RESEARCH LETTER METHODS ding to sta The protn-pre Alhediag For the both 100 e ,pH5.0.26 n data sets for B tained with the syn 心C th the re d scal )A e nce y Ex ted usi odata collected at led in res ge (G-V)c e ca ing()c ted fre ted and KA) 62 31.Ga nhibitio 18190 32 re gen 34 ctondatacoletedtn =80.200m2C phy.Acta 38 e)in 10mM Tris-HCl 39. 2eioninhcpcakiactioaswcrecoacemtnted 白拉部宽 e)(http:// pymol.org) 2002 2016 Macmillan Publishers Limited part of Springer Nature.All tiehts reserved RESEARCH Letter Methods CaVAb constructs and drugs. As originally defined, CaVAb was constructed by introducing the mutations E177D, S178D and M181D as a triple mutant into NaVAb19. This construct was used for all electrophysiological studies, except as noted in figure legends. In this work, we have also used CaVAb E177D S178D M181N, which has an identical structure and Ca2+-binding properties and has high Ca2+ selectivity19. It gives greater consistency of high-resolution crystal structures. We have also added the mutation W195Y, which substitutes the Y residue from the analogous position in mammalian CaV1.1 channels for W195 in CaVAb. This mutant gives better resolution of drugs bound at the dihydropyridine site. CaVAb E177D S178D M181N W195Y was used for all structural studies presented here, except as noted in the figure legends. Similar structural results were obtained for both versions of CaVAb. We found that amlodipine and other dihydropyridines (Figs. 1 and 2 and Extended Data Figs 1, 3 and 4) and verapamil and other pheny￾lalkylamines (Fig. 4 and Extended Data Fig. 6) effectively blocked CaVAb and gave high-resolution crystal structures; however, we were unable to prepare crystals with bound diltiazem for structural biology so we have not addressed the structure of the benzothiazepine receptor site in this work. Electrophysiology. All measurements were done in insect cells (Trichoplusia ni cells; High5). All CaVAb constructs used were made on the background of N49K mutation. Mutation N49K shifts the activation curve ~75mV to more positive potentials compared to wild-type CaVAb and abolishes the use-dependent inactiva￾tion as described previously19,31. All constructs showed good expression, allowing measurement of ionic currents 24–48h after infection. Whole-cell Ba2+ currents were recorded using an Axopatch 200 amplifier (Molecular Devices) with glass micropipettes (2–4MΩ). Capacitance was subtracted and 80–90% of series resist￾ance was compensated using internal amplifier circuitry. Extracellular solution contained in (mM) 10BaCl2, 140NMDG-methanesulphonate, 20HEPES, (pH7.4, adjusted with Ba(OH)2, [Ba2+]total=13mM). Intracellular solution contained in (mM) 105 CsF, 35 NaCl, 10 HEPES, 10 EGTA, (pH7.4, adjusted with CsOH). Current–voltage (I–V) relationships were recorded in response to steps to voltages ranging from -120 to +50mV in 10-mV increments from a holding potential of −120mV. Conductance-voltage (G–V) curves were calculated from the corre￾sponding (I–V) curves. Pulses were generated and currents were recorded using Pulse software controlling an Instrutech ITC18 interface (HEKA). Data were ana￾lysed using Igor Pro 6.2 (WaveMetrics). Sample sizes were chosen to give s.e.m. values of less than 10% of peak values based on prior experimental experience. Inhibition curves were fit with a Hill equation with nH=1.0 unless indicated oth￾erwise in the figure legends. Protein expression and purification. The pFastBac-Flag-CaVAb was used as the construct for producing homotetrameric model voltage-gated Ca2+ channel19. I199S, W195Y, and T206S constructs were generated via site-directed mutagen￾esis using QuickChange (Stratagene). Recombinant baculovirus were produced using the Bac-to-Bac system (Invitrogen), and T. ni insect cells were infected for large-scale protein purification. Cells were harvested 72h post-infection and re￾suspended in buffer A (50mM Tris-HCl, pH=8.0, 200mM NaCl) supplemented with protease inhibitors and DNase. After sonication, digitonin (EMD Biosciences) was added to 1%, and solubilization was carried out for 1–2h at 4 °C. Clarified supernatant was then incubated with anti-Flag M2-agarose resin (Sigma) for 1–2h at 4 °C with gentle mixing. Flag-resin was washed