Structure of the Type 1 Inositol 1,4,5-Trisphosphate Receptor Revealed by Electron Cryomicroscopy*

Irina I. Serysheva {ddagger} § , Dan J. Bare ||, Steven J. Ludtke §, Claudia S. Kettlun ||, Wah Chiu {ddagger} § and Gregory A. Mignery ||

From the {ddagger} Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030, § National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, || Department of Physiology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153

Received for publication, April 7, 2003 , and in revised form, April 21, 2003.
    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
The three-dimensional structure of the type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1) has been determined by electron cryomicroscopy and single-particle reconstruction. The receptor was immunoaffinity-purified and formed functional InsP3- and heparin-sensitive channels with a unitary conductance similar to native InsP3Rs. The channel structure exhibits the expected 4-fold symmetry and comprises two morphologically distinct regions: a large pinwheel and a smaller square. The pinwheel region has four radial curved spokes interconnected by a central core. The InsP3-binding core domain has been localized within each spoke of the pinwheel region by fitting its x-ray structure into our reconstruction. A structural mapping of the amino acid sequences to several functional domains is deduced within the structure of the InsP3R1 tetramer.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
The inositol 1,4,5-trisphosphate receptor (InsP3R)1 is a ligand-gated ion channel that modulates Ca2+ release from intracellular stores. The InsP3R is widely distributed in metazoans and plays a key role in diverse physiological functions. In mammals, three different InsP3R isoforms are expressed; each is encoded by a distinct gene. The three isoforms are homologous and share ~70% amino acid identity. Functional channels are composed of four subunits that tetramerize via determinants localized within the transmembrane regions and the cytosolic C terminus (13). Individual cell types often express more than one isoform, which may be present as homo- or heterotetrameric populations. The most characterized type 1 InsP3R (InsP3R1) forms largely homotetramers in cerebellum with Mr of 313,000 (2749 residues) for the monomer. Based on biochemical, molecular biological, and electrophysiological studies, each monomer subunit of the InsP3R1 is organized into three principal functional regions: an N-terminal InsP3-binding region (residues 226–578), a central (~1,700-residue) modulatory/coupling region, and a channel-forming region near the C terminus. The channel region contains six transmembrane segments interspersed within residues 2276–2590 that are critical for membrane targeting, oligomerization, and formation of the ion permeation pathway (14). Thus, both ends, the large N-terminal region (residues 1–2275) and the C-terminal region (residues 2590–2749), are exposed to the cytoplasm.

Electron micrographs of the negatively stained detergent-solubilized InsP3R1 and freeze-fracture deep etching electron microscopy of endoplasmic reticulum have been used to characterize the morphology of the InsP3R1 channel (5, 6). But the three-dimensional shape and dimensions of the channel particles were not determined. Two three-dimensional maps of the InsP3R1 were reported recently based on single-particle reconstruction from specimens prepared by ice embedding (7) and negative staining (8). There is a poor agreement between these two maps with respect to the overall appearance and dimensions of the channel structure. In this study, we present a 30-Å three-dimensional structure of the InsP3R1 determined by electron cryomicroscopy and single-particle reconstruction.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Purification of Bovine Type 1 InsP3R—Bovine cerebellar microsomal membranes were prepared as described previously (9) and solubilized in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 µM leupeptin, and 1 µM pepstatin (Buffer A) with 1.7% Triton X-100 at 4 °C. The solubilized microsomal protein was combined with immunoaffinity matrix prepared by conjugating a type 1 C-terminal (T1C) antibody to cyanogen bromide-activated Sepharose 4B beads (Amersham Biosciences) and equilibrated in Buffer A containing 1% Triton X-100 (10). The affinity matrix with bound InsP3R1 was washed with two alternating series of Buffer A containing 1% Triton X-100 and either 150 or 500 mM NaCl. A final wash was made with Buffer A containing 0.15% Triton X-100. InsP3R1 was eluted from the resin with 25 µM T1C immunogenic peptide (the 19 C-terminal amino acid residues of the rat type 1 InsP3R) in Buffer A. The sample was concentrated using a Vivaspin 6 concentrator (Vivascience Ltd., Binbrook, UK) and clarified of potential protein aggregates by centrifugation for 15 min at 16,000 x gav. Sucrose was added to the InsP3R1 sample to a final concentration of 5%. The purified protein was aliquoted and stored at–80 °C.

Biochemical and Functional Characterization of the Purified InsP3R1—Immunopurified InsP3R1 protein was resolved by 5% SDS-PAGE and evaluated by silver staining and immunoblotting with the T1C antibody as described previously (11).

Proteoliposomes containing immunopurified receptor were prepared by exchanging the detergent with 0.9% CHAPS by sedimentation over 5–20% sucrose density gradients followed by our dialysis reconstitution protocol (12). Proteoliposomes containing the InsP3R1 from sucrose gradient enriched CHAPS-soluble cerebellar microsomes (InsP3RGE) were prepared as described previously (9).

