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1. MATERIALS AND METHODS

Sample Preparation

The skin was removed from newborn mice and was placed in phosphate buffered saline

supplemented with 0.5 mM MgCl2 and 0.9 mM CaCl2. Pieces were cut ~2 mm in diameter and

immediately transferred into specimen carriers for high pressure freezing. Excess buffer was

removed with a paper point and the specimen chamber was filled with either 1-hexadecene, yeast

paste or 10% ficoll was added to eliminate air pockets; 1-hexadecene produced marginally better

results. Specimens were then frozen within 1 min in a high pressure freezer - either the Balzers

HPM 010 (Bal-Tec Products, Middlebury, CT) or the Leica EM PACT (Leica Microsystems Inc.,

Bannockburn, Ill.). In the latter case the operating pressure and rate of temperature change were

measured at ~2050 bar and ~17000°C/second, respectively. For freeze substitution, these

samples were placed in a solution of acetone containing 1% OsO4 and 0.1% uranyl acetate at -

99ºC for 6-8 hr, followed by -90ºC for 24-48 hr, -60ºC for 24 hr, and -30ºC for 18 hr;

temperature was maintained in a Balzers FSU 010 and, during transitions, was increased slowly:

2 hr for the first transition, 6 hr for the others. After slowly warming to 0ºC, the sample was

washed three times in pure acetone and warmed to room temperature. Tissue was carefully

separated from the sample carrier and transferred to a glass vial for infiltration with LX112 resin

(1:1 weight ratio of NMA and DDSA, Ladd Research Industries, Williston VT). Samples were

placed in flat molds and polymerization was done at 45ºC for 18-24 hr followed by 60ºC for 24-

48 hr. 30-70 nm sections were placed on 200 mesh fine bar hexagonal grids (Ted Pella, Inc.,

Redding CA) that were coated with formvar. These sections were stained with 3% uranyl acetate

in 70% methanol for 3 min, washed with water, stained with SATO lead stain for 2 min and again

rinsed with water. For tomographic data collection, sections were treated with 0.02% polylysine

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for 7-10 min, rinsed with water, and incubated 1-2 min with a solution of 10 nm colloidal gold

(Ted Pella, Inc., Redding CA) prior to a final water rinse. The colloidal gold was dispersed with

a bath sonicator for 10-15 min prior to application and its concentration was empirically adjusted

to provide a density of particles suitable for fiducial alignment. Finally, the section was coated

on both sides with 5-10 nm of evaporated carbon.

Electron Microscopy

Images were recorded with CM200FEG electron microscope (FEI Corp., Eindhoven,

Netherlands) equipped with a 1k x 1k Multiscan 794 CCD camera (Gatan Corp., Pleasanton CA)

using either the standard single-tilt specimen holder or a model 670 ultra high tilt holder (Gatan

Corp.). An automated system was used to collect the data consisting of EMACT (1) and

EMSCOPE (2). Samples were pre-irradiated with ~105 electrons/nm2 prior to collecting images

at 1º intervals either between +/-60º or +/-75º, depending of the holder. Dual axis tilt series were

obtained by removing the sample, rotating the grid ~90º, reinserting the sample and collecting a

second tilt series. Microscope magnification was 38 - 66kx, providing 0.55-0.93 nm/pixel after

binning the CCD images to 512x512. Defocus was 0.4-0.5 µm, which placed the first zero in the

contrast transfer function beyond the resolution limit of the images. The cumulative electron

dose during imaging was ~105 electrons/nm2.

Image analysis and 3D reconstruction

After assembling images into a single file with PRIISM (3), alignment was done with IMOD

(4) using 10-20 colloidal gold particles as fiducial markers. For dual-axis tilt series, each single-

tilt series was reconstructed individually with IMOD, aligned by cross-correlation and averaged

in real space. Although anisotropy in resolution is often a problem in tilt reconstructions by

electron microscopy, the use of dual-axis tilt series with full coverage up to +/-75 degrees made

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the corresponding artifacts negligible. Indeed, the definition of features is virtually

indistinguishable in X-Y and Y-Z sections. Fig S2 illustrates the effect of tilt range and of adding

the second tilt axis on anisotropy in the resolution. Effects of anisotropic resolution include the

presence of a halo, the elongated shapes of gold particles, and conical streaking in Y-Z sections;

all are quite evident in single-tilt axis reconstructions with a range of +/-60°. Increasing the tilt

range to +/-75°, or adding a second axis both produce significant improvements. The best results

are obtained by using dual-axis tomography with a range of +/-75º.

Segmentation and modeling

Overall, image tilt series were collected from 14 different desmosomes, 8 of which were

selected for tomographic reconstruction and 5 of which were used for delineation of the

molecular components. The reconstruction was segmented with AMIRA (TGS Inc., San Diego,

CA) in an iterative process that involved delineating molecular densities and fitting the x-ray

structure of C-cadherin 1L3W (5). Three videos are included as part of these supporting online

materials: the first two show intercellular densities as one scans through the sections composing

the 3D map of desmosomes "R" and "P", and the third shows the verification of the atomic fit

relative to the densities within the tomogram for desmosome "P". Even casual inspection of the

tomograms reveals distinct densities crossing the intercellular space, which are consistent with

the size of cadherin molecules (videos 1 and 2). Initially, this region of the tomogram was

displayed with a rather high density threshold to reveal numerous curved densities running

between the membrane surface and the midline. Depending on the threshold, a portion of these

densities were discontinuous, making the fit within a group of tangled molecules ambiguous.

These discontinuous densities were used for initial fitting of C-cadherin x-ray structures, starting

with well separated densities near the membrane surface and attempting to follow the relevant

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density into the midline. Fitting only involved rigid-body docking of the entire x-ray structure

without any changes of domain geometries or secondary structure. After fitting several

molecules within a local region, lower density thresholds were considered in refining the

molecular envelopes and the fitting was reevaluated to minimize molecular clashes and to

resolve ambiguities about density assignments within the midline. These assessments involved

scrolling back and forth through the original map to ensure that the original stain distribution

accurately represented the location of the molecules (video 3). This fitting accounted for the vast

majority of densities in the map, though some density remained near the membrane surface. It is

possible that this extra density corresponds to the E48 antigen, which is a GPI anchored protein

of 128 residues that is expressed in keratinocytes and squamous tumor cells (6) and that co-

purifies with desmosomes from bovine muzzle (7). This protein belongs to the Ly-6 family and

has been shown to function in selectin signaling pathway (8, 9). Although early immunolabeling

studies suggested its presence in the midline, its small size and GPI anchor are more likely to

place it next to the surface of the membrane.

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2. SUPPORTING TEXT

Comparisons of Cadherin Sequence

The N-terminal domain (EC1) has the highest sequence homology across the cadherin

family with N-cadherin, for example, showing 55% identity to C-cadherin and 37% identity to

desmosomal cadherins; identities for the entire extracellular region (EC1-EC5) are 44% between

N- and C-cadherin and 32-37% between these and the desmosomal cadherins (Table S1). This

high level of conservation is consistent with a common structure for classical and desmosomal

cadherins (10), especially considering that sequence identities amongst individual EC domains of

classical cadherins are considerably lower (e.g., 5-24% for C-cadherin, Table S2 ) yet have been

shown to adopt a consistent fold (5). This fold is likely stabilized by a conserved pattern of Pro,

Gly and hydrophobic core residues, which also extends to the more distantly related proteins in

the immunoglobulin family (10, 11). A lower conservation for the fifth extracellular domain

(EC5) reflects its unique features, which include a series of conserved cysteines and a substantial

truncation and genetic polymorphism in desmoglein (12, 13).

Flexibility between EC Domains

Local variations in the cadherin densities within our tomographic map suggest molecular

flexibility that is consistent with variability in the angle between individual EC domains in x-ray

structures. There is good evidence in the literature to support flexibility in the angles between

individual EC domains. In particular, the angle between these domains is variable, as shown

both by comparison of two-domain structures from N-and E-cadherins (14) and by pairwise

comparison of domains composing the EC1-EC5 structure of C-cadherin. The latter shows

interdomain angles from 106-137º with an ~180º disparity in the skew angle of EC3/EC4 relative

to the others (5). The linker regions between successive EC domains represent hinges for this

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flexibility and calcium binding by these linkers appears to modulate the magnitude of this

flexibility. As a result, calcium has documented roles in progressively straightening of the

molecule (15) and in reducing proteolytic susceptibility (16). On an atomic level, these roles

reflect the recruitment of side chains from the adjacent domains to these calcium binding sites,

the geometry of which enforces an obtuse angle between domains and structures the linker

peptide.

