DOI: 10.5061/dryad.fn2z34v7t
title: Structural models of <em>Cheiracanthium</em> <em>punctorium</em> spider toxins with putative defensive function (CSTX-type and phospholipase A2)

description: Spider venom is an important but evolutionarily poorly understudied
 functional trait. In this study, we have sequenced multiple venom gland
 transcriptomes from various spiders and analyzed their venom profile. The
 transcriptomes were generated via Illumina TruSeq RNA Sample Prep Kit v2
 or TruSeq Stranded mRNA Library Prep Kit (paired-end, 151-bp read length),
 sequenced with Macrogen (Korea) using different Illumina chemistries an
 assembled de novo using a pipeline incorporating Trinity v2.13.2/2.15.1 as
 well as rnaSPAdes v3.15.5. Identified toxin precursors were annotated
 using InterProScan v5.61-93.0/5.69-101.0 and a DIAMOND v2.0.15/2.1.9
 blastp search against the public available databases VenomZone,
 UniProtKB/Swiss-Prot Tox-Prot, UniProtKB/Swiss-Prot and UniProtKB/TrEMBL
 v2022_05/2024_04 was performed. Our analysis revealed the presence of
 novel double-domain toxins from the CSTX family and some phospholipase A2
 toxins sequence-wise resembling homolgs from honeybees and scorpions,
 indicating functional similarity. In order to explore the architecture of
 the CSTX toxins and whether the observed phospholipase A2 sequence
 similarities have structural repercussions, we utilized structural
 predictions. Therefore, Alphafold 3 in the Galaxy platform was used to
 generate 3D models of the proteins. Sequences were submitted online and
 structures were predicted using default settings. The resulting model was
 downloaded and visualized using ChimeraX. The results indeed suggested a
 high structural similarity of phospholipases from spider, honeybee and
 scorpion venom which renders them as likely functionally related,
 supposedly acting in defense and illustrated a complex structure of novel
 CSTX toxins. The dataset herein presented contains the models of the
 chosen spider toxins stemming from the Nurses thorn finger (Cheiracanthium
 punctorium), as well as the most similar ones from honeybees and
 scorpions.
description: RNA extraction and sequencing were outsourced to Macrogen.
 Following RNA extraction, libraries were constructed using the Illumina
 TruSeq RNA Sample Prep Kit v2 or TruSeq Stranded mRNA Library Prep Kit
 (paired-end, 151-bp read length). Quality was controlled by the
 verification of PCR-enriched fragment sizes using an Agilent Technologies
 2100 Bioanalyzer with a DNA 1000 chip. The library quantity was determined
 by qPCR using the rapid library standard quantification solution and
 calculator (Roche). Transcriptome data were processed
 using a modified version of our in-house assembly and annotation
 pipeline<sup>62</sup>. All input sequences were inspected
 using FastQC v0.11.9/0.12.1 (www.bioinformatics.babraham.ac.uk) before trimming with cutadapt v4.2/4.9. The trimmed reads were corrected using Rcorrector v1.0.5/1.0.7<sup>79</sup> and assembled de novo using a pipeline incorporating Trinity v2.13.2/2.15.1 with a minimum contig size of 30 bp and maximum read normalization of 50 and rnaSPAdes v3.15.5 with and without error corrected reads. All contigs were combined into a single assembly, in which transcripts from all assemblers were merged if they were identical. Reads were remapped to the assembly using HISAT2 v2.2.1 and expression values (transcripts per million, TPM) were calculated using StringTie v2.2.1/2.2.2. SAM and BAM files were converted using samtools v1.16.1/1.20. Open reading frames (ORFs) were then predicted with TransDecoder v5.5.0/5.7.1 (github.com/TransDecoder/TransDecoder) with a minimum length of 10 amino acids and provided for proteome analysis. Identified toxin precursors were annotated using InterProScan v5.61-93.0/5.69-101.0 and a DIAMOND v2.0.15/2.1.9 blastp search was performed. The E-value was set to a maximum of 1 x 10^-3^ in ultra-sensitive mode with all target sequences reported (--max-target-seqs 0). We then calculated the coverage of query and subject, and the similarity with the BLOSUM62 matrix using BioPython v1.81/1.83 for each hit. Sorting by similarity, query and subject coverage for each toxin candidate led to the resulting hit for the final analysis. Precursors without a predicted signal peptide by SignalP v6.0g/h in slow–sequential mode for eukarya were removed from our dataset. Annotated precursors were aligned to known venom components from the same putative family to verify our assignments using ClustalW in Geneious v10.2.6 (www.geneious.com). Alphafold 3 in the Galaxy platform was used to generate 3D models of the proteins. Sequences were submitted online and structures were predicted using default settings. The resulting model was downloaded and visualized using ChimeraX<sup>93</sup>, retrieved models are provided in the herein presented database.
description: # Structural models of *Cheiracanthium* *punctorium* spider toxins with
 putative defensive function (CSTX-type and phospholipase A2) Dataset DOI:
 [10.5061/dryad.fn2z34v7t](10.5061/dryad.fn2z34v7t) ## Description of the
 data and file structure In order to grasp the architecture of doule-domain
 CSTX toxins and to understand whether the sequence-wise suggested
 similarity of phospholipase A~2~ toxins from the Nurses thorn finger
 (C*heiracanthium punctorium*) are reminsicent to similarities in folding,
 structure and, hence, potentially function, we employed structural
 modelling of the sequenced transcripts. We modelled selected CSTX toxins
 (CPTX1a, CPTX 2d, CPTX 5a and CPTX5d) as well asidentified phospholipsae
 A~2~ toxins from *C. punctorium* (CPTX14a) and their most similar homologs
 from bees and scorpions (P00630 and Q6T178). The models (used to generate
 figure 3 and 5 from linked publication/preprint) are created via
 alphafold3 from sequence data under default parameters. ### Files and
 variables #### File: fold_cptx_1a_model_0.cif **Description:** Model of
 CPTX1a #### File: fold_cptx_2d_model_0.cif **Description:** Model of CPTX
 2d #### File: fold_cptx_5a_model_0.cif **Description:** Model of CPTX 5a
 #### File: fold_cptx_5b_model_0.cif **Description:** Model of CPTX 5b ####
 File: fold_cptx14a_model_0.cif **Description:** Model of Chepu-PLA2-1a
 #### File: fold_P00630_PA2_model_0.cif **Description:** Model of P00630
 #### File: fold_Q6T178_PA2_model_0.cif **Description:** Model of Q6T178 ##
 Code/software The data has been visualized using Chimera X
 ([https://www.cgl.ucsf.edu/chimerax/](https://www.cgl.ucsf.edu/chimerax/))
 but can be visualized in any of the commonly used protein viewers. ##
 Access information Other publicly accessible locations of the data: * Not
 applicable. Data was derived from the following sources: * Not applicable.