Supporting Information Smith et al. 10.1073/pnas.1103501108 SI Materials and Methods I To record NaV channel currents ( Na), pipettes contained Insect Bioassays. Dorsal unpaired median (DUM) neuron cell 135 mM CsF, 1 mM MgCl2, 34 mM NaCl, 10 mM Hepes, bodies were isolated as previously described (1), with modifica- 5 mM EGTA, and 3 mM ATP-Na2 (pH 7.35). The external solu- tions. Terminal abdominal ganglia were dissected and placed in tion contained 80 mM NaCl, 5 mM CsCl, 1.8 mM CaCl2,5mM sterile Ca2þ∕Mg2þ-free normal insect saline (NIS) containing: 4-AP, 50 mM tetraethlyammonium (TEA)-Cl, 10 mM Hepes, 180 mM NaCl, 3.1 mM KCl, 10 mM Hepes, 25 mM D glucose, 0.1 mM NiCl2, 1 mM CdCl2, and 0.01 mM verapamil (pH 7.4). 50 ∕ 50μ ∕ I penicillin ( IU mL), and streptomycin ( g mL), pH 7.4. To record CaV channel currents ( Ca), pipettes contained 10 mM The ganglia were desheathed and incubated in Ca2þ∕Mg2þ-free Na acetate, 110 mM Cs acetate, 50 mM TEA bromide, 2 mM NIS containing type IA collagenase (2 mg∕mL) for 38 min. Sub- ATP-Na2, 0.5 mM BaCl2, 10 mM EGTA, and 10 mM Hepes sequently, the ganglia were rinsed three times in NIS containing (pH 7.35). The external solution contained 160 mM Na acetate, 5 mM CaCl2 and 5% vol∕vol fetal bovine serum. Single cells were 30 mM TEA Br, 3 mM BaCl2, 10 mM Hepes, and 500 nM tetro- K I I mechanically isolated by trituration in NIS containing 5 mM dotoxin (pH 7.4). To record V channel currents [ KðDRÞ, KðAÞ, ∕ I CaCl2, 4 mM MgCl2, 20 mM D glucose, and 5% vol vol fetal calf and KðCaÞ], pipettes contained 135 mM KCl, 25 mM KF, 9 mM serum. The resulting suspension was incubated overnight to allow NaCl, 1 mM MgCl2, 0.1 mM CaCl2, 3 mM ATP-Na2,1mM adherence of neurons to glass coverslips previously coated with EGTA, and 10 mM Hepes (pH 7.4). External solutions contained concanavalin-A (2 mg∕mL). Tear-shaped DUM neurons with 150 mM NaCl, 30 mM KCl, 5 mM CaCl2, 4 mM MgCl2,10mM diameters larger than 45 μm were selected for experiments. HEPES, 10 mM D glucose, and 300 nM tetrodotoxin (pH 7.4). 1. Kubista H, et al. (2007) CSTX-1, a toxin from the venom of the hunting spider Cupiennius salei, is a selective blocker of L-type calcium channels in mammalian neurons. Neuropharmacology 52:1650–1662 Signal peptide Propeptide Mature peptide Fig. S1. Manual alignments of the signal and propeptide regions of U1-LITX-Lw1a and OcyC10. Areas shaded in gray are conserved. Smith et al. www.pnas.org/cgi/doi/10.1073/pnas.1103501108 1of4 35,000 4,174.92 30,000 100 90 4,173.92 80 4,175.90 25,000 70 60 4,176.89 50 4,172.92 20,000 40 4,177.85 30 15,000 Intentisty, cps 20 10 4,171.92 (arbitrary units) 10,000 0 5,000 4,170 4,172 4,174 4,176 4,178 4,180 Mass, amu 214nm A 0 -5,000 24 25 26 27 28 29 30 31 32 33 34 Retention time (min) Fig. S2. HPLC analysis of synthetic and native U1-liotoxin-Lw1a (U1-LITX-Lw1a). Analytical reversed-phase HPLC chromatograms of synthetic oxidized U1-LITX-Lw1a (magenta), native U1-LITX-Lw1a purified from L. waigiensis venom (cyan), and a mixture of the native and synthetic peptides (black). (Inset) An electrospray MS spectrum of synthetic U1-LITX-Lw1a showing isotopic resolution of masses. The observed mass of 4,171.917 Da is consistent with the calculated mass for the fully oxidized peptide of 4,172.072 Da. Smith et al. www.pnas.org/cgi/doi/10.1073/pnas.1103501108 2of4 A B Fig. S3. Disulfide-bond connectivity of U1-LITX-Lw1a. (A) The three theoretically possible disulfide-bond connectivities for U1-LITX-Lw1a. The native peptide was found to have the connectivity shown at the top. (B) MALDI-TOF spectra of peptide fragments resulting from a tryptic digest of U1-LITX-Lw1a, with disulfide bonds intact. The numbers above the masses in the mass spectrum correspond to the peptide digestion fragment numbers in the table below. The corresponding amino acid sequence is written next to each fragment number, along with the observed and calculated masses in the table on the right. Importantly, the masses of fragment numbers five to seven are consistent with two peptide digestion products connected only by an intact disulfide bond. These peptide digestion products conclusively show the disulfide-bond arrangement. Smith et al. www.pnas.org/cgi/doi/10.1073/pnas.1103501108 3of4 Signal peptide Propeptide Mature peptide Fig. S4. Alignment of U1-LITX-Lw1a and the calcines. Disulfide pairings are indicated above the sequences with square brackets. The arginine residue essential for ryanodine receptor activity is indicated with an arrow. Sequences for the upstream regions of imperatoxin-A, maurocalcine, and hemicalcin are not available. Table S1. Statistical analysis of U1-LITX-Lw1a structures Experimental restraints* Interproton distance restraints Intraresidue 192 Sequential 264 Medium range, i − j < 5 107 Long range, i − j ≥ 5 246 Hydrogen-bond restraints† 12 Disulfide-bond restraints 6 Dihedral-angle restraints, φ, ψ, χ1 4 Total number of restraints per residue 23.1 rms deviation from mean coordinate structure, Å Backbone atoms (residues 2–36) 0.24 ± 0.08 All heavy atoms (residues 2–36) 0.67 ± 0.07 Stereochemical quality‡ Residues in most favored Ramachandran region, % 83.2 ± 2.1 Ramachandran outliers, % 0 ± 0 Unfavorable sidechain rotamers, % 9.1 ± 3.7 Clashscore, all atoms§ 11.2 ± 1.3 Overall MolProbity score 2.95 ± 0.15 All statistics are given as mean ± SD. *Only structurally relevant restraints, as defined by CYANA, are included. †Two restraints were used per hydrogen bond. ‡According to MolProbity (http://molprobity.biochem.duke.edu). §Defined as the number of steric overlaps >0.4Å per thousand atoms. Smith et al. www.pnas.org/cgi/doi/10.1073/pnas.1103501108 4of4.