with ten column volumes of buffer B (buffer A supplemented with 0.12% digitonin) and eluted with buffer B supplemented with 0.1mgml−1 Flag peptide. The eluant was concentrated and then passed over a Superdex 200 column (GE Healthcare) in 10mM Tris-HCl pH= 8.0, 100mM NaCl and 0.12% digitonin. The peak fractions were concentrated using a Vivaspin 30K centrifugal device. Crystallization and data collection. CaVAb and the W195Y mutant were concen￾trated to ~20mgml−1 and reconstituted into DMPC:CHAPSO (Anatrace) bicelles according to standard protocols16,17,32,33. The protein-bicelle preparation and a well solution containing 1.8–2.0M ammonium sulphate, 100mM Na-citrate pH=5.0 was mixed in 1:1 ratio and set up in a hanging-drop vapour-diffusion format. All the antagonist complex crystals were obtained through co-crystallization by incubating the protein-bicelle with 100μM antagonist overnight before setting up crystallization trials. For the UK-59811 complex, both 100μM and 200μM antagonist were used for UK-59811/CaVAb crystals. Crystals were cryoprotected by soaking in 0.1M Na-acetate, pH 5.0, 26% glucose, 2.0M ammonium sulphate, and 5mM Ca2+. Crystals were plunged into liquid nitrogen and maintained at 100K during all data collection procedures. The anomalous diffraction data sets for Br were collected at 0.9194Å, and the anomalous data sets for Ca2+ were collected at 1.75Å with the same synchrotron radiation source (Advanced Light Source, BL8.2.1). To optimize the anomalous scattering signal, the data sets were collected by using the ‘inverse beam strategy’ with the wedge size of 5°. Structure determination, refinement, and analysis. X-ray diffraction data were integrated and scaled with the HKL2000 package34 and further processed with the CCP4 package35. The structure of CaVAb and its antagonist complex were solved by molecular replacement using an individual subunit of the CaVAb struc￾ture (PDB code 4MS2) as the search template. The data sets were processed in P21221 space group, in which there are four molecules in one asymmetric unit. Crystallography and NMR System software36 were used for refinement of coordi￾nates and B-factors. Final models were obtained after several cycles of refinement with REFMAC37 and PHENIX38 plus manual re-building using COOT39. The geometries of the final structural models of CaVAb and its antagonist complexes were verified using PROCHECK40. Divalent cations were identified by anoma￾lous difference Fourier maps calculated using data collected at wavelengths of 1.75Å for Ca2+. The Br atoms of UK-59811 and Br-verapamil were identified by anomalous difference Fourier maps calculated using data collected at wavelengths of 0.9194Å. Procedures accounting for merohedral twinning were performed during structural refinement of amlodipine, nimodipine, and Br-verapamil data sets. Detailed crystallographic data and refinement statistics for all constructs are shown in Extended Data Table 1. All structural figures were prepared with PyMol41. 31. Gamal El-Din, T. M., Martinez, G. Q., Payandeh, J., Scheuer, T. & Catterall, W. A. A gating charge interaction required for late slow inactivation of the bacterial sodium channel NavAb. J. Gen. Physiol. 142, 181–190 (2013). 32. Faham, S. & Bowie, J. U. Bicelle crystallization: a new method for crystallizing membrane proteins yields a monomeric bacteriorhodopsin structure. J. Mol. Biol. 316, 1–6 (2002). 33. Faham, S. et al. Crystallization of bacteriorhodopsin from bicelle formulations at room temperature. Protein Sci. 14, 836–840 (2005). 34. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997). 35. Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994). 36. Brünger, A. T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998). 37. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997). 38. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010). 39. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004). 40. Laskowski, R. A., Moss, D. S. & Thornton, J. M. Main-chain bond lengths and bond angles in protein structures. J. Mol. Biol. 231, 1049–1067 (1993). 41. DeLano, W. L. PyMol molecular viewer (V.1.2r3pre) (http://www.pymol.org) (2002). © 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved