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1 Generation of protein lattices by fusing proteins with matching rotational symmetry John C. Sinclair, Karen M. Davies, Catherine Vénien-Bryan, Martin E. M. Noble Supplementary Information Protein Sequences Linkers are in blue type, underlined. Peptide fusions are in red type, bold. The Calmodulin N-terminal fusion is shown in green. DsRed-Express-Streptag I MTMITPSLHACRSTLEDPRVPVATMASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEG RPYEGTQTAKLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWE RVMNFEDGGVVTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPR DGVLKGEIHKALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVE QYERAEGRHHLFLRSAWRHPQFGG ALAD-Streptag I MTMGSHHHHHHPLIPMTDLIQRPRRLRKSPALRAMFEETTLSLNDLVLPIFVEEEIDDY KAVEAMPGVMRIPEKHLAREIERIANAGIRSVMTFGISHHTDETGSDAWREDGLVARMS RICKQTVPEMIVMSDTCFCEYTSHGHCGVLCEHGVDNDATLENLGKQAVVAAAAGADFI APSAAMDGQVQAIRQALDAAGFKDTAIMSYSTKFASSFYGPFREAAGSALKGDRKSYQM NPMNRREAIRESLLDEAQGANCLMVKPAGAYLDIVRELRERTELPIGAYQVSGEYAMIK FAALAGAIDEEKVVLESLGSIKRAGADLIFSYFALDLAEKKILRRSAWRHPQFGG ALAD-Lac21E MTMGSHHHHHHPLIPMTDLIQRPRRLRKSPALRAMFEETTLSLNDLVLPIFVEEEIDDY KAVEAMPGVMRIPEKHLAREIERIANAGIRSVMTFGISHHTDETGSDAWREDGLVARMS RICKQTVPEMIVMSDTCFCEYTSHGHCGVLCEHGVDNDATLENLGKQAVVAAAAGADFI APSAAMDGQVQAIRQALDAAGFKDTAIMSYSTKFASSFYGPFREAAGSALKGDRKSYQM NPMNRREAIRESLLDEAQGANCLMVKPAGAYLDIVRELRERTELPIGAYQVSGEYAMIK FAALAGAIDEEKVVLESLGSIKRAGADLIFSYFALDLAEKKILRRSMEELADSLEELAR QVEELESA ALAD-Lac21K MTMGSHHHHHHPLIPMTDLIQRPRRLRKSPALRAMFEETTLSLNDLVLPIFVEEEIDDY KAVEAMPGVMRIPEKHLAREIERIANAGIRSVMTFGISHHTDETGSDAWREDGLVARMS RICKQTVPEMIVMSDTCFCEYTSHGHCGVLCEHGVDNDATLENLGKQAVVAAAAGADFI APSAAMDGQVQAIRQALDAAGFKDTAIMSYSTKFASSFYGPFREAAGSALKGDRKSYQM NATURE NANOTECHNOLOGY 1

2 NPMNRREAIRESLLDEAQGANCLMVKPAGAYLDIVRELRERTELPIGAYQVSGEYAMIK FAALAGAIDEEKVVLESLGSIKRAGADLIFSYFALDLAEKKILRRSMKKLADSLKKLAR QVKKLESA SHS-Streptag I MTMITPSLFERMFKEFFATPMTGTTMIQSSTGIQISGKGFMPISIIEGDQHIKVIAWLP GVNKEDIILNAVGDTLEIRAKRSPLMITESERIIYSEIPEEEEIYRTIKLPATVKEENA SAKFENGVLSVILPKAESSIKKGINIEGSAWRHPQFGG Calmodulin-ALAD-Streptag I MTMGSADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINE VDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLG EKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAKPLIPMTDLIQRPRRLRKSPALRAM FEETTLSLNDLVLPIFVEEEIDDYKAVEAMPGVMRIPEKHLAREIERIANAGIRSVMTF GISHHTDETGSDAWREDGLVARMSRICKQTVPEMIVMSDTCFCEYTSHGHCGVLCEHGV DNDATLENLGKQAVVAAAAGADFIAPSAAMDGQVQAIRQALDAAGFKDTAIMSYSTKFA SSFYGPFREAAGSALKGDRKSYQMNPMNRREAIRESLLDEAQGANCLMVKPAGAYLDIV RELRERTELPIGAYQVSGEYAMIKFAALAGAIDEEKVVLESLGSIKRAGADLIFSYFAL DLAEKKILRRSAWRHPQFGG 2 NATURE NANOTECHNOLOGY

