cDNAs and Protein expression
Full-length arfaptin2 in the expression vector pGEX6P2 was a gift from Steve Gamblin (National Institute for Medical Research, London, UK), and the cDNA for Drosophila amphiphysin was a gift from Cahir O’Kane (Univ. of Cambridge, Cambridge, UK). cDNA for centaurinbeta2 (KIAA0041) was a gift from the Kazusa DNA research institute, Chiba, Japan. cDNAs for human BRAP1/Bin2 (5722815), mouse nadrin2 (3500822) were gifts from the I.M.A.G.E. consortium. Oligophrenin1 was cloned from a rat brain library, and amphiphysin1 (residues 1-377) and 2 (residues 1-422) were cloned from a rat brain library as previously described (S1). Derivatives and mutants were subcloned into pGEX4T1 or 4T2, expressed in E. coli BL21 cells, purified by glutathione-affinity, cleaved with thrombin, and further purified by ion exchange chromatography before use. Clathrin was purified from rat brain as described on www2.mrc-lmb.cam.ac.uk/groups/hmm/techniqs/CCVprep.htm. CD spectra were measured at 20°C in 5mM HEPES, pH 7.4, 150mM NaCl using a Jobin Yvon CD6.

Protein crystallization
Drosophila amphiphysin (residues 1-245) was expressed as an NH2-terminal GST fusion protein in minimal medium supplemented with selenomethionine. The protein was purified as above. Crystals were grown by vapour diffusion from a 2.5mg/ml protein solution in 18% PEG4000, 200mM NaCl, 1mM DTT, 20mM HEPES pH 7.4 mixed with the well solution 18% PEG 4000, 0.2M ammonium acetate, 100mM sodium citrate pH 6.0. Crystals were equilibrated in 20% glycerol for cooling to 100K. The crystal asymmetric unit contains one molecule, and the crystals belong to spacegroup P3121, cell dimensions a = b = 49.6Å, c = 190.3Å, g = 120°. A single-wavelength X-ray diffraction dataset was collected to 2.6Å resolution at the Se edge at ESRF beamline ID29 (Table S1). Images were integrated with Mosflm (S2) and scaled with Scala (S3). Two SeMet sites were located using the anomalous differences with the program Shake-and-Bake (S4). An additional two anomalous scattering sites at positions of the cysteine sulphur atoms were added during phasing by Sharp (S5). Phases were improved by solvent flattening with Solomon (S6). The model was built with O (S7) and refined with Refmac (S8).

Analytical ultracentrifugation
Sedimentation equilibrium experiments were performed in a Beckman Optima XL-A analytical ultracentrifuge with an An60-Ti rotor, in 10mM Tris Cl, pH 7.4, 200mM NaCl, 1mM TCEP. Sedimentation was at 11,000 rev/min, 20.0°C, with initial overspeeding at 18,000 rev/min for 6hr, to reduce the time to reach equilibrium (S9). Long sample columns were used, with cells loaded at a variety of initial concentrations. Scans (averaging 10 readings) were taken at 280nm at 24hr intervals, until no movement of the distribution was visible, when final scans (averaging 100 readings) were taken and assumed to be operationally at equilibrium. The rotor was then accelerated to pull the macromolecule away from the meniscus, and further scans taken to provide initial estimates of the baseline for each cell. Data were analyzed as described in detail in the supplementary information to (S10).

Sequence alignments
We first obtained a general BAR sequence alignment by overlaying the Drosophila amphiphysin and arfaptin2 BAR structures (see Figs. 1E and S5). Candidate BAR domain proteins were identified using repeated iterations of Psi-BLAST (www.ncbi.nlm.nih.gov/BLAST/) against the arfaptin and amphiphysin BAR domains. The BAR domains in these proteins were tested by aligning them with the Drosophila amphiphysin and arfaptin BAR domains using the clustalW function in MacVector, with an open gap penalty of 100, extend gap penalty of 0.5 and delay divergent setting of 40%. Generally when the third protein led to a correct alignment of the amphiphysin and arfaptin BARs, some charge conservation and a repeat of hydrophobic residues (green in Fig 1E) could be detected in the third BAR. Finally, candidate BARs were checked for a-helical content using Coils and Multicoil tools available at http://us.expasy.org/tools/. There should be caution in simple predictions of BAR domains given that a-helical repeats and coiled-coils are common in proteins.

Liposome sedimentation, tubulation, and lipid monolayer assays
Liposomes consisting either of 40% phosphatidylcholine, 40% phosphatidylethanolamine, 10% cholesterol, and 10% PtdIns(4,5)P2 (Avanti Polar Lipids), total bovine brain lipids (Folch fraction 1, sigma B1502; referred to in text as brain liposomes) or total liver lipids (Avanti Polar Lipids) were resuspended at 1mg/ml in 20mM HEPES, pH 7.4, 150mM NaCl (200mM NaCl for Drosophila amphiphysin), 1mM DTT and sized by extrusion (S11). For sedimentation assays, 5mM protein was incubated with 0.6mg/ml liposomes in 100 ml of buffer for several minutes before sedimentation at 140000xg for 15 minutes in a Beckman TLA100 rotor. After spinning, supernatants were removed immediately, and pellets were resuspended in an equal volume of buffer. Proteins were subjected to SDS-PAGE, visualized by Coomassie stain, photographed with a Bio-Rad XRS system, and quantified using ImageQuant (Molecular Dynamics). For tubulation assays, proteins were incubated as above, spread on glow-discharged carbon-coated grids and negatively stained with 5% uranyl acetate. Mutations tested are described in the text, with the exception of arginines 65 and 68 in Drosophila amphiphysin, which were also mutated to glutamate but had no effect on binding or tubulation. Clathrin recruitment and lipid monolayer assays were performed at 20°C in 25mM HEPES pH 7.4, 125mM potassium acetate, 5mM magnesium acetate, 1mM DTT. Monolayers composed of either brain lipids or synthetic lipid mixes (which gave identical results) were formed on a 40ml buffer drop in a Teflon well. A carbon-coated grid was placed on the droplet, proteins were injected underneath, and after 60min the grid was removed and negatively stained. A schematic of the procedure and a detailed protocol can be found on www2.mrc-lmb.cam.ac.uk/groups/hmm/techniqs/techniqs.htm.

Transfections and immunofluorescence
COS-7 cells were transfected with pCMV-myc vectors using GeneJuiceTM (Novagen) according to the manufacturer's protocol. 28 h after transfection, cells were transferred to serum-free medium and incubated with biotinylated transferrin for 15 min before fixation with 4% PFA. Transfected cells were detected using a polyclonal anti-Myc tag antibody (Cell Signalling), biotinylated transferrin was detected with labelled avidin, amphiphysin1 and arfaptin were detected with rabbit antisera raised against purified proteins, and TGN46 was detected with an anti-TGN46 antibody (Serotech). Cells were mounted and imaged using a Radiance confocal system (Bio-Rad Laboratories). It should be noted that the amphiphysin constructs are not NH2-terminally tagged as this prevents the tubulation in vivo (Fig. S3A), although in vitro an NH2-terminal histidine- or glutathione-S-transferase-tag does not prevent tubulation.


Supplemental references and notes (click link for .pdf)
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