Materials and Methods

IgV- and TCRV-Region Gene Sequences
A database of nucleic acid sequences for germline-encoded CDR1 and 2 and frameworks 1, 2, and 3 of functional Ig V-regions was extracted from www.ncbi.nlm.nih.gov/projects/igblast and compiled as described (2). All available mouse and human V genes were used in the analyses. Framework regions (FRs) and CDR sequences were defined using the Kabat and/or IMGT definitions as indicated in the text or figure legends (20–22). The framework regions (FRs) 1–3 or CDR1 and 2 sequences were fused to form a continuous sequence, and codon frequencies were calculated by the function provided at http://www.kazusa.or.jp/codon/. This approach was made possible by the fact that CDR and FR definitions begin and end with intact codons. IgVH genes from cartilaginous fishes were extracted from http://www.imgt.org/. All 12 functional genomic DNA sequences available at the time of the analyses were used to determine the average observed/expected ratios of AGY and TCN Ser codons among germline-encoded CDRs. The following sequences were used in the analyses: Ginglymostoma cirratum (IGHV2S1*01, IGHV2S2*01, IGHV2S3*01, and IGHV2S4*01), Heterodontus francisci (IGHV1S1*01, IGHV1S15*01, IGHV1S3*01, IGHV1S4*01), Leucoraja erinacea (IGHV1S3*01, IGHV1S4*01, and IGHV1S5*01), and Hydrolagus colliei (IGHV1S3*01). Nucleotide sequences encoding mouse TCRV-region CDRs (IMGT definition) were also extracted from functional V genes at http://www.imgt.org/ (20). In cases where a V gene had multiple alleles, the first listed allele was analyzed.

Sequence Analyses
Observed over expected ratios were calculated by dividing the codon observed frequency (described above) by the expected frequency obtained from the codon use table for the species at http://www.kazusa.or.jp/codon/. Reading frame frequencies for CDR AGY triplets were determined manually, with the provision that any non-Ser AGY triplet that overlapped a FR–CDR boundary was conservatively included in the corresponding non-coding CDR Ser reading frame.

Antiviral Antibody Sequences
Sequences of antiviral Abs were obtained from http://www.ncbi.nlm.nih.gov/nuccore/. The influenza antibody sequences were originally described by Wrammert et al. (23) and Li et al. (24). The search criteria used for the other antiviral Abs were “virus AND antibody AND Homo sapiens AND range: 300–800 bp” using the nucleotide database at PubMed. Sequences were chosen based on their order of appearance. The GI numbers for analyzed sequences are: Rhinovirus: 475389817, 475389820, 475389822, 475389827, 475389830, 475389834, 475389838, 475389842, 475389846, 475389853, 475389856, and 475389859. Avian Influenza: 269273439, 269273440, 269273441, 269273442, 269273443, 269273444, 269273448, 269273449, 269273450, 269273451, 269273452, 226894290, 226894291, 226894292, 226894293, 226894294, 226894295, 226894299, 226894300, 226894301, 226894302, 226894303, 311361464, 311361465, 311361466, 311361467, 311361468, and 311361469. West Nile: 207046350, 207046351, 207046352, 207046353, 207046354, and 207046355. Dengue: 46009632727, 46009632730, 46009632735, and 46009632737. Hepatitis A, B, and C: 7012696, 7012699, 7012701, 7012704, 7012706, 7012709, 18042112, 18042114, 18042116, 18042118, 4837672, 4837674, 4837676, 4837678, 4837680, 4837682, 4837684, 4837686, 4837688, 4837690, 4837692, 4837694, 4837696, 4837698, 29650296, 29650298, 29650303, 29650314, 29650328, 29650334, 29650337, 29650339, 76443955, 76443957, 76443959, 76443961, 76443963, 2578112092, 2578112094, 2578112096, 2578112098, 184921, 184922, 184923, 184924, 186113, 186114, 185815, 185816, 809552, 809550, 809551, 809553, 809554, 3928209, 1657318, 1657324, 1657320, 1657326, 1657322, and 1657328. Sequences were aligned using http://www.ncbi.nlm.nih.gov/igblast/, and missense mutations were determined by alignment against the closest predicted germline IgV-region gene.

Immune Complex Crystal Structures
Structures of Ab–Ag complexes were acquired from the database at http://www.rcsb.org/pdb/home/home.do. The search criterion used was “antibody–antigen,” and the inclusion criterion was that the Ag had to be proteinaceous. Only Ab sequences from human or mouse were analyzed. Sequences were downloaded based on their order of appearance in the RCSB protein data bank (pdb) database. Duplicate structures were excluded from analysis. Contact residues between Ab and Ag were calculated using ncont from the CCP4 program suite with an atom to atom cutoff distance of 4 Å (25). When calculating, Ag contacts, the complete Ab heavy and light chain sequences (as written in the individual pdb files) along with the complete Ag sequence were used in the search parameter. The heavy and light chain search sequences were restricted to amino acid side-chain atoms only while contact residues in the Ag were not restricted. If more than one Ab–Ag complex was present in the asymmetric unit, only one complex was included in the analysis. A total of 46 M. musculus and 26 H. sapiens immune complexes crystal structures were analyzed and the pdb files for these are 4ot1, 4rrp, 4tsc, 4v1d, 4xak, 4xvu, 4zs6, 5c0s, 2dd8, 3gbn, 3lzf, 3sdy, 4fqi, 4hkx, 4m5z, 4o58, 4py8, 4r8w, 4xnm, 4yjz, 5a3i, 5dum, 4dgv, 4mwf, 4n0y, 4uta, 1eo8, 1nca, 1nma, 1qfu, 2aep, 2b2x, 2xqy, 2nr6, 2ypv, 3gi9, 3hb3, 3i50, 3mj9, 3o0r, 3rv, 3v7a, 3wfb, 3wfc, 4aei, 4cad, 4etg, 4ffv, 4gag, 4gms, 4hlz_2, 4k2u, 4lqf, 4u0r, 4m1g, 4m48, 4mhh, 4n8c, 4oii, 4okv, 4plj, 4qnp, 4qww, 4rgn, 4rgo, 4tuk, 4u6h, 4xpa, 5c0n, 5dj8, 5dlm, and 5en2.

Statistical Analysis
Statistical analyses were performed using GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego, CA, USA, www.graphpad.com.

Box Plot-Whiskers Graphs
Box plots with notches were created using the web tool at http://boxplot.tyerslab.com/. Center line shows the median; box limit indicates the 25th and 75th percentiles as determined by R software; whiskers extend to minimum and maximum of the values; crosses indicate sample means (26, 27). The notches are defined as ±1.58× interquartile range per square root (n) and represent the 95% confidence interval for each median.