3 Image analysis The image analysis presented in Figure 3 was performed using programs distributed with the 2dx suite. The CTF was fitted using the program CTFFIND3 and corresponded to a defocus of 711 nm, with an astigmatism of 115nm. The diffraction lattice was determined using the Get Lattice option within 2dx, and was refined using MMLATREF. Unbending was performed using CCUNBEND. Following CTF correction using 2dx_ctfapplyk (phase flipping only), diffraction spot IQ values are consistent with diffraction beyond 18 Å resolution (Figure S1A,B). Analysis of phase residuals using the program ALLSPACE confirmed the assignment of space group P422, which yielded a phase residual of 5.3 o on 184 spots in the Fourier Transform, compared with a target value of 7.5 for this space group. This phase residual is likely to derive from a small tilt (~0.57 o ) in the lattice, as determined from the variation of defocus across the sample using the program CTFTILT. A comparison of the unsymmetrized (P1) and symmetrised (P422) maps is presented in figure S2. Figure S1. IQ plots of reflections used in the image reconstruction in Figure 3. A) IQ plot after CTF correction (no resolution limit applied). Rings correspond to zero-values in the CTF. B) Resolution plot from the measured lattice. Rings are drawn at resolutions of 36, 24, 18 and 15 Å. NATURE NANOTECHNOLOGY 3

4 Figure S2. Comparison of A) unsymmetrised and B) symmetrized (P422) backprojections. 4 NATURE NANOTECHNOLOGY

5 Atomic Force Microscopy An ALAD-Streptag I/streptavidin 2D crysalin was captured on a carbon-coated copper electron microscopy grid and fixed with uranyl acetate, as described for preparing samples for transmission electron microscopy. The grid was subsequently imaged using a JPK Nanowizard II AFM system. Crysalin lattices are clearly visible beneath the layer of inorganic stain. A B Figure S3. AFM images showing the height (A) and lateral deflection (B) recorded for regions containing ALAD-Streptag I/streptavidin crysalins. NATURE NANOTECHNOLOGY 5

6 Incorporation of a genetically fused calmodulin moiety into a 2D crysalin lattice. 7nm Figure S4. Crysalin lattice incorporating a genetically fused calmodulin moiety. The ALAD-Streptag I construct presented in Figure 2 was expressed with an N-terminally fused calmodulin domain. The purified calmodulin-alad-streptag I component was mixed with streptavidin and prepared for transmission electron microscopy under identical conditions to those used for the parent ALAD-Streptag I/streptavidin crysalin. Crystalline patches of lattice were observed with a size and apparent regularity comparable with that of the parent lattice. 6 NATURE NANOTECHNOLOGY

7 3D crysalins Our preliminary designs for self-assembling binary 3D lattices have produced particles that assemble according to the crysalin design principle. Figure S5 illustrates pseudocrystals that form when SHS-Streptag I fusion protein is mixed with streptavidin under appropriate conditions. Materials formed in this way are sensitive to trace amounts of desthiobiotin. This sensitivity to a highly specific inhibitor of a protein:protein interaction demonstrates not only that both components are present, but also that their association is mediated through the intended interface. However, we have yet to observe X-ray diffraction from any of our 3D crysalin systems. In other self-assembling protein systems, the development of long-range order has been hindered by the proliferation of poorly ordered, kinetically trapped states (e.g. Teixeira, L.M. et al., Entropically driven selfassembly of Lysinibacillus sphaericus S-layer proteins analyzed under various environmental conditions. Macromol Biosci 10 (2), (2009)). We are therefore exploring the use of thermal and chemical annealing strategies to promote the growth of diffraction quality crystals. Figure S5. Pseudo-crystals produced by mixing two components (SHS-Streptag I and streptavidin) in the presence of urea. The urea concentration was reduced over time from 3M to 1.8M by controlled vapour diffusion. NATURE NANOTECHNOLOGY 7