GAP JUNCTION STRUCTURES. II. Analysis of the X-Ray Diffraction Data

Published Online: 1 August, 1977 Supp Inf: http://di.rg/10.1083/jcb.74.2.629 Dwnladed frm jcb.rupress.rg n Nvember 7, 2018 GAP JUNCTION STRUCTURES II. Analysis f the X-Ray Diffractin Data LEE MAKOWSKI, D. L. D. CASPAR, W. C. PHILLIPS, and D. A. GOODENOUGH Frm the Rsenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02154 and the Department f Anatmy, Harvard Medical Schl, Bstn, Massachusetts 02115 ABSTRACT Mdels fr the spatial distributin f prtein, lipid and water in gap junctin structures have been cnstructed frm the results f the analysis f X-ray diffractin data described here and the electrn micrscpe and chemical data presented in the preceding paper (Caspar, D. L. D., D. A. Gdenugh, L. Makwski, and W. C. Phillips. 1977. 74:605-628). The cntinuus intensity distributin n the meridian f the X-ray diffractin pattern was measured, and crrected fr the effects f the partially rdered stacking and partial rientatin f the junctins in the X-ray specimens. The electrn density distributin in the directin perpendicular t the plane f the junctin was calculated frm the meridinal intensity data. Determinatin f the interference functin fr the stacking f the junctins imprved the accuracy f the electrn density prfile. The pair-crrelatin functin, which prvides infrmatin abut the packing f junctins in the specimen, was calculated frm the interference functin. The intensities f the hexagnal lattice reflectins n the equatr f the X-ray pattern were used in crdinatin with the electrn micrscpe data t calculate the tw-dimensinal electrn density prjectin nt the plane f the membrane. Differences in the structure f the cnnexns as seen in the meridinal prfiles and equatrial prjectins were shwn t be crrelated t changes in lattice cnstant. The parts f the junctin structure which are variable have been distinguished frm the invariant parts by cmparisn f X-ray data frm different specimens. The cmbinatin f these results with electrn micrscpe and chemical data prvides lw-reslutin threedimensinal representatins f the structures f gap junctins. The structural variatins detailed in the preceding paper (5) establish that we are lking at nt ne structure f the gap junctin, but at a family f structures. By bserving clsely related states f a mlecular assembly, it is ften pssible t infer smething abut the way that transitins ccur between states. These mlecular rearrangements may be significant in the functinal activity f the structure. Furthermre, plymrphism prvides a cnstraint n the interpretatin f the diffractin patterns. Fr example, the cnnexn structure is likely t be similar in arrays with different lattice cnstants, althugh the intensities in the X-ray patterns may be quite different. TItE JOURNAL OF CELL BIOLOGY 9 VOLUME 74, 1977 9 pages 629-645 629

Frm analysis f the X-ray intensity measurements, the distributin f scattering density within the gap junctin can be determined. Prtein, lipid, and water have distinguishable scattering densities. The electrn density f hydrated prtein and lipid plar grups is abut 0.40 e/,~ 3 (electrns per cubic angstrm), that f lipid hydrcarbns is abut 0.27 e//~ 3, and that f water is 0.33 e/]k 3. It is, therefre, pssible t use scattering density maps t distinguish the spatial distributin f the principal chemical cnstituents within the junctin. In this way, the diffracted X-ray intensity measurements can be used t relate the mrphlgical features bserved in the electrn micrgraphs t the chemical cmpsitin f the.junctins. Disrder in the gap.junctin lattice limits the structural detail that can be seen by crystallgraphic analysis f X-ray diffractin patterns r electrn micrgraphs. Averaging ver many identical units results in images that are blurred by disrder. The effect f disrder in any peridic array n its X-ray r electrn diffractin pattern r n the ptical diffractin frm an electrn micrgraph is t reduce the intensity f higher-angle reflectins. Beynd sme limiting reslutin determined by the average distance that the units are irregularly displaced, reflectins disappear int the diffuse scattering backgrund frm the disrdered structure. Since each reflectin may be cnsidered a piece f infrmatin abut the structure, a reductin in the number f bserved reflectins crrespnds t a lss f infrmatin abut the structure. Electrn micrscpe images f individual macrmlecular units can be btained, but nly at a radiatin dse that destrys the bilgical structure. What is usually lked at in an electrn micrgraph is a stained fssil r a cast f the structure. With lw electrn dses which yield very faint, nisy micrgraphs, Unwin and Hendersn (14) btained high-reslutin images f the prtein in the tw dimensinally crystalline purple membrane by averaging ver many units as is dne with X-ray crystallgraphy. They btained the phase infrmatin that is lst in a diffractin pattem frm the averaged image f the rdered structure. Electrn diffractin patterns that we have recrded frm gap junctins under lw dse cnditins d nt, hwever, extend t the reslutin f the X-ray diffractin patterns; n mre than tw r three reflectins were bserved. The X-ray diffractin patterns presented in the preced- ing paper prvide the highest-reslutin structural infrmatin that we have been able t btain abut the arrangement f prtein and lipid in the gap junctin. Lack f phase infrmatin in the X-ray diffractin patterns themselves des nt present a significant prblem in the analysis f the gap junctin structure. At the reslutin at which we are wrking, sufficient infrmatin can be btained frm a cmbinatin f electrn micrscpy and a priri knwledge f bilayer structure t reliably phase the data. The principal reasn why a phase prblem des nt arise is the small amunt f data. All pssible phase chices can be used t calculate structures which can then be checked fr cnsistency with the electrn micrscpe images. The majr prblem in the data analysis is the measurement f the diffracted intensity. In rder t extract the signal frm the nise in the X-ray patterns and t reliably separate meridinal and equatrial diffractin, methds were used which were develped riginally fr image analysis f electrn micrgraphs and fr signal prcessing in electrical engineering. These methds prvided a way f extracting infrmatin abut the structure frm diffractin patterns f disriented gap junctin specimens in which there is a significant amunt f disrder. The use f results frm X-ray diffractin, electrn micrscpy, and chemical analysis tgether t characterize the structure prvides mre infrmatin than simply adding up cnclusins derived independently frm the different techniques. The X-ray scattering density maps, calculated using cnstraints frm electrn micrgraphs, imply a distributin f lipid and prtein that must be there in the prprtins indicated by the chemical analyses. Requiring that the interpretatins f the different bits f infrmatin abut the structure be self-cnsistent reduces the ambiguities inherent in the separate bservatins. MATERIALS AND METHODS Islatin f gap junctins frm muse liver, electrn micrscpy, and X-ray diffractin were carried ut as reprted in the preceding paper (5). The X-ray phtgraphs were densitmetered with an Optrnics rtating drum scanner (Optrnics Internatinal, Inc., Chelmsfrd, Mass.). Optical densities were usually measured n a 100 ~m raster, althugh a 25 ~m raster was ccasinally used. The resulting square grid f ptical density measurements was averaged ver 10 ~ arcs at cnstant radius frm the center f the diffractin patterns, prducing a set f data n a plar grid. Back- 630 THE JOURNAL OF CELL BIOLOGY " VOLUME 74, 1977

