Supporting InformationShi et al. 10.1073/pnas.1218203110SI MethodsExpression, Purification, and Reconstitution of the Voltage-GatedPotassium Channel from Aeropyrum Pernix and Its Mutants. Ex-pression,purification, and reconstitutionofwild-typevoltage-gatedpotassium channel from Aeropyrum pernix (KvAP), KvAPΔ36, andthe other KvAP mutants were performed as described previously(1). All mutations were confirmed by sequencing. The KvAPΔ36lacks the C-terminal 36 residues and is cys-less and fully functional.For making cysteine mutants, the cys-less KvAP mutant (C247S)was used as the “wild type” in this study. In lipid bilayers, C247Swas found to function the same as the native wild-type KvAP.The expression and purification of the cysteine mutants fol-

lowed the published procedures with some modifications. Proteinexpression was induced in the presence of 2.0 mM β-mercap-toethanol (βME). After the cells were harvested by centrifugationat 10,000 × g, they were resuspended in a buffer containing 50mM Tris·HCl, pH 8.0, 100 mM NaCl, and 2.0 mM βME toprotect the introduced cysteine residues in the expressed pro-teins. The resuspended bacterial cells were left in a plastic tubeburied in ice overnight. On the second day, the bacteria werelysed by microprobe sonication. Unbroken cells and cell debriswere removed by centrifugation at 10,000 × g for 15 min. Thesupernatant was collected and centrifuged at 200,000 × g for 1 hto obtain the membrane pellet. The pelleted membranes wereresuspended in a Dounce homogenizer, and then solubilized with40 mM decyl-maltoside (DM). Purification of the cysteine mu-tants was performed in buffers supplemented with 2.0 mM βME.The full procedure for KvAP reconstitution has been described

previously (1). Briefly, the purified KvAP mutants in 5.0 mMDM, 20 mM Tris·HCl, pH 8.0, 100 mM KCl, and 2.0 mM βMEwere concentrated to ∼1.0 mg/mL, and mixed with equal volumeof 10 mg/mL detergent-treated phosphatidylethanolamine (PE)/phosphatidylglycerol (PG) [1-palmitoyl-2-oleoyl-sn-phosphati-dylethanolamine (POPE)/1-palmitoyl-2-oleoyl-sn-phosphatidyl-glycerol (POPG) = 3:1 by weight with 10 mM DM]. The mixtureswere incubated at room temperature for 2 h before being set upfor dialysis against 10 mM Hepes/KOH, pH 7.4, and 150 mMKCl and 2.0 mM βME. In some cases, the last three changes ofdialysis buffer did not contain βME to allow slow air oxidizationin membranes (∼30 h). Our procedure consistently led to highincorporation efficiency of the channels into lipid bilayers. Thesucrose gradient separation of the vesicles indicated comigrationof all protein with the vesicles without detectable precipitation ofthe channel protein at the bottom of a 0–45% (wt/vol) gradient.Treatment with a solution of 1.0 M Na2CO3, pH 11.0, failed tostrip any protein out of the vesicles.

Preparation of the KvAP/Fv Complexes for 2D Crystallization.Recombinant 33H1 Fv protein was expressed and purified asreported previously (1). It was mixed with KvAPΔ36 protein ina molar ratio of 5:1. The complex was purified through a Super-dex 200 column in a buffer containing 30 mM β-octyl-glucoside,20 mM Tris·HCl, pH 8.0, and 100 mM KCl. The peak fractionswere collected and concentrated to ∼2.0 mg/mLFor 2D crystallization, lipids at 10.0 mg/mL were fully solubi-

lized in 45mMDM in a buffer (10mMTris·HCl, pH 8.0) or water.KvAPΔ36/Fv complexes were mixed with the lipids in differentratios [the protein–lipid ratio (PLR), and 1.2–1.5 usually led togood crystals] and incubated overnight before being dialyzedagainst a buffer containing 100 mM sodium citrate, pH 6.35,100 mM KCl, 3.0% (vol/vol) glycerol, and 0.020% (wt/vol) NaN3.PLR below 1.0 never led to good crystals. This makes sense when

we consider the areas occupied by lipids versus proteins in the 2Dcrystals. In the final project map, the total area per unit cell is175 × 175 Å2, and the eight subunits of the channel proteinwould occupy ∼15,000 Å2. The estimated number of lipid mol-ecules per unit cell is ∼320. The estimated PLR (in weight) basedon the unit cell in the crystal is therefore ∼0.9, close to what weused for crystallization.Different lipids were tested. 1-Palmitoyl-2-oleoyl-sn-phosphati-

dylcholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidicacid (POPA) (3:1) mixture, dimyristoyl-sn-phosphatidylcholine(DMPC)/dimyristoyl-sn-glycero-phosphatidyl acid (DMPA) (3:1)mixture and POPA, and DMPA all yielded crystals with the samepacking and lattice parameters. The PLR for different lipids was inthe same range of ∼1.2. Different regimes of slow removal of de-tergents were used to improve crystal quality, and dialysis wastypically complete in a week. Screening of the 2D crystals wasdone by negative-stain EM. Images of good crystals stained with6.0% (wt/vol) ammonium molybdate, pH 6.4, and 0.50% (wt/vol)trehalose normally showed strong and sharp reflections to theresolution limit of negative stain EM (∼20 Å).When crystals were fractionated in 10–55% sucrose gradients,