Flexibility in the Intermolecular EC1 Domain Interface

A different kind of structural flexibility is evident in the intermolecular EC1 interactions that

we have identified from our desmosome maps. For molecules involved in W-, S- and λ-shapes,

there is considerable variability in the angle between interacting molecules (Fig. S5). Similar

variability was seen in the W-shape in x-ray structures from various crystal forms displaying

intermolecular angles ranging between 54 and 88º (5). This observation is consistent with the

innate flexibility in the N-terminus of EC1, which is further manifested by its variable

disposition in the x-ray structures. Specifically, although the short β-strand at the N-terminus

was involved in stabilizing the first strand dimer seen in EC1 constructs of N-cadherin (17), the

latest C-cadherin structure shows no such N-terminal strand involved in the strand-dimer

exchange (5). In fact, the first 6 residues (DWVIPP) do not form hydrogen bonds to any other

piece of secondary structure. Furthermore, the main chain of these N-terminal residues is also

unconstrained in structures of EC1/EC2 constructs from both E- and N-cadherins, where the

critical Trp is either disordered or inserted into its own hydrophobic pocket, respectively (15,

18). Thus, the primary stabilizing element of the EC1 intermolecular interface would appear to

be the Trp side chain within the hydrophobic pocket.

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3. SUPPORTING FIGURES

Fig. S1. Projection image of an ultra-thin (~35 nm) section from skin prepared with high

pressure freezing and freeze sustitution. Although this section is too thin for the tomographic

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analysis of cadherin packing, the intermediate filaments and extracellular domains of cadherins

are more clearly seen in the projection image due to minimal superposition. In many cases,

individual cadherin molecules can be seen crossing from the membrane to the midline. The

white bar corresponds to 500 nm.

Fig. S2. Determinants of isotropic resolution. (A-B) X-Y slices from samples K and R,

respectively (Table 1). Sample K has a tilt range of +/-60° and white rings are visible around the

gold particles, reflecting artifacts from missing data (i.e., tilt angles between 60-90°). Sample R

has a tilt range of +/-75° and as a result these artefacts are much diminished. Images were

recorded at 38kx and 66kx respectively, accounting for the apparent difference in gold particle

size. (C-D) Y-Z sections from single-axis tilt series with tilt range +/- 60° show significant

artifacts from the missing wedge of data, namely a white zone around the gold particles at the

top of the section and conical tails coming down at 30° angles from each gold particle. (E-F) Y-Z

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sections from dual-axis tilt series with tilt range +/- 60° showing a reduction of the above-

mentioned artifacts. (G-H) Y-Z sections from single axis tilt series with tilt range +/- 75 degrees.

Main artifacts are a white zone around the gold particles and a slightly elongated shape of these

spherical particles, which reflects anisotropic resolution. (I-J) Y-Z sections from dual-axis tilt

series with tilt range +/-75°. Artifacts are minimal with round gold particles and minimal white

zone. The resolution of features within the section also appears to be isotropic, which allows for

the most accurate delineation and fitting of individual molecules.

Fig. S3. Fitting of molecules to desmosome P. (A) An alternative configuration for a 4-way

molecular networks that consists of a λ-shape plus one additional molecule in trans. (B-C)

Groups of molecules at the midline consist of two or more networks that provide the potential for

a variety of alternative, uncharacterized intermolecular interactions.

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Fig. S4. C-cadherin molecules fitted to desmosome P. (A) α-carbon trace of 136 molecules after

fitting to the densities shown in Fig. 2A. (B-C) EC5 domains on apposing extracellular

membrane surfaces show a tendency to form pairs (some of which are marked with red circles).

Molecules along the upper, left border of this surface are missing because this region was not

fitted with the x-ray structure.

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Fig. S5. Intermolecular interactions between EC1 domains have a variable geometry. (A)

Archetypal W-, S- and λ-shapes with molecules 0 and 1 representing a W-shape with

symmetrical Trp2 strand exchange. Molecules 2 and 3 form alternative S-shapes with molecule

0: 2 acts as a donor of Trp2, whereas 3 acts as an acceptor of Trp2. Similarly, molecules 4 and 5

form alternative λ-shapes with molecule 0, with 4 acting as Trp2 donor and 5 as Trp2 acceptor.

(B) Range of orientations of molecules assigned to the W-shape with the red pair corresponding

closely to that seen in the C-cadherin x-ray structure (5). For the most part, these molecules are

related by a rotation within the plane of the image. (C-D) Range of orientations for molecules

acting as Trp2 donors for the S- and λ-shapes with the magenta and green molecules

corresponding to those in (A). These molecules are roughly related by rotations about a vertical

axis running through the molecular interface.

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4. SUPPORTING TABLES

Table S1. Sequence identities between cadherins and their EC1 domains.

Numbers below the diagonal correspond to % sequence identity for the entire extracellular

portion of the molecule, whereas numbers below the diagonal refer to only the EC1 domain.