grund was estimated frm the ptical density measured at the psitins f the zers f meridinal diffractin. When verlap f meridian and equatr was significant, cntributins frm the tw culd ften be separated by recgnitin f the distinctive peak shapes f the tw surces f diffracted intensity. Where ambiguities in backgrund determinatin arse, the ptical densities at a given radius frm the center f the diffractin pattern were analyzed as a functin f angle abut the center f the pattern. Angular decnvlutin f the data fr a disrientatin functin was ccasinally used, but culd prvide reliable backgrund determinatin nly n the very best riented specimens. After backgrund subtractin, the cntinuus diffractin n the meridian was crrected fr disrientatin and the Lrentz factr by multiplying the measured intensities by the square f the reciprcal space radius R = 2 sins/h, where 20 is the angle f scattering and ~. is the wavelength f the X rays used (1.54/~). The abslute value f the cntinuus transfrm was btained by taking the square rt f the resultant, where I(R) is the backgrund crrected signal. The structure factrs fr the equatrial reflectins were btained in a similar manner, first crrecting the reflectins fr multiplicity (ah,), as well as disrientatin and the Lrentz factr (R~), f F~kl = X/m~/~, where ahk is the multiplicity crrectin fr the reflectin (h, k), lwhenh = 0rk = 0rh =k ahk = ]. 1/2 Otherwise In these equatins, ne factr f R is due t disrientatin, and ne is the Lrentz factr. ANALYSIS OF X-RAY DATA Meridinal Diffractin Meridinal diffractin is due t electrn density fluctuatins perpendicular t the plane f the gap junctin, and cmplete analysis f this diffractin leads t the calculatin f the ne-dimensinal electrn density distributin alng this directin. Sme diffractin phtgraphs were f sufficient quality t allw the calculatin f the interference functin and the pair-crrelatin functin f the junctins stacked in the sample. As mentined in the preceding paper (5), significant differences in meridinal diffractin were bserved amng different specimens. In this sectin, the cmplete analysis f the meridinal diffractin frm speci- men E153 is described. In later sectins, the meridinal diffractin frm different specimens will be cmpared. A densitmeter trace f the meridinal diffractin frm specimen E153 is shwn in Fig. 1. The mdulus f the cntinuus transfrm, 11~, is determined by subtractin f backgrund and crrectin fr gemetric factrs. In rder t calculate the electrn density prfile frm this functin, the cntinuus transfrm must be phased, and its value at very small diffractin angles must be determined. Mre accurate calculatin f the electrn density prfile can be made by crrecting the data fr interference effects due t the stacking f junctins in the specimen. This crrectin als leads t an estimate f the pair-crrelatin functin fr the junctins in the specimen. The backgrund was subtracted by assuming that the minima f the meridinal diffractin are ndes f the transfrm. That this is, in fact, the case can be shwn by the minimum wavelength principle as discussed belw. Only tw ambiguities arise in this prcedure. Assuming that the minimum f abut 0.04 A -1 is a zer leads t an unexpected "bump" in an therwise mntnically decreasing backgrund. By pltting the ptical density (which has been measured n a plar grid relative t the center f the diffractin pattern) as a functin f angle at this radius n the diffractin phtgraph, it was fund that bth the meridian and equatr were minima f ptical density. The maximum at this radius crrespnds t the psitin f a lattice line assciated with the (1,0) r (2,0) reflectin. The bump can be seen in the backgrund n bth meridinal and equatrial densitmeter traces and is clearly due t an ffequatrial diffractin peak. The presence f this ff-equatrial peak lwers the reliability f the backgrund subtractin in its neighbrhd, but since its prfile must be smth and its magnitude is relatively lw, the errrs related t its presence are nt likely t be t great. The ther ambiguity in backgrund subtractin is in the regin f very lw intensity centered at 0.06 A-1. Here, the nise in the data is cmparable t the diffracted intensity and chice f backgrund may be in errr by an amunt crrespnding t the estimated signal. The questin as t hw many ndes there are in this regin f the diffractin pattern presents the nly ambiguity in phasing, and the phase chices culd be biased by the way the backgrund is subtracted. Chsing phases fr the meridinal diffractin MAKOWSKI, CASPAR, PHILLIPS, AND GOODENOUGH Gap Junctin Structures. H 631

Film 4 0 >- ---_ ------ i I i I = I i I i I 0,02 0.04 0.06 0.08 0,10 R'2 Sin #/~. FIGURE 1 Densitmeter trace f the meridian f the X-ray diffractin pattern frm specimen E153 shwn in Fig. 10a f the preceding paper. The reciprcal space crdinate R is defined in terms f the scattering angle 28, and the wavelength h f the X-rays used which was 1.54,~. At small scattering angles, the distance, R, is simply prprtinal t distance measured n the film. The ptical densities were measured n a 100-/zm grid fr Film 1 and a 25-p,m grid fr Film 4, and then averaged n arcs -*5 ~ frm the meridian. Fur films were expsed simultaneusly. Since each film absrbs abut 30% f the incident radiatin, Film 1 receives abut 27 times the expsure f Film 4. This allws measurement f a wide range f intensities with a single expsure. Traces taken frm Film 4 and Film 1 are shwn. In rder t shw the high-angle detail, the intensity scale n the tracing frm Film 1 is magnified ( 5) in the third trace shwn. The brken line represents the backgrund drawn t cnnect the minima identified as zers f the junctin transfrm. nce the backgrund is subtracted is relatively straightfrward. Since the gap junctin is centrsymmetric, the transfrm must be a real functin, and the phase chice is reduced t a sign chice. The minimum wavelength principle (3) is sufficient t determine the sign relatins amng adjacent peaks. Accrding t this principle, tw adjacent peaks in the diffractin frm a centrsymmetric structure f lateral extent, d, in real space must be f ppsite sign if the peak separatin in reciprcal space is less than 2/d. Electrn micrgraphs f gap junctins shw that the width is abut 150/~; therefre, any tw diffractin peaks clser than (1/75),~-1 t each ther must be f ppsite sign. This cnditin requires a sign change at all bserved minima in the intensity except the regin f lw intensity abut 0.06 ~-1. There is clearly a zer at abut 0.052 /~--1 Between 0.058 ~-~ and 0.062/k -~ there may be ne r tw zers. Clse inspectin f the densitmeter traces suggests that tw is mst likely. Hwever. since the magnitude f the nise in this regin is cmparable t the intensity f diffractin, a mre reliable demnstratin is necessary. The number f zers in this regin is imprtant since it will affect the assignment f signs fr the last tw diffractin fringes. These fringes cntribute significant high-reslutin detail t the calculated density prfile. Knwledge f the width f the junctin was used t reslve the ambiguity abut the number f zers in the regin abut 0.06/~-1 in the diffractin pattern. The electrn density prfiles fr the tw pssible chices f signs were calculated. These prfiles were truncated at -+90/~ frm the center since the maximum thickness f the junctin is definitely less than 180/k. The diffractin pattern was recalculated frm the truncated prfiles. The truncatin remves the cntributin f high spatial frequency nise and interference effects in the diffractin pattern. Cmparisn f the recalculated intensity with that measured shwed 632 THE JOURNAL r CELL BIOLOGY' VOLUME 74, 1977

that the structure crrespnding t nly ne zer in this regin wuld give rise t diffractin much strnger than that actually detected at spacings f abut 0.06 A -~. Only the chice f tw zers was cnsistent with the bserved intensity. Neither the uncertainties in measurement f backgrund nr the presence f interference effects substantially affect this analysis. In this regin f the pattern, diffracted intensity is hardly greater than backgrund; thus backgrund is accurately measured. Interference is a multiplicative effect which is negligible in regins f lw intensity. We cndude frm this analysis that there are tw zers between 0.058 and 0.062/~-1. The central maximum must be psitive since the density f the gap junctin is greater than that f water. All phase relatinships between adjacent peaks have therefre been established ut t a spacing f 0.1 ~,-]. The phasing f the meridihal diffractin determined by these methds is shwn in Fig. 2. The central maximum was measured int a spacing f 0.0042 A -~ using a lng camera (300 mm specimen-t-film distance). The central maximum was then extraplated int zer angle using the Shannn sampling therem (11). Care was taken t insure that the interference effects discussed belw did nt affect this estimate. With the sign chice in Fig. 2, the electrn density prfile shwn in Fig. 3 was calculated using the extraplated central maximum. The measured X-ray diffractin intensity is, in general, the prduct f a term cntaining infrmatin abut the structure f the scattering unit and a term due t interference f diffractin frm different scattering units in the specimen. The prperties f the interference functin are discussed in the appendix. Interference intrduces higher spatial frequencies int the diffractin pattern than wuld be present in diffractin frm a single unit. Examinatin f the electrn density prfile in Fig. 3 shws that significant ripple is present at dis- A rr tl n. I- 1.5 1.0 ~ ~ O ~ 0 0 0 O0 0 0 0 0 I 0.5 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 R = 2 Sin elk FIGURE 2 Meridinal diffractin amplitude and Furier transfrm f the membrane electrn density prfile crrected fr interference effects. The pen circles in the tp graph are measured frm the densitmeter traces frm specimen E153 (Fig. 1) and phased as described in the text. The slid line is the cntinuus transfrm crrected fr the interference effects due t stacking f the junctins. This curve is the Furier transfrm f the electrn density prfile in slid line in Fig. 3. The bttm graph, T(R), is a plt f the square rt f the interference functin as defined in the Appendix. This functin is the rati f the data pints and cntinuus curve pltted abve. The pen circles indicate T(R) as experimentally determined. The cntinuus curve is the T(R) calculated frm the pair-crrelatin functin shwn in Fig. 4. Since the experimental errr in T(R) is inversely prprtinal t the magnitude f F(R), data pints near the zers f F(R) are indeterminant and have been mitted frm this graph. IVIAKOWSKI, CASPAR, PHILLIPS, AND GOODENOUGH Gap Junctin Structures. H 633