they always were seen in the layers between 35% and 55% su-crose. During crystallization, we never saw any significant por-tion of Fv molecules fall off the crystals. This is not surprisingbecause our estimated Fv-binding affinity is below 0.10 nM andthe Fv molecules mediated the crystal packing in the lattice.Indeed, when the crystals were treated with 0.8–1.0 M Na2CO3,pH 11.0, for 30 min at room temperature, a significant fraction ofthe Fv molecules were stripped off from the crystals. This sug-gested that the crystals were not formed in collapsed vesicleswhere all Fv molecules were inside the vesicles and all channelshad their extracellular sides facing the interior of the vesicles. Inour reconstitution system, unidirectional insertion was neverbeen achieved. Therefore, we concluded that our crystals werenot formed through the unidirectionally inserted channel/Fvcomplexes in the collapsed vesicles, where all of the Fv mole-cules would be trapped inside the vesicles.

Data Collection from KvAP 2D Crystals and Data Analysis. Cryoprotectionof the crystals. One of the major difficulties we encountered inpreparing our 2D crystals for cryo-EM study was to retain enoughcrystals on the surface of carbon-coated grids. We experimentedwith many different conditions and found that mildly hydrophobiccarbon surfaces were able to retain roughly 20–40 crystals per gridusing a modified back-injection procedure (2). Our current pro-cedure works to an acceptable level, but there is room for fur-ther improvement. Briefly, carbon-coated 200-mesh molybdenumgrids were prepared in the laboratory, aged by baking at 80 °Cfor 2–3 h in a small oven, and cooled down to room tempera-ture before being used. Care was taken to keep the carbon filmsflat (3, 4). Fresh crystals were loaded to the backside of the gridwhere the atomically flat carbon surface is. The crystals wereallowed to stick to the carbon film for 10–15 min in a wetchamber. After the excess crystal solution was removed, 2.0 μLof embedding medium containing 0.75% (wt/vol) tannic acid(pH 6.0 with KOH) and 10% (wt/vol) trehalose were added andmixed with the crystal (5–7). After a minute or two, the grid wasblotted against two layers of No. 4 Whatman filter paper for ∼10s, and then plunged directly into liquid nitrogen. The grids wereeither examined immediately or stored in liquid nitrogen untilEM examination.

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For all of the images used for calculating the projection mapin this manuscript, the grids were immediately transferred intoa cryoholder where the carbon-coated sides of the grids faceddown in the specimen slot. Once inside the microscope, thecrystals encountered the electrons before the carbon films un-derneath. This procedure also helped us determine the handed-ness of the cryo-EM map. In a right-handed coordinate system inwhich the z-axis is parallel to the moving direction of the elec-trons, the carbon films will be in the background of the calculatedprojection map in the contour plot we generated (see below).Data collection from cryo samples. For cryo-EM data collection, thegrids were loaded into a Gatan 626 cryoholder or an OxfordCT3500 cryoholder, and inserted into a JEOL JEM2200FS fieldemission gun (FEG) microscope that was operated at 200,000 V.Due to the uneven embedding of the 2D crystals, only crystalsshowing thin embedding media were selected for imaging. Theselected crystals always had sharp square corners, suggesting thatthese corners were from the single sheets, not vesicles. Drying theembedding media tended to push the crystals against the surfaceof the carbon films such that the molecules with a large ecto-domain facing the carbon films could be slightly deformed andresolved at a lower resolution. Such sample-carbon film in-teraction has been reported previously (8) and probably con-tributed to the loss of screw symmetry in many of our imagedcrystals. In fact, even the good crystals showing strong P4212symmetry still exhibited a clear difference in resolution betweenthe two oppositely oriented molecules in each unit cell (Fig. 1E).The cryo-EM images were recorded at 50,000× magnificationwith defocus values varying from −0.3 to −1.0 μm. When thecrystals were tilted for imaging, we used defocus levels higherthan −1.0 μm to compensate for the defocus gradient across theimaged area. The dose rate of the electron beam was adjusted to15–20 electrons per Å2 per second, and normally the exposureswere taken in 0.70 s. An energy filter of 35 eV was inserted forzero-loss imaging. The films were selected in a SIRA optical dif-fractometer (Michelson Diagnostics), and those showing obviousdrift or strong astigmatism, or lacking diffraction spots beyond15 Å were eliminated. Good negatives were scanned in a Photo-scan flat-bed scanner (Z/I Imaging) at 7.0-μm steps yielding acalibrated pixel size of 1.42 Å.Processing of the cryo-EM images of the 2D crystals. Analysis of theimages of the 2D crystals followed a standard procedure using theMRC package (9). Individual images (still in negative contrastand the protein being black) were checked and the best areaswere boxed out individually with a dimension of 4,000–5,000pixels. Three to four runs of unbending were performed, and thephases were corrected after contrast transfer function (CTF)application (contrast reversed). ALLSPACE search typicallyindicated square lattices (10, 11), either P4 or P4212 (Table S2).By examining the systematic absences along the h and k axes,only images showing good P4212 symmetry in the ALLSPACEsearch were selected for further analysis. The same images werealso analyzed in 2dx package for comparison (12). The best set ofeight images yielded good merging statistics to 6.5–7.0 Å, eventhough some strong spots were seen to 5.0 Å (Table S1 and Fig.S3). Therefore, we conservatively truncated the data to 7.0 Å andcalculated a projection map to that resolution. A negative Bfactor of −700 Å2 was used to sharpen the map (13).Handedness determination.Thehandedness of the channelmoleculesin the projectionmapwas determined in three different ways. First,as explained above, the sample was loaded in a consistent fashionsuch that the z-axis went from the sample to the carbon film. In thescanned images that were ported into theMRCpackage, the z-axisunder the MRC convention ran opposite to the electron beamdirection; to correct for this, we mirrored the map in the final stepwhen preparing for presentation. Second, the symmetry of ourcrystals suggested that the projection map contained channelmolecules facing two different orientations as showed in Fig. 1E.

