SUPPLEMENTARY INFORMATION

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1 doi: /nature06147 SUPPLEMENTARY INFORMATION Figure S1 The genomic and domain structure of Dscam. The Dscam gene comprises 24 exons, encoding a signal peptide (SP), 10 IgSF domains, 6 fibronectin type III (FnIII) domains, a transmembrane segment (TM) and a cytoplasmic domain. Exon-4, -6, -9, and -17 have 12, 48, 33 and 2 alternatives, respectively. The splicing process at the RNA level generates 38,016 possible protein isoforms that differ in sequence in D2, D3 and D7 domains, as well as in two distinct transmembrane segments. The part of the Dscam molecule that is covered by the X-ray structure is indicated by a red bar at the genomic DNA and mrna level, and a red box at the protein level. Figure S2 Electron microscopy of the Dscam D1-D8 construct. (a) Raw image of negatively stained particles of the D1-D8 construct. (b) Representative class averages show that the molecule can 1

2 adopt different conformations but retains the horse-shoe configuration of the N-terminal D1-D4 domains. Scale bars in a and b correspond to 50 nm and 10 nm, respectively. Figure S3 Raw image from an electron micrograph of negatively stained Dscam D1-D molecules. The arrows point at individual particles absorbed on a carbon layered grid. The scale bar corresponds to 25 nm. 2

3 Figure S4 Structure-based sequence alignment of D1-D41.34 and D1-D49.9. Each domain is designated, and for D2 and D3, the sequences for both D1-D41.34 and D1-D49.9 are depicted. Secondary structure elements are shown as arrows (b strands) and coils (a helices) for D1-D Residues that are situated on exons 4 and 6 are colored, so that conserved residues are boxed in cyan, variable residues are colored green and hypervariable residues are colored red following the classification of Figure 1c.

4 doi: /nature06147 SUPPLEMENTARY INFORMATION Figure S5: The intra-molecular domain interfaces of (a) D2/D3 and (b) D1/D4. In a, only residues contributing to hydrogen bonds in the interface are depicted, and colored cyan when conserved and green when variable. The interface of axonin is superimposed in b, and residues contributed to the interface are colored cyan when belonging to axonin and red when belonging to Dscam. Figure S6: Superposition of Dscam with axonin and hemolin. Ca carbon trace of Dscam (red), with axonin (green) and hemolin (cyan) superimposed using secondary structure elements (Krissinel E, Henrick K (2004). Acta Crystallogr. D60, ). 4

5 Figure S7: Unique structural elements coincide with the most variable regions of exon-4 and exon-6. The C-terminal part of exon-4 is shown for D1-D in a and D1-D4 9.9 in b. Residues are colored according to the color code as described in Figure 1, and strands are marked. Hydrogen bonds that contribute to the D 0 configuration in a are drawn in grey, and the residues involved in the tyrosine corner (sidechain of Tyr154 and mainchain of Phe149, as well as a water molecule) are indicated. The insertion of two residues in b leads to a bulge in the C -D 0 unit that is stabilized by the sidechain of Leu161. The N- terminal part of exon-6 is shown for D1-D in c. and D1-D4 9.9 in d. 5

6 Figure S8: Mutations in epitope I but not epitope II disrupt homophilic binding. Binding of wild type Dscam (right panel) and 8 different constructs bearing mutations in either epitope I (left panel, magenta frame) or epitope II (blue frame) to Cos cells expressing wild type Dscam isoform (in all panels). Red fluorescent beads were either coated with recombinant wild type isoform (wt), with wild type isoform or with mutant isoforms. Note, additional controls also tested the inverse configurations with mutant receptor forms expressed in COS cells and beads coated with wild type protein (Supplemental figure 9). Between 1 and 4 point mutations were introduced into D2 sequences (encoded by exon 4) and D3 sequences (encoded by exon 6) of the full-length extracellular domain of Dscam. Quantification of all binding experiments is given in figure 3, main text. 6