Bilyer Gp Bilayer g / / w g, I I ~ ~-~ J ~ - - f ~ I O0 50 0 50 I O0 r (~.) FIGURE 3 Electrn density prfile f gap junctins. The cntinuus curve is the electrn density prfile crrected fr the interference functin. The high density peaks in the electrn density prfile crrespnd t the psitins f the plar grups f the bilayer lipids. The plar grups are separated by 42/~. acrss the bilayers and 45 A acrss the gap. The lw density minimum in the center f the bilayer is ccupied mainly by lipid hydrcarbn and prtein. The electrn density f this regin is much higher than wuld be expected fr pure lipid hydrcarbns. There must be a significant prtein cntent in this regin t raise the average electrn density t nearly that f water. The center f the gap has an electrn density cnsiderably greater than that f water, indicating that a significant fractin f the gap must als be ccupied by prtein. The electrn density prfile calculated directly frm the data frm specimen E153 is shwn by the brken line. This curve differs frm the crrected electrn density prfile nly at distances greater than the thickness f the junctin. The difference between these tw curves is the nly infrmatin available fr the calculatin f the interference functin which is the reasn fr the high experimental errr invlved in its determinatin. The ripple beynd the junctin bundary represents an averaged density prjectin f the neighbring junctin units. If the stacking arrangement f junctins were crystalline, the prjectin f the neighbring unit wuld be a mirrr image f the junctin shwn in the slid line. tances greater than 90.~ frm the center f symmetry f the junctin. Sme f this ripple is due t experimental errr, but mst f it is due t interference effects. By crrecting the diffractin data fr this effect, a mre accurate electrn density distributin may be btained and the pair-crrelatin functin calculated. The interference functin may be determined frm electrn micrgraphs f X-ray specimens using ptical diffractin as reprted in the preceding paper (5). In this study, the ptical transfrms were used nly as a guide t aid in the evaluatin f the X-ray results. In the ptical transfrm f the tracings shwn in the preceding paper, the diffractin fringes due t interference have a wavelength f abut 150 A. The interference damps ut slwly, with eight r mre fringes visible in strngly expsed patterns. As will be shwn belw, these prperties are cmpletely cnsistent with the interference functin determined frm the X-ray data. The interference functin was estimated frm the X-ray data by assuming that all calculated electrn density greater than 90 A frm the center f symmetry f the junctin was due t interference effects as discussed in the Appendix. The electrn density prfile was then refined using the real space cnstraint that the pair-crrelatin functin must be zer inside the interval _+140 abut the rigin. This is due t the fact that iunctins, which appear t be abut 150/~ acrss frm the electrn micrscpy, cannt cme clser than 150 /~ frm each ther, center-t-center. The value f 140 ~ was chsen fr the refinement t allw fr pssible interleaving f the junctins and t allw fr the finite reslutin f the data. The refinement has little effect n the electrn density prfile as shwn in Fig. 3. Hwever, it results in the calculatin f the interference functin shwn in Fig. 2 and the pair-crrelatin functin shwn in Fig. 4. The electrn density prfile in Fig. 3 shws that the gap junctin exhibits a typical lipid bilayer prfile with the electrn density elevated by a large amunt f prtein which spans the bilayer. The high electrn density in the gap regin is als indicative f high prtein cntent in the space between membrane bilayers. 634 THE JOURNAL OF CELL BIOLOGY" VOLUME 74, 1977

The pair-crrelatin functin shwn in Fig. 4 is sharply peaked between 150-160 /~. This indicates that the junctins in the pellet are capable f I J I I I I00 ZOO 300 400 SO0 FmURE 4 Pair-crrelatin functin crrespnding t the interference functin shwn in Fig. 2. The uncertainty in its determinatin is relatively high, but the general features are likely t be crrect. The high peak at 150-170/~ indicates that pairing f junctins is a highly favred interactin. Integratin f this peak shws that abut 26% f junctins have a nearest neighbr between 150 and 170/~ frm their center. The asympttic value f the pair-crrelatin functin is the number density f the specimen which is equal t 0.0014/~-1. T cnvert this t vlume fractin, it is multiplied by the extent f the junctins (abut 180 A) t btain a vlume fractin f abut 0.25. interleaving by 20-30/~ since the maximum extent f a single junctin frm the crrected density prfile (Fig. 3) is abut 180/~,. The value f the pair-crrelatin functin at large distances frm the rigin shuld equal the number density f the junctins in the specimen. Cnverting this t vlume density, the derived pair-crrelatin functin indicates that abut 25% f the specimen vlume is ccupied by gap junctins. This is cnsistent with the estimated water cntent f the centrifuge pellets. Equatrial Diffractin A densitmeter trace f the equatrial diffractin is shwn in Fig. 5. The hexagnal lattice f cnnexns in the gap.junctin gives rise t lattice sampling f the diffractin n the equatr. The first fur reflectins indexed as (1,0), (1,1), (2,0), and (2,1) are much strnger than any f the thers. The (1,1) and (2,0) are superimpsed n a strng cntributin frm the meridian. The (2,1) reflectin is brader than the ther reflectins, and shifted t a higher radius than wuld be expected frm the psitins f the ther equatrial / E 153 Film 3 Filml ~ Film I {xs) I I 0 \\ I * I * I, I * I * I 0.02 0.04 0.06 0.08 0.10 0.12 R=2 Sin 0/), FIGURE 5 Densitmeter traces f the equatr f diffractin pattern frm specimen E153. Optical densities were averaged n 10 ~ arcs. Vertical bars mark the calculated psitins fr crystalline reflectins frm a hexagnal lattice with lattice cnstant f 86.7 A. The backgrund indicated by the brken line defines the crystalline diffractin maxima. The backgrund is higher than that fr the meridian due t the presence f a mnchrmatr streak. Significant cntributin f meridnal diffractin t the ptical density n the equatr can be seen under the (1,1) and (2,0) reflectins. Althugh the first fur rders f diffractin are easily bserved, the lattice reflectins at higher angles are indistinct, being replaced by a brad cntinuus diffractin centered at abut 0.1.~-k The psitin f the (2,1) deviates slightly frm that calculated due t a significant ff-equatrial cntributin t the reflectin. MAKOWSKI, CASPAR, PHILLIPS, AND GOODENOUGH Gap Junctin Structures. 11 63S