We cut out part of the map in the middle of the unit cell andcompared it with the projection map of the X-ray structure of theKvAP pore (Fig. 2A), as well as its mirror. The middle portion ofthe EM map matched well with the KvAP pore structure, muchbetter than the pore domain from the KvAP map that was mir-rored along the y-axis. This comparison suggested that the mole-cule in the middle of the unit cell (Fig. 1D) probably has thecorrect handedness. Third, as mentioned above, the carbon–sample interaction tends to affect the molecules with their largeectodomains facing the carbon film. We know that the carbon filmis in the background of our projection map and our KvAP/Fvcomplex has a lot of mass on the extracellular side of the channeland very little mass on the intracellular side. Thus, the molecule inthe middle of the unit cell has its extracellular side facing thereader, not the carbon film, whereas the four molecules sur-rounding it at the four corners have their extracellular side facingthe carbon. The contact of the Fv molecules with the carbon filmwould have some disturbance to the structure of the voltage sensordomains (VSDs) connected to them. Therefore, it is not surprisingthat the molecule in the middle has its density peaks in the voltagesensor ring better resolved than those in the four molecules at thefour corners. Together, these three lines of examination ensureda high level of certainty in determining the chirality of the channelmolecule in the projection maps (Fig. 1 D and E).The KvAP crystals revealed detailed information about the

transmembrane portion of the channel, much more than theprevious electron crystallographic analysis of the Kv1.2 α/βcomplex and the MlotiK channel (14, 15). The crystallization ofthe Kv1.2 α/β complex was likely mediated by interactions be-tween the bulky β-subunits on the intracellular side of thechannel. The calculated map for Kv1.2 complex contained fairlystrong contributions from the β-subunits and did not showclearcut details of the helical arrangement in the transmembranedomain of the α-subunit. Similarly, the calculated projection mapof the full-length MlotiK in the 2D crystals in the absence andpresence of cAMP suggested that the nucleotide-binding do-mains made significant contributions to the density, and thedensities corresponding to the voltage sensor helices were notclear. This made it difficult to discern the helical arrangement ofthe voltage sensor domains.Examining other possible packing and map calculations for comparison.Because the structure of the voltage sensor ring seen in the cryo-EMprojectionmapappeared significantly different from theX-raystructures in detergents, wewanted to test whether various packingofX-ray structures would yield a projectionmap that is the same asourexperimental projectionmapof theKvAP inmembranes. First,we checked whether the 2D crystals were made of two layers ofchannel molecules that took the four-helix bundle structures androtated against each other by 45° to create a ring. In general, thetwo layers of molecules could generate four possible arrange-ments (Fig. S5). Twomolecules could be in parallel or antiparallelorientations, and could be perfectly aligned or rotated by 45°against each other. Using the homologous model of the KvAP,which was generated based on the Kv1.2 structure by aligning keyresidues in the voltage sensor domains, we built four types ofmodels and calculated their projection maps to 7.0 Å. The pro-jections from two antiparallel molecules led to mirror symmetryalong four different axes—vertical, horizontal, and two diagonalaxes (Fig. S5 C andD, red lines). They were clearly different fromwhat was observed in our experimental projection map because ofthe obvious handedness in the latter (Fig. 1D and Fig. S4B). Thus,they were excluded.Theprojections fromthe twoparallelmolecules gave rise to ring-

shaped structureswhen theywere rotated against each other by 45°(Fig. S5B). However, their projection is significantly differentfrom our experimental projection map of the KvAP in mem-branes. Their projection has two rings of density beyond the poredomain, instead of only one ring of density surrounding the pore

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domain as in our experimental map. The pore region in the modellacks key features seen in both the projection map of KcsA andour map (compare Fig. S5B with Fig. 1D; see also ref. 16).Two more considerations led us to exclude the possibility of

crystal twinning or the existence of double layers that could skewour results. First, the 45° rotation of one layer with respect to theother would introduce C8 symmetry in both the pore domain andthe two rings of density surrounding the pore. Such symmetrywas not observed in our experimental map. Second, all channelsin our experiments had full occupancy of their voltage sensordomains by Fv molecules to maintain uniformity in channelconformation. A 45° rotation between the two parallel moleculesshould create a ring of the density corresponding to the eight Fvmolecules. In our experimental map, there are only four prom-inent densities corresponding to the Fv molecules (Fig. 1 D andE and Fig. S7).Thus, we are left with one possibility (Fig. S5A). When the two

parallel molecules are well aligned, their projection is exactly thesame as that of the single layer. This does not change our con-clusion on how the individual helices in the voltage sensor do-mains are arranged, nor does it affect our conclusion about theformation of the voltage sensor ring.Nevertheless, we conducted further examination because it

would be important for building our 3D dataset. To check whetherour crystals were two-layered stacks in perfect alignment, we tookimages of crystals tilted at 10°, 15°, 30°, and 45°, hoping to be ableto observe the lattice lines that correspond to the thickness of thetwo layers, which should be ∼80–120 Å, much shorter than the175-Å lattice dimensions of single-layered crystals. However,when we indexed all of the tilted images, the lattice parametersindicated single layered crystals. For example, Fig. S6 shows onelattice calculated from the image of a crystal tilted at a nominalangle of 40°. The lattice parameters were 145 × 152 Å2 with anintersection angle of 140.2°. The exact tilt angle turned out to be48°. These data suggested that many, if not all, of the crystals weresingle-layered.Twinningwithin single-layered crystals can generate artifacts.

In such mosaic crystals, two or more patches of crystals aremerged together by rotating against each other, either along themembrane normal or along an in-membrane axis. If the rotationwas along the membrane normal, we would expect to see two ormore different lattices in the final image. This was not observedin our images. If the rotation between patches were along an in-membrane axis, the merged image would have features corre-sponding to two antiparallel molecules overlapping with eachother with some rotation between them (similar to Fig. S5Dexcept that the angles may not be exactly 45°). We would expectthis arrangement to introduce four mirror axes and a Fv-den-sity ring in the final projection map. However, we did not ob-serve these mirror axes in our experimental map, nor the Fv-density ring.Furthermore, when we inspected the high-contrast images of

crystals, (e.g.,FigS7,proteindensity inwhite), the strong features inthe images that correspond to the Fv molecules outside themembranes (X-shaped) were well aligned (it is easier to see themalong the red line). There is no significant rotation (∼45°) betweenneighboring lattice units that would be required to make a voltagesensor ring. However, we took multiple images from differentareas of large 10- to 20-μmcrystals.We found that the images fromthe same large crystals always had the same lattice parameters andlattice orientation, and gave rise to similar projections with varyingresolutions, suggesting that our large crystals did not consist ofsmall pieces of crystals randomly rotated against each other. Al-ternatively, when we analyzed multiple 1,000 × 1,000 pieces froma large 5,000 × 5,000 crystal image, the lattice parameters and theorientations from these small pieces were all very close to eachother. In addition, in our crystallization process the orientationalrandomness of the channel molecules in a membrane was ex-

pected, and the sharp edges of our crystals suggested that themerge of the small crystals was likely along these sharp edges andwould maintain macroscopic crystallinity.When we surveyed literature for different 2D crystals, we

noticed that the detergent-induced fusion of the bacteriorho-dopsin 2D crystals led to clear twinning in the membranes (17).The main reason for this was likely that in those experimentspatches of 2D bacteriorhodopsin crystals (purple membranes)were fused and the crystals were not grown out of fully solubi-lized proteins.Twining of 2D crystals can also occur when two crystals attach to

eachother in opposite orientations.The randomattachment of twolayers would be easily discovered as there would be two differentlattices. However, the exact interdigitation between the unit cellsfrom two layers may lead to a rotation (screw) axis relating the twolayers as seen previously (8, 18, 19). This arrangement is equivalentto the vesicular form of crystals where two layers from one vesiclehave collapsed against each other and are arranged into a lattice.Such well-ordered crystals might be treated as single crystals.However, based on the PLR estimation from our projection mapand our Fv-stripping experiment, we do not think this was the case.For instance, if two interdigitated layers of KvAP/Fv moleculescame together to generate the lattices we observed, the estimatedPLR would be ∼0.32, much smaller than our estimate of 0.9 basedon single-layer crystals. In our experiments, PLR of 1.2–1.5 wasrequired to produce 2D crystals. Moreover, the interdigitatedlayers would sandwich the Fv molecules between two membranes,thus not allowing the Fv–carbon interaction that would lead to thedifferent resolutions for the two views in Fig. 1E. Based on theseconsiderations, we concluded that we did not obtain interdigitatedstacked two-layered crystals.Finally, we askedwhether it was possible that, in the 2D crystals,

