae produces victorin, a host-selective toxin (HST) essential for pathogenicity to certain oat cultivars with resistance against crown rust. Victorin is a mixture of highly modified heterodetic cyclic hexapeptides, previously assumed to be synthesized by a nonribosomal peptide synthetase. Herein, we demonstrate that victorin is a member of the ribosomally synthesized and posttranslationally modified peptide (RiPP) family of natural products. Analysis of a newly generated long-read assembly of the C. victoriae genome revealed three copies of precursor peptide genes (vicA1–3) with variable numbers of “GLKLAF” core peptide repeats corresponding to the victorin peptide backbone. vicA1–3 are located in repeat-rich gene-sparse regions of the genome and are loosely clustered with putative victorin biosynthetic genes, which are supported by the discovery of compact gene clusters harboring corresponding homologs in two distantly related plant-associated Sordariomycete fungi. Deletion of at least one copy of vicA resulted in strongly diminished victorin production. Deletion of a gene encoding a DUF3328 protein (VicYb) abolished the production altogether, supporting its predicted role in oxidative cyclization of the core peptide. In addition, we uncovered a copper amine oxidase (CAO) encoded by vicK, in which its deletion led to the accumulation of new glycine-containing victorin derivatives. The role of VicK in oxidative deamination of the N-terminal glycyl moiety of the hexapeptides to the active glyoxylate forms was confirmed in vitro. This study finally unraveled the genetic and molecular bases for biosynthesis of one of the first discovered HSTs and expanded our understanding of underexplored fungal RiPPs.

Fungi are prolific producers of secondary metabolites (SMs), which are bioactive small molecules with important applications in medicine, agriculture and other industries. The backbones of a large proportion of fungal SMs are generated through the action of large, multi-domain megasynth(et)ases such as polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). The structure of these backbones is determined by the domain architecture of the corresponding megasynth(et)ase, and thus accurate annotation and classification of these architectures is an important step in linking SMs to their biosynthetic origins in the genome. Here we report synthaser, a Python package leveraging the NCBI's conserved domain search tool for remote prediction and classification of fungal megasynth(et)ase domain architectures. Synthaser is capable of batch sequence analysis, and produces rich textual output and interactive visualisations which allow for quick assessment of the megasynth(et)ase diversity of a fungal genome. Synthaser uses a hierarchical rule-based classification system, which can be extensively customised by the user through a web application (http://gamcil.github.io/synthaser). We show that synthaser provides more accurate domain architecture predictions than comparable tools which rely on curated profile hidden Markov model (pHMM)-based approaches; the utilisation of the NCBI conserved domain database also allows for significantly greater flexibility compared to pHMM approaches. In addition, we demonstrate how synthaser can be applied to large scale genome mining pipelines through the construction of an Aspergillus PKS similarity network. Synthaser is an easy to use tool that represents a significant upgrade to previous domain architecture analysis tools. It is freely available under a MIT license from PyPI (https://pypi.org/project/synthaser) and GitHub (https://github.com/gamcil/synthaser).

Human and are duplicated genes that encode innate immune proteins restricting poxviruses and lentiviruses, such as HIV, and implicated in life-threatening genetic diseases and cancer. Here, we combined structural similarity searches, phylogenetics and population genomics with experimental assays of SAMD9/9L functions to resolve the evolutionary and functional dynamics of these immune proteins, spanning from prokaryotes to primates. We discovered structural analogs of SAMD9/9L in the anti-bacteriophage defense system Avs, resulting from convergent evolution. Further, the predicted nuclease active site was conserved in bacterial analogs and was essential for cell death functions, suggesting a fundamental role in defense across different life kingdoms. Despite this ancestral immunity, we identified genomic signatures of evolutionary arms-races in mammals, with remarkable gene copy number variations targeted by natural selection. We further unveiled that the absence of in bonobos corresponds to a recent gene loss still segregating in the population. Finally, we found that chimp and bonobo SAMD9Ls have enhanced anti-HIV-1 functions, and that bonobo-specific SAMD9L polymorphisms confer increased anti-HIV-1 activity to human SAMD9L without compromising its effect on cell translation. These SAMD9/9L adaptations likely resulted from strong viral selective pressures, including by primate lentiviruses, and could contribute to lentiviral resistance in bonobos. Altogether, this study elucidates the interplay between ancient immune convergence across kingdoms and species-specific adaptations within the Avs9 and SAMD9/9L antiviral shared immunity.