reflectins. This was a cnsistent prperty f all diffractin patterns measured, and is mst prbably due t strng diffractin falling slightly ff the equatr n the lattice line crrespnding t the (2,1) reflectin. A slight "bump" in the backgrund between the (2,1) and (3,0) reflectins cmes frm ff-equatrial diffractin as discussed in the analysis f the meridinal diffractin. Beynd the (3,0), the reflectins are very weak and nly estimates f their intensity can be made. The bserved intensities frm specimens E153 and F38 are pltted in Fig. 6. Fr the very weak, higher index reflectins, an estimate f the maximum pssible intensity cnsistent with the bserved data is given. Nte, hwever, that the intensities f sme f these reflectins are nt significantly different frm backgrund. The minimum intensity fr these reflectins is zer, and the maximum is set by the nise level which determines the uncertainty in the backgrund. At higher angles the sampled diffractin is replaced by a brad band f cntinuus diffractin centered at abut 10,~ spacing n the equatr. This cntinuus diffractin is typical f diffractin frm a-helices. Measurement f the ptical density as a functin f angle abut the center f the diffractin pattern shws that the disrientatin f this band is apprximately the same as that frm ther reflectins. This suggests that the band f intensity is cming frm a-helices riented within 20 ~ f the perpendicular t the plane f the junctin. An analysis f the hexagnal lattice reflectins IFI li E 155 F38 I = I = ~..a 0.02 0.04 0.06 R= 2 SinS/X FIGURE 6 Magnitude f the structure factrs f equatrial lattice reflectins fr specimens E153 (9 and F38 (A). The lattice cnstant f E153 is 86.7/~, and f F38, 82 A, thus the F38 reflectins ccur at larger spacings. leads t the calculatin f the prjectin f the electrn density nt the plane f the junctin. Electrn micrscpy prvides a guide in the chices f phases fr the equatrial diffractin. As discussed in the preceding paper, electrn micrscpe and bichemical evidence shws that the cnnexn, abut 30 ~ in radius, is centered n the sixfld axis f the unit cell. Mst f the rest f the unit cell must be made up f junctin lipid. The meridinal data indicate that the lipid is in a bilayer arrangement. The equatrial prjectin f a lipid bilayer is expected t be flat since n largescale lateral rder which culd prduce significant density fluctuatin is likely in the lipid packing. The prjected lipid density will be significantly less than that f prtein. These facts prvide cnstraints n the frm f the electrn density prjectin. It must be relatively flat utside 35/~ radius frm the sixfld axis with much higher density inside that radius. Finding a phase chice that satisfies these cnstraints demnstrates that the equatrial X-ray pattern is cnsistent with ther infrmatin abut the structure. Furthermre, having determined the phases, the X-ray data prvide infrmatin abut the distributin f prtein within the cnnexn. The P6m symmetry f the junctin in prjectin cnstrains the equatrial reflectins t be real. As fr the meridian, the phase chice is reduced t a sign chice, and nly a few electrn density prjectins need t be examined. Fr the first five reflectins, all pssible electrn density prjectins were calculated. Thse with unacceptable features, such as a peak n the threefld axis, were discarded, and nly ne phase chice was fund t be cnsistent with all the structural cnstraints f the electrn micrscpe data. The prjectins calculated fr specimens E153 and F38 using this phase chice are shwn in Fig. 7. Phase assignments fr higher-rder reflectins were ambiguus. Hwever, because f the very lw intensities f these reflectins, the prjectins calculated including the higher reslutin data are similar t the general frm f thse shwn in Fig. 7. Several imprtant features f these electrn density prjectins are wrth pinting ut. First, the very lw electrn density at the sixfld axis was nt presumed frm the micrscpe data. It is a cnsistent feature f prjectins which satisfy the necessary cnstraints f the electrn micrgraphs. Lw density near the sixfld axis suggests that the prtein des nt fill the center f the cnnexn. Secnd, the diameter f the high den- 636 THE JOURNAL OF CELL BIOLOGY" VOLUME 74, 1977

FmURE 7 Electrn density prjectins f specimens E153 and F38. The lw-reslutin prjectins f E153 (a) and F38 (b) were calculated frm the first five reflectins f the equatrial data using the phase cmbinatin (+... ). This is the nly phase cmbinatin which prduced electrn density prjectins cnsistent with the structural infrmatin btained frm electrn micrscpy. The lattice cnstant f E153 is 86.7 A; that f F38 is 82.0/~. The data used t calculate these prjectins extended ut t a spacing f 25 A,. In these prjectins, the cnnexns appear circularly symmetric with a diameter f abut 50 A, and a lw electrn density cre abut 20 A in radius. sity regin in these prjectins is less than that f the stain-excluding regins seen in micrgraphs. This may indicate that the diameter f the cnnexn in the gap regin is greater than that f the prtin extending thrugh the bilayer. Finally, there is sme difference between the prjectins frm E153 and F38, suggesting a slightly different structure fr the tw specimens which may be crrelated with the difference in lattice cnstant. This is particularly evident between 30 and 40 A frm the sixfld axis, where the electrn density f F38 is greater than that fr E153. This is mre clearly illustrated in the crss sectins f these prjectins shwn in Fig. 10. INTERPRETATION OF THE RESULTS The Structure The electrn density prfile and equatrial prjectin prvide an accurate, lw-reslutin image f the gap junctin ultrastructure. The prfile in Fig. 3 was calculated using X-ray data extending t a spacing f abut 10 A. It shws a distributin f scattering density typical f that expected fr a pair f lipid bilayers with their average electrn density elevated relative t the slvent density by prtein which extends thrugh bth bilayers and the extracellular gap. The high density peaks f the lipid plar head grups are separated by 45,~ acrss the gap and 42,~ acrss the bilayer. The shape f the membrane prfile is very similar t that determined fr pure lipid bilayers. This means that the cntributin frm prtein t the average scattering density must be nearly cnstant acrss the width f the bilayers. The gap junctin bilayer prfile is asymmetric. Between the plar head grup peak and hydrcarbn minimum there is a step in the electrn density distributin. In the extracellular half f the bilayer, this "step" is much higher than that in the cytplasmic half. This asymmetry may be due t a nn-unifrm prtein distributin. Hwever, it crrespnds clsely t a similar asymmetry in myelin (6) which has been attributed t an asymmetric chlesterl distributin. The higher density step in myelin is als in the extracellular half f the bilayer. The width f the plar peak n the cytplasmic side f the bilayer is significantly greater than that MAKOWSKI, CASPAR, PHILLIPS, AND GOODENOUGH Gap Junctin Structures. H 637

n the extracellular surface. This suggests that additinal prtein may be assciated with this inner surface f the membrane. The pair-crrelatin functin shws that islated junctins can pack with a separatin f 150-160/~. Prtein n the cytplasmic surface f the junctin must be capable f interleaving with prtein n adjacent junctins t make this clse packing pssible. The electrn density prjectins in Fig. 7 were calculated using data which extended t a spacing f 25 A,. In these prjectins, darker shading crrespnds t higher average scattering density. A lipid bilayer will have a lw electrn density in these prjectins, nly slightly greater than that f slvent. Thus, the dark areas are an image f the prtein distributin. This image is smewhat different frm the ne prvided by the electrn micrgraphs f negatively stained gap.junctins presented in the preceding paper (5). In the filtered images in Figs. 2 and 3 f that paper, lightly shaded areas are stain-excluding regins, and these were interpreted t crrespnd t the distributin f prtein within the extracellular gap. These stain-excluding regins appear t ccupy a much larger prtin f the unit cell than the average prtein distributin seen in the equatrial prjectins. This cmbinatin f results frm electrn micrscpy and X-ray diffractin suggests that the prtein within the gap ccupies a larger fractin f the unit cell area than it des within the bilayer. The dimensins f the prtein within the gap can be estimated frm the electrn micrgraphs f negatively stained specimens. The mean diameter f the cnnexn spanning the bilayer can be estimated frm the end n prjectin calculated frm the equatrial diffractin data. The diameter f the cnnexn within the gap is 60-70 A, (Figs. 2 and 3 f the preceeding paper [5]); whereas the mean prjected diameter is 50-55 A, (Fig. 7). The end n electrn density prjectins f the cnnexn have a regin f lw density abut 20 A, in diameter centered n the sixfld axis. This crrespnds t a heavily staining regin in micrgraphs f negatively stained gap junctins. Frm its lwer electrn density, there can be little prtein here. The high electrn density regin between 10 ~ and 25 A, frm the sixfld axis must cntain mst f the junctin prtein. A substantial fractin f this prtein is within the lipid bilayer. Beynd 25 A frm the sixfld axis, the electrn density cntinues t decrease t a minimum at the threefld axis. There is relatively little prtein n the threefld axis, and the bserved electrn density there is prbably mainly due t lipid and slvent. The chemical interpretatin f these prjectins will be discussed further belw. Structural Variatins Althugh all the electrn density prfiles and prjectins calculated are similar, there are sme significant differences. Figure 8 shws ne example f these differences. This figure cmpares the lw-reslutin electrn density prfiles f specimens E153 and F38. Althugh the width f the bilayers in the tw specimens is abut the same, the F38 bilayer is 4 A clser t the center f the gap than that f E153. The electrn density at the middle f the bilayer is substantially larger in F38, suggesting that the prtein t lipid rati within the bilayer is greater in this specimen. F38 has a much smaller lattice cnstant than E153 (82 A, cmpared t 87 ~ fr E153). A larger prtein t lipid [ I 10 20 30 40 50 60 70 80 d() FIGURE 8 Lw-reslutin electrn density prfiles calculated frm the meridians f specimens E153 and F38. The peak psitins f the plar head grups are marked. Althugh the bilayer thickness is the same fr the tw specimens, the distance f the bilayer frm the center f the gap is abut 4,~ less fr F38. The electrn density at the center f the bilayer is higher in F38. This is a result f the smaller lattice cnstant in this specimen which is assciated with a lwer lipid t prtein rati in specimen F38 cmpared t E153. I 638 THE JOURNAL OF CELL BIOLOGY" VOLUME 74. 1977

rati results because a decrease in lattice cnstant crrespnds t a decrease in the lipid cntent f the junctin if the crss-sectinal area f the prtein is invariant. F38 als has substantially higher electrn density in the center f the gap. By making assumptins abut the electrn densities f prtein, lipid hydrcarbn and water, the amunt f prtein in the center f the gap and in the bilayer can be calculated. The electrn densities assumed are 0.40 e//~ a fr hydrated prtein, 0.27 e/a 3 fr lipid hydrcarbn, and 0.33 e/ A 3 fr water. Assuming that the center f the bilayer is ccupied by prtein and lipid hydrcarbn nly, the prtein cntent there is calculated t crrespnd t a prtein cylinder with a radius f 26 A, fr E153 and 28/~ fr F38. This is cnsistent with the prtein distributins shwn in the equatrial prjectins in Fig. 7, and shws that, t within experimental errr, the amunt f prtein within the bilayers f these tw specimens is the same. Assuming that nly prtein and water ccupy the center f the gap, the same calculatin can be made fr this prtin f the junctin. The prtein cntent in the gap crrespnds t a prtein cylinder abut 30 A in radius fr E153 and abut 39,~, in radius fr F38. Examinatin f the electrn density prjectin fr F38 in Fig. 7, r the crss sectins f this prjectin pltted in Fig. 10, shws that there is a high density regin extending ut t abut 40 A frm the sixfld axis which is less prnunced in the E153 prjectin. The electrn density prfiles lcalize this extra density bserved in the prjectin t the gap between bilayers. It appears that when the gap decreases in width, the prtein and lipid mve twards the gap as a unit and the extra prtein in the gap spreads ut ccupying a cnsiderably larger fractin f the gap area. The vlume f prtein within the gap, hwever, appears t remain the same. Our X-ray diffractin measurements shw that there is a strng crrelatin between the width f the extracellular gap and the lattice cnstant. In Fig. 9 the psitins f the secnd, third, and furth meridinal zers are pltted vs. lattice cnstant fr 33 specimens. When the lattice cnstant decreases there will be an increase in the prtein t lipid rati in the junctin lattice. Hwever, this will have very little effect n the psitins f the meridinal zers unless the width f the junctin changes. A series f mdel calculatins were dne which indicated that the change in the psitins f the meridinal zers is due t a change in the gap I1-1) 0.029 0.020 0.02~ 0.021 0.019 0.018 0.017 0.011 9.** #* ~ 0.010 9 u. W 0.009 r t i t! 11 sa /3 84 /s 86 /7 /s 00 a 9 lattice cnstant (g) FIGURE 9 A plt f the psitins f meridinal zers vs. lattice cnstant fr thirty-three specimens. The slid lines are the results f a mdel building calculatin described in the text. Several partiauy dried specimens were examined which had very lw values fr the psitin f the first meridinal zer measured. These specimens are included in the figure and are respnsible fr the relatively brad scatter in the bserved values f the psitin f this zer. width. This was cnsistent with several electrn density prfiles which were calculated fr specimens with different lattice cnstants. In the mdel which mst satisfactrily reprduced the psitins f the meridinal zers, the gap width decreased by 1,~ fr every 1.3 /~ decrease in lattice cnstant. Fr this mdel the bilayer prfile was held invariant and, as the gap was narrwed, the vlume f prtein within the gap was maintained cnstant as suggested by the cmparisn f the E153 and F38 prfiles in Fig. 8. The psitins f the zers predicted by this mdel are pltted in Fig. 9. There is, hwever, cnsiderable scatter in the individual measurements. Fr example, cmparing specimen F38 with E153 there is an 8 ~, decrease in gap width crrelated with nly a 5 A decrease in lattice cnstant. Structural Mdels Frm the X-ray diffractin, electrn micrscpe and chemical data, mdels fr the spatial distribu- 9 MAKOWSI~, CASPAR, PHILLIPS, AND GOODENOUGH Gap Junctin Structures. H 639