7 Figure S9: Surface expression of mutant Dscam isoforms in transfected COS cells is normal. a) Cos cells were co-transfected with GFP and either wild-type Dscam (grey box), epitope II mutant Dscam (blue box) or epitope I mutant Dscam (magenta box). Transfected cells were incubated with red fluorescent beads coated with wild-type Dscam isoform Only cells transfected with wild-type and epitope II mutant isoforms bound beads efficiently (left side panels). Cells transfected with the epitope I mutant V218P show some binding (arrow), but overall significantly less than a wildtype isoform (see figure 3). B) Surface expression of Dscam isoforms was tested by co-transfecting COS cells with GFP and different Dscam constructs (GFP: green, as in a, above). Cells were fixed and stained with a rabbit anti-dscam antibody (secondary antibody anti- Rabbit Cy3: red) directed against the extracellular Ig-domains D1-D4 of Dscam. The top row panels show the two-channel overlay of GFP and Cy3 signals, the bottom row shows the Dscam expression only. Dscam is expressed throughout the surface of the Cos cells and in cell processes. Untransfected cells or the secondary antibody alone show no staining (not shown). 7

8 Figure S10 (Following pages): Sequence Logos for Exon-6 orthologues. Only D. melanogaster isoforms for which at least 8 other orthologues could be found are shown. Note that the inclusion of the small sample correction results in different maximal bit scores for different logos depending on the number of incorporated species. 8

9 Exon 6.01 Exon 6.02 Exon 6.03 Exon 6.05 Exon 6.06 Exon

10 Exon 6.08 Exon 6.09 Exon 6.10 Exon 6.12 Exon 6.13 Exon 6.14 Exon

11 Exon 6.16 Exon 6.17 Exon 6.18 Exon 6.19 Exon 6.21 Exon 6.22 Exon

12 Exon 6.24 Exon 6.25 Exon 6.26 Exon 6.27 Exon 6.28 Exon 6.29 Exon

13 Exon 6.31 Exon 6.32 Exon 6.33 Exon 6.34 Exon 6.35 Exon 6.36 Exon

14 Exon 6.40 Exon 6.41 Exon 6.44 Exon 6.45 Exon 6.46 Exon 6.47 Exon

15 Figure S11: Multiple sequence alignment of orthologues of exon-4.01 of D. melanogaster. Residues that form intermolecular hydrogen bonds in the dimer (see figure 3c) are shown in red. Conserved amino acids are shaded dark. Similar amino acids are shaded light. The sequence for Anopheles gambiae was obtained using Flyblast ( and Aedes aegypti from the Broad institute database ( = 15

16 Figure S12 (Following pages): Sequence logos of all exon-4 orthologues. For each isoform of Drosophila melanogaster, exon-4 orthologues from 12 species of the Drosophila subgroup were aligned and sequence logos were generated with Weblogo. The maximum bit score in each logo indicates a conserved residue in all sequences. 16

17 Exon 4.01 Exon 4.02 Exon 4.03 Exon 4.04 Exon 4.05 Exon

18 Exon 4.07 Exon 4.08 Exon 4.09 Exon 4.10 Exon 4.11 Exon

19 Table S1: Data collection, phasing and refinement statistics for Dscam D1-D and D1-D D1-D D1-D D1-D4 9.9 K 2 PtCl 4 derivative Data collection Space group P C222 1 C2 I Cell dimensions a, b, c (Å) 99.2, 99.2, 99.8, 166.8, 277.8, 70.5, 146.7, 146.7, α, β, γ (º) 90.0, 90.0, , 90.0, , 105.1, , 90.0, 90.0, Peak Inflection Remote Wavelength (Å) Resolution (Å) R sym or R merge (0.85) (0.23) 0.11 (0.26) 0.11 (0.26) 0.15 (0.52) (0.51)* I/σI 23.4 (1.9) 13.3 (2.0) 10.9 (2.9) 38.6 (15.0) 41.5 (13.9) 28.3 (7.5) Completeness (%) 97.8 (91.8) 96.2 (93.7) 86.0 (55.0) 99.9 (100.0) 99.8 (100.0) 99.8 (100.0) Redundancy Refinement Resolution (Å) No. reflections R work/ R free 0.173/ / /0.303 No. atoms Protein Ligand/ion Water B-factors Protein Ligand/ion Water R.m.s deviations Bond lengths (Å) Bond angles (º) *Highest resolution shell is shown in parenthesis. 19