the channel pore domains were well aligned along the latticepoints, but the voltage sensor domains had static disorder. Thiswould mean that the voltage sensor domains in different latticesrandomly took twoormoredifferent conformations, theaverageofwhich could give rise to the voltage sensor ring. There aremultiplelines of evidence arguing against this. First, each voltage sensordomain in our crystals was bound by one Fv molecule. The Fv-mediated packing of crystals fixed the orientation of the voltagesensors (mainly the S3/S4 paddles). In the high-contrast images oftrehalose-embedded crystals (for example, see Fig. S7), theectodomains, contributed by the Fv molecules interacting witheach other, were well aligned across a long distance, meaning thatthe S3/S4 paddles, bound to the Fv, have to be equally well po-sitioned in membrane. Thus, we concluded that, at the currentresolution, there is little or no static disorder for the S3/S4 voltagesensor paddles in our crystals inmembranes. Second, when the S3/S4 helices are fixed in position, the chance for the S1/S2 to adopttwo or more statically disordered conformations becomes veryslim. This is because the S1 and S2 are known to be long α-helicesthat are stable when integrated in a lipid bilayer. It is well estab-lished that the S2 interacts with the S3/S4 through strong elec-trostatic interactions (20). This is further evidenced by the factthat, in the “up” state, the S1 is packaged well against the poredomain. These interactions minimize the likelihood of static dis-order in the S1/S2. Third, it has been demonstrated that the S3/S4voltage sensor paddle undergoes significant conformationalchanges inmembranes (21).When we screened for the 2D crystalsof the KvAP alone in membrane, we obtained lattices that onlydiffracted to ∼20 Å and were not able to improve the crystals anyfurther. The conformational heterogeneity in the S3/S4 paddlethat switches the voltage sensor domains among the Cx, O, and Istates (Fig. S1) appeared to be the main reason behind our failedattempts to achieve better crystallization. Once we realized thispossibility, we used the Fv to stabilize the channels in the in-activated (I) state and thus minimized conformational flexibilityof the VSDs. Using this approach, we obtained high-quality

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crystals within a few rounds of optimization in several parametersand the resolution of the crystals quickly surpassed 10 Å. Fourth,in the projection map (Fig. 1D), the two strongest peaks in onevoltage sensor domain, i.e., one-quarter of the voltage sensor ring,are between the two neighboring Fv-binding sites. Because the S3/S4 helices have to go with the Fv domain, these two strongestpeaks must be attributed to the other two integral helices, the S1and S2. If there were static disorder in these two helices, theywould not form the strongest peaks in the voltage sensor ring.Finally, when we compared the projection map in membraneswith the cryo-EMmap of the KvAP/Fab complex in detergents [inDM; see Jiang et al. (22)], we noticed that the S1/S2 in the latterwere also the strongest densities in the transmembrane domain,much stronger than the S3/S4 paddle, suggesting the stablepacking of the S1/S2 against the pore domain. Even though thesingle particle reconstruction for channels in detergents is stilldifferent from the projection map from 2D crystals, the samepattern of density variation among the S1/S2 and the S3/S4 re-appeared in the experimental projection map in membrane, albeitwith better resolution and clarity. All these considerations led usto conclude that, at the working resolution, the Fv binding ap-parently minimized the conformational flexibility for both thevoltage sensor paddle and the S1/S2, and that our data do notsupport the overlapping of two or more conformations of thefour-helix bundled voltage sensor domains to create a voltagesensor ring.Helix assignment in the projection map. The multiple lines of evidencediscussed above made the peak assignment in the voltage sensorring fairly straightforward. Because the loops between VSDhelices of KvAP are fairly short, strong density peaks in thevoltage sensor ring need to be assigned to the transmembranehelices. When we looked at the projection map from the extra-cellular side, the molecule did not show mirror symmetry (Fig.1D). When the X-ray model was overlaid to the projection mapsuch that the S3/S4 sit next to the Fv domain, the two strongpeaks between two neighboring Fv-binding sites stand out andcannot be accounted for by the VSD four-helix bundles. Becausethe tip of the S3/S4 voltage sensor paddle has to be positionednext to the Fv, the two strong peaks must be assigned to theother two integral transmembrane helices, the S1 and the S2.The S2 helix needs to be close to the S3/S4 paddle to align itsnegatively charged residues with the positively charged residueson the S4. This allows us to determine the relative positions of S1and S2. In membranes, the S4 is known to be tilted and posi-tioned right next to S5 when the voltage sensor domain is in theup position (23, 24). Therefore, we assigned the weaker elon-gated peak next to the pore domain to the tilted S4. The re-maining strong peak next to the Fv domain has to come from theS3 helix (mainly the S3b as the S3a is very short). There is alsoa weak peak between the assigned S2 and S3. Either the S2–S3linker or the short S3a may contribute to this weak peak. Thus, inour assignment the S2, S3, and S4 form a loose triangular bundle(Fig. 2).Processing the electron diffraction data. For electron diffraction, thecrystals were prepared as described above, and diffraction datawere collected in an FEI Tecnai 20 electron microscope equippedwith a FEG and a bottom-mounted Gatan slow-scan CCDcamera. Diffraction data were recorded using ∼5 electrons per Å2

per second, a 70-μm selection area aperture, and a diffractioncamera length of 3,500 mm. The unit cell dimensions and theresolution of the diffraction were calibrated using aquaporin0 crystals (25). Typically, good recordings from the KvAP/Fvcrystals showed strong and sharp spots up to ∼5-Å resolution.Electron diffraction patterns were processed in the XDP suite ofprograms as described previously (26).Estimating the resolution of the projection map. In cryo-EM maps, it ispossible to have heterogeneous resolutions in different parts ofthe samemap, which is similar to the local ambiguities in 3Dmaps