tin f the prtein, lipid, and water in gap junctin structures were cnstructed. The mdels derived fr specimens E153 and F38 are shwn in Fig. 10. The chemical mdels were derived in the fllwing way. The meridinal prfiles were put n an abslute scale by assuming that the electrn density f the plar head grup peak is 0.40 e/~ 3 and that that f slvent is 0.33 e//~ 3. The amunt f prtein in the gap and in the bilayer was deter- mined by assuming that the gap cntained nly prtein and water, and that the center f the bilayer cntained nly prtein and lipid hydrcarbn. The similarity f the bilayer prfile t previusly derived prfiles in pure lipid systems supprts the assumptin that the distributin f prtein in the bilayer is relatively unifrm acrss its width. Electrn density extending int the cytplasm frm the bilayer surface was attributed t 3000 -- 2000,% < 1000 Wter Wter Lipid Hydrcarbn 2000 Lipid Plar Grups 1000 Lip,,.t.,ll Lipid Plar Grubs -- Hydrcarbn Prtein Prtein 20 40 60 80 r (~1 20 40 60 I 8O m *< 0.40 I 0,35 0.35 w 20 ~ 60 SO r(~,) 20 40 60 80 r (A) 9 E 153 @ F38 Water J Water 5O 50 - ~ Prtein Prtein,T- I Lipid Lipid O J 20 40 r(~,) ;" I _ E 153 ~ 0.40 I w 0.35 IT,, - 20 40 r(~) I 0 I ' I, 20 40 r{~) 0.40 i ~ 0.33 20 40 r(~) \ F38 640 THE JOURNAL OF CELL BIOLOGY" VOLUME 74, 1977

additinal prtein. The ttal prtein vlume was calculated t be in the range f 180-200,000 A 3 fr bth specimens E153 and F38. Similar calculatins were made fr the equatrial prjectins by assuming that the center f the cnnexn has the electrn density f slvent and that the peak lcated 17-18 /~ frm the sixfld axis has the electrn density f prtein. At lw reslutin, cylindrically symmetric mdels are cnsistent with the electrn density prjectins. The prtein vlume calculated frm the equatrial prjectins frm E153 and F38 fall in the range f 190-210,000 A3, cnsistent with that derived frm the meridian. The meridinal and equatrial mdels were tested fr cnsistency by cmparing the ttal prtein and ttal lipid vlumes derived. Details f the tw prjectins were als cmpared. Fr instance, frm a cmparisn f meridinal prfiles, it was fund that the electrn density in the gap regin f specimen F38 is much higher than that f E153. This difference crrespnds t a difference in the equatrial prjectins f these tw specimens 30-40 A frm the center f the cnnexn. The crre- spndence between these tw bservatins presents an image f the variability f the prtein distributin within the gap. The meridinal mdels als explain the difference in the images f the cnnexn in the equatrial prjectins and in the filtered images f negatively stained junctins. Prtein ccupies a larger fractin f the area in the gap than in the bilayer. The variability in the staining prperties f islated junctins (5) als appears cnsistent with these mdels. Fr example, there wuld be much less rm fr stain t accumulate within the gap f the junctins in specimen F38 than in specimen E153. The prtein t lipid rati can als be calculated frm the chemical mdels. In the mdels fr specimen E153, the junctin is 52% prtein by weight and in F38 it is 57% by weight. This is cnsistent with the 1:1 weight rati f prtein t lipid estimated frm the nitrgen and phsphrus cntent and the buyant density f islated gap junctins as reprted in the preceding paper (5). The ml wt f prtein in a cnnexn as estimated frm these mdels is in the range f 140-170,000. If the cnnexn is a hexamer, it is made up f FIGURE 10 Diagram representing the relative psitins f the chemical cmpnents f gap junctins. The distributin f prtein, lipid, and water perpendicular t the plane f the junctins is represented by blck diagrams fr specimens E153 (A) and F38 (B). The electrn density prfiles resulting frm these distributins are superimpsed n the lw-reslutin electrn density prfiles fr these tw specimens. The distance crdinate, r, in (A) and (B) is distance frm the center f the gap measured nrmal t the junctin plane. The elecrn density f the prfiles is built up frm the partial areas f prtein, lipid, and water fr the tw specimens. The abslute electrn density males are based n setting the electrn density f water t 0.33 e/a. a, f hydrated lipid plar grups and prtein t 0.4 e/.~ a, and f lipid hydrcarbns t 0.27 e/,~ a. The lipids in the step regins include cntributin frm chlesterl and have smewhat higher electrn densities. The prtein cntent f the tw specimens is the same, but the lipid cntent f F38 is lwer because f the decrease in the area per unit cell with smaller lattice cnstant. In (C) and (D), the distributin f prtein lipid, and water parallel t the plane f the junctins is represented by blck diagrams f the chemical distributin alng a line extending ut frm the center f a cnnexn. The distance crdinate, r, in (C) and (D) is radius frm the sixfld axis. Fr simplicity, a circularly symmetric mdel has been cnstructed. The electrn density prjectins fr specimen E153 (C) and F38 (D) are built up frm the partial thicknesses f the cmpnents being prjected. The electrn densities calculated frm the chemical distributins are cmpared t crss sectins f the electrn density prjectins shwn in Fig. 7. The smth slid line is the electrn density alng a line cnnecting tw adjacent sixfld axes in these prjectins. The brken line is the electrn density distributin alng a line cnnecting a sixfld axis and a threefld axis. The vlume f prtein in the mdels fr bth meridinal prfiles and bth equatrial prjectins is the same. Althugh its distributin is different in the tw specimens, its distributin in the prfile is cnsistent with its equatrial prjectin in the individual specimens. The lipid distributins pstulated fr the prfiles are als cnsistent with the prjectins. There is mre lipid in specimen E153 than F38 because f the difference in lattice cnstant. Frm these mdels, the weight rati f prtein t lipid in the gap junctin is abut 1:1, prtein accunting fr 52% f the dry weight frm the mdel fr specimen E153 and 57% f the dry weight frm the mdel fr specimen F38. Thus, the distributin f chemical cnstituents as defined by these mdels is cnsistent with the results frm measurements f nitrgen and phsphrus cmpsitins and buyant density reprted in the preceding paper (5). ~takowski, CASPAR, PHILLIPS, AND GOODENOUGH Gap Junctin Structures. II 641

