Additional file 2 of Putative ligand binding sites of two functionally characterized bark beetle odorant receptors
Creators: name: Yuvaraj, Jothi K.
name: Roberts, Rebecca E.
name: Sonntag, Yonathan
name: Hou, Xiao-Qing
name: Grosse-Wilde, Ewald
name: Machara, Aleš
name: Zhang, Dan-Dan
name: Hansson, Bill S.
name: Johanson, Urban
name: Löfstedt, Christer
name: Andersson, Martin N.
Publisher: name: figshare
Year: 2021
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 2: Supplementary Table 2. Compounds used for characterization of Ips typographus odorant receptors (ORs) and Orco, including their purities, source information, and examples of main biological origins. Supplementary Table 3. Assessment of the completeness of antennal transcriptome Trinity and CLC assemblies using the Benchmarking Universal Single-Copy Orthologs (BUSCOv3) tool performed against the Insecta odb9 dataset ( https://busco.ezlab.org/ ).Supplementary Figure 1. Protein detection of Ips typographus odorant receptors (ORs; V5-tagged) and Orco (myc-tagged) from TREx/HEK293 cells by Western blot. Upper left panel: Detection of wildtype ItypOR46 and ItypOR49 (two cell lines). Upper right panel: detection of Orco in the same cell lines. Lower panel: detection of three versions of mutated ItypOR46 proteins and wildtype (WT) ItypOR46 (included as control). Proteins were only detected from cells induced (+) to express ItypORs and Orco, and not from non-induced (−) control cells, indicating proper regulation by the repression system. Supplementary Figure 2. Response of TREx/HEK293 cells expressing ItypOR46 and ItypOrco to all stimuli (30 μM) and vehicle control in the screening experiment (n = 3 biological replicates, each including 3 technical replicates, i.e., ntotal = 9). (+)-Induction: response of cells induced to express ItypOrco and ItypOR46; (−)-Induction: response of non-induced control cells. Data represent mean responses ± SEM. Supplementary Figure 3. Response of TREx/HEK293 cells expressing ItypOR49 and ItypOrco to all stimuli (30 μM) and vehicle control in the screening experiment (n = 3 biological replicates, each including 3 technical replicates, i.e., ntotal = 9). (+)-Induction: response of cells induced to express ItypOrco and ItypOR49; (−)-Induction: response of non-induced control cells. Data represent mean responses ± SEM. Supplementary Figure 4. A) Current traces of two oocytes expressing ItypOrco/ItypOR49, indicating responses to the Orco agonist VUAA1 and minute responses to racemic ipsdienol. B) Current trace of oocyte expressing myc-tagged ItypOrco and V5-tagged ItypOR46 showing the same specificity as non-tagged versions of these receptors in this system. Supplementary Figure 5. Energy minimized model of ItypOR49 (blue cartoon) showing the extended extracellular loop 2 (EL2) overlaid with the published Orco Cryo-EM map (mesh). Homology models of ItypOR46 and ItypOR49 were built using the Orco structure (PDB ID 6c70) as template. The part of EL2 that was not included in the published structure (residues 156–170) was modeled ab initio. An extended β-sheet fold in this part of the loop is supported by the density in the Cryo-EM map. In neither of the models the putative binding cleft is blocked by this part of EL2. The RMSDs between the Cα of the template and energy minimized models for ItypOR46 and ItypOR49 (excluding the ab initio modeled parts) are 0.958 Å and 0.880 Å, respectively. Supplementary Figure 6. Chemical structures and IUPAC names of compounds included in the molecular docking analyses against ItypOR46 and ItypOR49. Supplementary Figure 7. Response of TREx/HEK293 cells expressing A) ItypOR46Tyr84Phe, B) ItypOR46Tyr84Ala, and C) ItypOR46Thr205Ala and ItypOrco to all stimuli (30 μM) and vehicle control in the screening experiment (n = 2 biological replicates, each including 3 technical replicates, i.e., ntotal = 6). (+)-Induction: response of cells induced to express ItypOrco and the mutated ItypOR46; (−)-Induction: response of non-induced control cells. Data represent mean responses ± SEM. Asterisks (* p &lt; 0.05; ** p &lt; 0.01) in B) indicate significant differences between induced and non-induced cells. Supplementary Methods. Description of the chemical synthesis of ipsenol and ipsdienol as well as their pure enantiomers.