by X-ray crystallography. In our projection map, the extracellularview is clearly better resolved than the intracellular view (Fig. 1E).As we discussed above, it is likely the interaction with the carbonfilm that hurt our resolution of the voltage sensor ring from theintracellular view. If we truncate the data to 8.5–9.0 Å, the high-resolution noise is suppressed and the intracellular view achievesslightly better resolution of the individual peaks in the voltagesensor ring, suggesting that the intracellular view is probably re-solved to this resolution. However, the extracellular view is wellresolved such that we can count the peaks individually and makeassignments based on available information about the channel.The resolution of the neighboring helices is quite clear, suggest-ing that the resolution is better than 7.5–8.0 Å. Indeed, when welooked at the spots from the unbending of individual images wefound strong spots (low IQ, high signal-to-noise ratio), evenly to7.0 Å with some IQ3 spots in the resolution shell between 7.0 and5.0 Å (Fig. S3). Therefore, we think that a conservative estimateof the resolution in our projection map is ∼7.0 Å, consistent withthe statistics of the phase residual calculation (Table S1). Thismay improve with better ways to increase the cushion betweenthe crystals and the carbon films.Fv-stabilized conformation versus other conformations. The availablestructural and functional information about KvAP supports ourinterpretation of the projectionmap. The question is howmuch ofthis truly reflects channels freely diffusing in membranes. Weknow that the Fv-bound channels are in the “inactivated” state inmembranes (1) and that the coupling between the voltage sensordomains and the pore domain remains intact in lipid mem-branes. This is already quite a helpful scenario. The key questionthen becomes how much effect the Fv molecule has on thechannel. We tested the predictions based on the voltage sensorring structure by collecting biochemical and electrophysiologicaldata from channels freely diffusing in membranes. Our data ina systematic manner agreed with the voltage sensor ring.It is important to note that the observation that the voltage

sensor ring is the most stable conformation for the channels in theinactivated state does not exclude the possibility that in a differentconformational state the channel may take on the four-helixbundle structure, or some other similar structures. It remains tobe investigated whether the lateral movement proposed in Fig. 5plays a role in the voltage-driven motion of the voltage sensordomains in a membrane-embedded channel. More experimentalevidence is needed to elucidate the structures of the voltagesensors in other gating conformations of a voltage-gated ionchannel in membrane (Fig. S1A).

Electrical Recordings from Reconstituted Channels in Lipid Bilayers.Bilayer recordings from reconstituted channels were obtained aspreviously described (1). The vesicles containing the KvAP and itsmutants were fused into the black lipid membranes, which are themodel bilayers formed across holes of 100–300 μm in diameterthat are optically black due to destructive interference. Voltage-gated ionic currents were recorded by delivering short depola-rizing pulses from a holding potential of −80 mV. All cysteinemutants that were tested showed robust voltage-dependent cur-rents under reducing conditions. At 0 mV, the mutant channelsachieved a high open probability to generate macroscopic cur-rents, suggesting that they are all able to reach the open state inthe absence of any transmembrane voltage bias. Because thecysteine mutants were used to test the proximity between sites inthe voltage sensor domains in the up conformation and the cross-linked states are stable, we concluded that as long as they are ableto generate sizeable ionic currents, the cysteine mutants must beable to reach the up conformation of the voltage sensor domains.Consistent with this conclusion, we also noticed that withoutproper reduction all mutants in the VSDs that showed clearlyinterchannel or intrachannel cross-linking usually had very low orno channel activity in the lipid bilayers.

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Cross-Linking Experiments for Cysteine Mutants in PhospholipidVesicles. Vesicles with reconstituted channels were first reducedby 5.0 mMDTT for 30 min at room temperature, diluted 20× witha buffer containing no DTT, and pelleted down by ultracentrifu-gation at 200,000 × g for 45 min. The vesicle pellets were re-suspended and centrifuged again to minimize the residual DTT.After the second spin, the vesicles were resuspended in a smallvolume (∼50 μL) of buffer that contained 10 mM Hepes, pH 8.0,and 150 mM KCl and left at room temperature for 1–2 h. The airoxidization of the cysteines through this procedure was relativelyweak, but usually avoided significant multichannel aggregates andprotein loss as we often observed when using strong oxidants suchas copper phenanthroline (10-30 μM) or bifunctional cross-linkerssuch as 1,1-methanediyl bismethanethiosulfonate (MTS-1-MTS,10–20 μM). After weak oxidization, 5.0 mM N-ethylmaleimide(NEM) was introduced into the vesicles to block all available -SHgroups before the vesicles were solubilized with 50 mM decylmaltoside. The proteins extracted from the vesicles were filtered