prtein units with a ml wt f 23-28,000. The structural mdels cnstructed in this way are cnsistent with the X-ray diffractin, electrn micrscpe, and chemical data. Many f the cnclusins f this study are summarized in Fig. 11, which is a drawing f the gap junctin structures. The mrphlgical units are cmpsed f a dimer f hexamers f the cnnexin mlecule; ne hexamer is assciated with each membrane. The hexamers frm a prtein tube with an uter radius f abut 26 A and an inner radius f abut 10 ~. An aqueus channel extends mst r all f the way thrugh the cnnexn frmed by the six prtein mlecules. The hexamers are surrunded by the lipids f the membrane bilayers. Each cnnexn is cnnected acrss the gap t a secnd hexamer, the pair making up a single mrphlgical unit. The prtein within the gap has been bserved in significantly different cnfiguratins as represented by the tw drawings in Fig. 11. The prtein in the gap is capable f maintaining the cnnectin between cells ver differences in gap width f at least 8 A. The changes in gap width are crrelated with changes in the average distance between cnnexns in the lattice. DISCUSSION There are tw key facts abut the gap junctins which must be cnsidered in any structural study. The first is the prpsed functin f these structures as a pathway fr intercellular cmmunica- tin. The secnd is their existence as a differentiated regin f membrane which maintains chemical hmgeneity and structural integrity in the fluid, hetergeneus envirnment f cell plasma membranes. All evidence frm physilgy, electrn micrscpy, and X-ray diffractin is cnsistent with the existence f an aqueus channel extending thrugh the cnnexns. Physilgical experiments indicate that these channels are pen t passage f small mlecules between cytplasms, but that the channels can clse in respnse t changes in the cell envirnment (1). Indeed, it seems necessary fr the survival f the rganism that the junctins be able t seal in respnse t changes in cytplasmic cnditins, e.g., the death f ne f the adjining cells. Electrn micrscpy and X-ray diffractin data indicate that the channel is 8-10 A in radius, but cannt demnstrate the cntinuity f the channel ver the entire width f the junctin. Electrn micrscpy has revealed structural changes in the large subunit gap junctins f the crayfish that are crrelated with the state f electrical cupling between cells (10). These studies shw that when the junctins were subject t cnditins which resulted in uncupling, the gap width and lattice cnstant f the junctins decreased. This was accmpanied by an increase in the curvature f the junctin membranes. The similarity between these bservatins and the structural variatins bserved by X-ray diffractin FIGURE 11 Gap junctin structures. These drawings illustrate the variatin in the junctin structure revealed by X-ray diffractin studies. The upper drawing crrespnds t specimen E153 with an 87-/~ lattice cnstant, and the lwer t specimen F38 with an 82-/~ lattice cnstant. The 42/~ dimensin fr the membrane bilayer thickness crrespnds t the separatin f the peaks in the electrn density prfiles, which mark the mean psitins f the lipid plar grups. The verall thickness f the bilayer is abut 10/~ greater. The gap in F38 is 8/~, smaller than that in E153 as measured frm the electrn density prfiles. The 60-/~ diameter cnnexns are hexamers f a prtein mlecule abut 80/~ lng and 20/~ wide. The prtein subunits f each cnnexn are arrayed t frm an axial channel with a maximum diameter f abut 20/1~. The sectined view f the cnnexn indicates that the prtin f the prtein traversing the bilayer is smewhat narrwer than the ends f the mlecule n the cytplasmic and extracellular sides. Cperative variatins in the side-t-side distances between cnnexns in the lattice are linked t changes in the gap separatin. Cnfrmatinal changes appear t ccur in the part f the prtein spanning the gap while the interir prtins f the cnnexn mlecule seem t be relatively invariant. Changes in lattice cnstant f 5 /k and mre require either highly flexible links between the prtein mlecules r indirect cupling thrugh the lipid phase. Prtein-prtein cntacts have nt been illustrated since there is n direct evidence fr such interactins. The cnnexn units which traverse the pair f membranes are shwn flating in the twdimensinal lipid layers. This representatin f the gap junctin membrane structure can be viewed as a liquid crystalline versin f the fluid msaic mdel fr cell membranes. The bilayer structure in the gap junctin is very similar t that seen by X-ray diffractin in ther membranes. The crdinated cnfrmatinal changes in the cnnexn at the level f the gap, schematically illustrated in the drawings, culd be related t the regulatin f intercellular cmmunicatin. 642 THE JOURNAL OF CELL BIOLOGY" VOLUME 74, 1977

~'~KOWSKI, CASPAR, PHILLIPS, AND GOODENOUGH Gap Junctin Structures. H 643