Alignments and identity determination were peformed by CLUSTALW. Sequences were all

from mouse, except C-cadherin which was from Xenopus. Accession numbers were N-cadherin:

AAH22107, C-cadherin: IJXLCP, Desmocollin 1: NP_038532 and Desmoglein 2: NP_031909.

N-cadherin C-cadherin Desmocollin Desmoglein

N-cadherin - 55 37 37

C-cadherin 44 - 37 34

Desmocollin 34 37 - 35

Desmoglein 32 32 31 -

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Table S2. Sequence identities amongst individual EC domains of N- and C-cadherin.

Numbers above the diagonal correspond to % sequence identity for EC domains of C-cadherin,

whereas those below the diagonal refer to N-cadherin. Sequence information and alignment

methods are described in Table II.

EC1 EC2 EC3 EC4 EC5

EC1 - 24 10 20 13

EC2 30 - 7 20 5

EC3 20 14 - 15 11

EC4 21 15 10 - 5

EC5 6 11 2 11 -

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5. SUPPORTING REFERENCES AND NOTES

1. J. C. Fung et al., J. Struc. Biol. 116, 181 (1996).

2. N. Kisseberth, M. Whittaker, D. Weber, C. S. Potter, B. Carragher, J. Struc. Biol. 120, 309

(1997).

3. H. Chen, D. D. Hughes, T. A. Chan, J. W. Sedat, D. A. Agard, J. Struc. Biol. 116, 56 (1996).

4. J. R. Kremer, D. N. Mastronarde, J. R. McIntosh, J. Struc. Biol. 116, 71 (1996).

5. T. J. Boggon et al., Science 296, 1308 (2002).

6. R. H. Brakenhoff et al., J. Cell Biol. 129, 1677 (1995).

7. A. H. Schrijvers et al., Exp. Cell Res. 196, 264 (1991).

8. R. Eshel et al., J. Biol. Chem. 275, 12833 (2000).

9. R. Eshel et al., Int. J. Cancer 98, 803 (2002).

10. L. Shapiro, P. D. Kwong, A. M. Fannon, D. R. Colman, W. A. Hendrickson, Proc. Natl.

Acad. Sci. U.S.A. 92, 6793 (1995).

11. M. Overduin et al., Science 267, 386 (1995).

12. F. Nollet, P. Kools, F. van Roy, J. Mol. Biol. 299, 551 (2000).

13. S. Puttagunta, M. Mathur, P. Cowin, J. Biol. Chem. 269, 1949 (1994).

14. K. Tamura, W. S. Shan, W. A. Hendrickson, D. R. Colman, L. Shapiro, Neuron 20, 1153

(1998).

15. O. Pertz et al., EMBO J. 18, 1738 (1999).

16. M. Takeichi, J. Cell. Biol. 75, 464 (1977).

17. L. Shapiro et al., Nature 374, 327 (1995).

18. B. Nagar, M. Overduin, M. Ikura, J. M. Rini, Nature 380, 360 (1996).

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6. CAPTIONS TO MOVIES

Video 1. Scanning through the sections composing the tomographic map of desmosome

"R". This same map was used for Fig. 1e,f. These sections have been cut parallel to the untilted

sample, same as Fig. 1e and the movie therefore starts and ends with the upper and lower faces

of the section. Densities have been segmented with colored lines both in the intercellular space

and in the cytoplasmic plaque; colors used are blue for cadherins, red for the membrane, orange

and light green for the two zones composing the cytoplasmic plaque, and dark green for the

intermediate filaments. Discrete densities are clearly seen crossing the intercellular space that

correspond to cadherin molecules. The large black circles at one end of the section correspond to

the colloidal gold particles that were deposited on one surface of the section.

Video 2. Scanning through the sections composing the tomographic map of desmosome "P".

This map was used for extensive molecular delineation and fitting depicted in Fig. 2. These

sections have been cut parallel to the untilted sample as in Fig. 1e and in video 1. Segmentation

is shown in the forward half of the movie for the membrane (cyan) and a significant number of

individual cadherin molecules (various colors). The colloidal gold particles are visible at one

surface of the section and their larger size reflects the higher magnification used for desmosome

"P" relative to "R" (see Table 1).

Video 3. This video illustrates the use of Amira to compare segmented densities as well as

fitted coordinates with the original densities in the original tomogram. Sections through the

tomogram appear as black and white and are scanned backward and forward to intersect with the

3D shapes of segmented densities and fitted coordinates. Although the movie starts with the

majority of desmosome P, it focuses on several molecules toward one end of the desmosome,

which includes some of the same molecules shown in Fig. 2b. As expected, high densities are

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present in the map wherever the section intersects the 3D shapes. This is shown for both X-Y

and X-Z sections.