through a 0.22-μm filter before injection into a gel filtrationcolumn, (Agilent SEC-5 5.0-μm beads and 150-Å pores), in anAgilent 1260 HPLC system. In our analysis, the cross-linkedoctamers (formed by two channels; ∼300 kDa) and individualtetrameric channels (∼150 kDa) were well separated chro-matographically (baseline separation; Fig. 4). For gel analysis,the fractions (0.20 mL each) corresponding to each peak werecollected, concentrated to ∼30 μL, and run on 12% non-reducing SDS/PAGE gels, which were silver-stained to visualizeprotein bands. To confirm that the cross-linking was due to thedisulfides, the same samples in parallel were reduced with 5.0mM DTT and quenched with 15 mM NEM before being run onreducing SDS/PAGE gels. All cross-linked mutants we studiedhere were found to be reducible by DTT. For double-cysteinemutants, the intrachannel cross-linking was compared and lis-ted in Table S3. For single-cysteine mutants, the interchannelcross-linking was compared and listed in Table S4.

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Fig. S1. Overview of voltage-gated ion channels with KvAP as the chosen example. (A) A gating scheme for KvAP. Depolarization switches four VSDs from“down” to “up” while the pore remains closed. Because of the tight coupling between the VSDs and the pore domain during each step of the gating transition,we labeled the different up conformations of VSDs and the different “closed” conformations of the pore domain using subscripts. When all four VSDs are up(up2), a concerted step opens the channel pore and the pore domain then becomes inactivated, probably due to a change in the selectivity filter region (closed3).(B) The topology of one channel subunit (Left) and the arrangement of four monomers into the tetrameric channel (Right). The arrangement of the four VSDsfollows the X-ray structures as viewed from the extracellular side. (C) Structure of KvAP with the voltage sensor domains modeled after the VSD of Kv1.2/0.1chimera as viewed from the extracellular side. (D) Electrical recordings from the cysteine-less mutant (Left) and in the presence of Fv (Right). Purified monomericBSA has no effect on the channel. Fv binds to the channel when the voltage sensor domain is in the up conformations (0 mV). The Fv binding kept the channels ina well-defined inactivated state in which the channel pores are closed due to steady-state inactivation and the voltage sensors are in the up conformation (up4).

Fig. S2. Two-dimensional crystals of KvAP contain membrane-embedded channels. A small fraction of our 2D crystals were incubated with 1.0 M sodiumcarbonate and fractionated on a sucrose gradient as indicated. The Fv was stripped while the channels migrated with the vesicles to the bottom of the gradient(45% sucrose). The data suggest that KvAP is membrane-embedded in the 2D crystals, whereas Fv molecules are not. Control experiment without sodiumcarbonate is presented at the bottom. Similar results were obtained with 0–55% sucrose gradient with the channels running above the 55% region.

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Fig. S3. Electron crystallography of KvAP 2D crystals. CTF-corrected IQ plot of the data from a representative 2D crystal. Strong spots are observed ata resolution far better than 7 Å. The concentric rings are Thon rings from CTF fitting. The edge of the plot is 5.0 Å, and the fourth CTF ring is at ∼7.0 Å.

Fig. S4. Validation of the structural model of KvAP in membranes. (A) The proposed molecular model of KvAP in membranes as viewed from the intracellularside. Pore domain is in blue, three voltage sensors are in gray, and one voltage sensor has individually colored S1–S4 helices as indicated. This model was builtthrough iterative cycles of rigid body refinement against the experimental map (gray mesh in B) until the proposed structure matched the experimental map asshown with gold ribbons in B (also intracellular view).

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Fig. S5. Projections from overlapped four-helix bundles in two channels. (A and B) Two channel models of KvAP, based on the Kv1.2 chimera model by aligningkey residues in the voltage sensors, were positioned in parallel along the fourfold axis as if they were in two separate membranes stacked on each other. Thesecond molecule has a rotation of 0° (A) or 45° (B). Projection maps were calculated to 7.0 Å. (C and D) Two models were positioned in antiparallel along thefourfold axis with a rotation of 0° (C) and 45° (D). Maps were calculated to 7.0 Å. The four red lines indicate the four mirror axes in the two projection maps.

Fig. S6. Single lattice in the cryo-EM image of a tilted crystal. (A) A small piece of the cryo-EM image from an embedded crystal that was tilted at a nominal40°. Two sets of parallel lines could be discerned and informed the position of the lattice vectors. (B) Central part of the Fourier transform with selected latticelines as indicated (red dashed lines). Some spots are indexed. The lattice accounts for all visible spots.

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Fig. S7. The arrangement of individual units in a 2D crystal. The crystals were mixed with 3.0% trehalose and then loaded onto the carbon surface. Afterincubation and blotting, they were frozen and imaged. The trehalose embedding made possible the good resolution of the X-shaped ectodomains (Fvmolecules). The contrast of the image was reversed so that the protein density is white. Along the red dashed line, we can see that the X-shaped Fv densitiesare well aligned and ordered in the same angle against the red line, suggesting that the voltage sensor paddle (S3/S4) must be arranged in the same order.

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Fig. S8. Effect of strong oxidization on the KvAP T47C/G108C mutant. The channel-containing vesicles were reduced, washed to remove the reducing agents,and treated with 20 μM copper phenanthroline for 10 min at room temperature. NEM at 5.0 mM was added to quench all free –SH groups. The vesicles wereextracted and analyzed by gel filtration FPLC (A). The fraction containing the peak of the tetrameric channel was collected, concentrated, and analyzed ina 12% Coomassie blue-stained nonreducing SDS/PAGE gel (B). More than 70% of the double-mutant protein (∼0.60 mg used in total) was lost during thesample concentrating and filtration step before injection into the size-exclusion column, suggesting that the strong oxidation induced heavy aggregation. Inthe remaining tetramers, there were cross-linked dimers, trimers, and even faint traces of tetramers on the gel. Under the same conditions, strong oxidationled to the complete aggregation (and loss) of T47C/L118C mutant proteins. These observations made us elect to use weak air oxidation in our analysis (Figs. 3and 4, and Tables S3 and S4).

Table S1. Statistics for merging the structural factors from eightimages of 2D crystals

Resolution range, Å

200–14 14–9.9 9.9–8.1 8.1–7.0 Total

No. of comparisons 65 63 59 47 234Phase errors, degrees* 23.6 27.1 39.2 39 31.6

*The phase error is expressed as the phase residual with respect to the targetphase of 0° or 180°. Only reflections with an IQ ≤6 were included in thecalculations.

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Table S2. Statistics for the ALLSPACE search in one of the crystal images (no. 1238)

SPACEGROUP

Phase resid(No)vs. other spots(90 random)

Phase resid(No) vs.theoretical (45 random) OX OY TX TY

Target residualbased on

statistics takingFriedel weightinto account

1 p1 41.0 794 31.6 7942 p2 64.8! 397 32.4 794 −19.0 34.5 0.00 0.00 63.23b p12_b 45.5! 181 13.3 22 160.9 168.0 0.00 0.00 42.43a p12_a 43.5! 180 28.9 20 −102.0 −145.6 0.00 0.00 42.24b p121_b 42.9* 181 15.0 22 71.0 108.0 0.00 0.00 42.44a p121_a 43.7! 180 34.5 20 −84.0 124.5 0.00 0.00 42.25b c12_b 45.5! 181 13.3 22 160.9 168.0 0.00 0.00 42.45a c12_a 43.5! 180 28.9 20 −102.0 −145.6 0.00 0.00 42.26 p222 55.1! 758 32.4 794 −19.0 −145.5 0.00 0.00 52.67b p2221b 54.9! 758 32.1 794 70.9 124.4 0.00 0.00 52.67a p2221a 54.3! 758 32.1 794 70.9 124.4 0.00 0.00 52.68 p22121 54.6! 758 32.4 794 161.0 34.5 0.00 0.00 52.69 c222 55.1! 758 32.4 794 −19.0 −145.5 0.00 0.00 52.610 p4 54.3! 757 32.4 794 161.0 34.6 0.00 0.00 52.611 p422 46.7* 1,423 32.4 794 −18.8 34.5 0.00 0.00 47.212 p4212 46.5* 1,423 32.4 794 −18.9 34.5 0.00 0.00 47.2

* = acceptable; ! = should be considered; ` = possibility.

Table S3. Intrachannel cross-linking in double-cysteine KvAP mutants reconstituted in POPE/POPG (3:1) vesicles after weak air oxidation

Mutated sites(helix no.)

Dimerization withintetrameric channels

Equivalent sitesin Shaker

L50C(S1)-G108C (S3) − F252-V331Q49C(S1)-G108C(S3) − E251-V331M48C(S1)-G108C(S3) − P250-V331T47C(S1)-G108C(S3) + L249-V331T47C(S1)-L110C(S3) − L249-E333T47C(S1)-A111C(S3) + L249-V334E45C(S1)-G108C(S4) − E247-V331T47C(S1)-F116C(S4) − L249-L361T47C(S1)-L118C(S4) + L249-V363

Notes: Besides these, multiple other double-cysteine mutants were prepared but did not express well for ourexperiments.

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Table S4. Interchannel disulfide formation of KvAP mutants withsingle-cysteine residues introduced at different locations of S1,S2, S3, S4, and S5

Mutated sites(helix no.)

Dimerization betweenmutant tetramers

Equivalent sitesin Shaker

V 39C(S1) + I241E45C(S1) − E247T47C(S1) − L249M48C(S1) + (weak) P250Q49C(S1) + (weak) E251L50C(S1) + (weak) F252Y59C(S2) − F280L60C(S2) − L281E62C(S2) + E283G108C(S3) − V331L110C(S3) + E333A111C(S3) −/+ (very weak) E334G114C(S4) + A359L115C(S4) + I360F116C(S4) + (weak) L361R117C(S4) + R362L118C(S4) −/+ (very weak) V363R120C(S4) + R365L121C (S4) − L366L122C(S4) − V367F124C(S4) + V369L125C(S4) − F370F166C(S5) − S412A167C(S5) − A413I170C(S5) − F416V171C(S5) − A417

Notes: To minimize the formation of multichannel aggregates, air oxidi-zation was used to form the cross-linked dimers in channel mutants in mem-branes. The cysteine residues were blocked by NEM before the channels invesicles were extracted in detergents for further analysis.

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