Electrophysiological characterization of the purified receptor using planar lipid bilayers was performed essentially as described previously (10). Planar lipid bilayers were formed across a 150-µm-diameter aperture. Lipid bilayer-forming solution contained a 7:3 mixture of phosphatidylethanolamine and phosphatidylcholine in decane (Avanti Polar Lipids, Pelham, AL). Proteoliposomes containing immunopure InsP3R type 1 or InsP3RGE were added to the cis side (the cytosolic side of the ER) of the bilayer. The trans side (the lumenal side of the ER) of the bilayer was held to virtual ground. Standard solutions contained 220 mM CsCH3SO3 cis and 20 mM CsCH3SO3 trans,20mM HEPES (pH 7.4), 1 mM EGTA and CaCl2 ([Ca2+]free = 250 nM). The [Ca2+]free was calculated using the Max-Chelator program (13) and corroborated with Ca2+-specific microelectrodes (14). Channel sidedness was determined by the sensitivity to InsP3. Heparin (50 µg/ml, Sigma) was used to inhibit the channel. Unitary currents were recorded using a conventional patch clamp amplifier (Axopatch 200B, Axon Instruments, Union City, CA). The current signal was digitized at 5–10 kHz with a 32-bit analog to digital/digital to analog converter (Digidata 1322A, Axon Instruments) and filtered at 1 kHz. Unitary current measurement and data processing were performed using commercially available software packages (pClamp version 8.1, Axon Instruments and Origin, Microcal).

Electron Cryomicroscopy—The vitrified samples of the purified InsP3R1 were prepared as described previously (15, 16) and transferred using a GATAN cryoholder into a JEOL1200 electron microscope operated at 100-kV accelerating voltage. Images of InsP3R1 particles were recorded on Eastman Kodak Co. SO163 film under low dose conditions (~10 electrons/Å2) at a magnification of x40,000 and at the defocus range of 1.8–3.5 µm underfocus.

Three-dimensional Reconstruction of InsP3R—Micrographs were digitized using a Zeiss SCAI scanner with a final digitized pixel size of 3.5. ~9,500 individual particle images were selected manually, and the contrast transfer function (CTF) was determined using EMAN (17, 18). The initial three-dimensional model produced by either EMAN or IMAGIC (19) yielded the same overall shape. The final structure was refined from the initial model using a novel procedure. In brief, the overall refinement methodology is the same as a typical EMAN refinement: projection-based particle classification followed by generating a CTF-corrected average for each class and then constructing a new three-dimensional model from the class averages. The difference in the new procedure is the method of projection-matching technique for classification. The validity of this new methodology was confirmed by yielding an identical structure from lower contrast images of the Ca2+ release channel as determined previously (20). Resolution was determined to be ~30 Å based on the 0.5 criterion in Fourier shell correlation of two independent reconstructions. No tilt experiments were performed, so the absolute handedness of the model is undetermined.

Docking of X-ray Structure of InsP3-binding Domain—A density map of InsP3-binding domain at 30-Å resolution was generated from its x-ray structure, Protein Data Bank code 1N4K [PDB] (21), using EMAN (17). FOLDHUNTER (22) was used to dock the crystal structure of the InsP3-binding domain into the cryomicroscopy reconstruction. Visualization of the fitting was done using IRIS EXPLORER (NAG, Downers Grove, IL) and CHIMERA (23).


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Biochemical Purity and Functional Integrity of the Purified InsP3R1—The InsP3R1 was detergent-solubilized and purified to homogeneity by a one-step procedure using immunoaffinity chromatography. SDS-PAGE and Western blotting with an InsP3R1 antibody (T1C) revealed a band with the apparent Mr of ~300,000 (Fig. 1A). Sedimentation behavior of the immunopurified InsP3R1 in 5–20% sucrose density gradients was consistent with those of tetramers (data not shown). The functional integrity of the purified channel complex was confirmed in single-channel recordings of the InsP3R1 reconstituted into planar lipid bilayers (Fig. 1B). In the absence of agonist (InsP3) the channel was quiescent with rare opening events. Addition of 2 µM InsP3 to the cis chamber resulted in channel activity exhibiting frequent and rapid opening events. The InsP3-activated channels were blocked in the presence of 50 µg/ml heparin.



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FIG. 1.
Immunopurification of the type 1 InsP3R. A, silver-stained 5% SDS-polyacrylamide gel of purified InsP3R1 (lane 2) and corresponding immunoblot with the T1C antibody (lane 1). B, representative current traces recorded from lipid bilayer with reconstituted proteoliposomes containing immunopurified InsP3R1. Open events are shown as upward deflections. The channel is closed after addition of heparin (50 µg/ml) to the cis chamber. C, current-voltage curves of immunopure InsP3R (open circles) and gradient enriched InsP3RGE (solid squares) from CHAPS-solubilized cerebellar microsomes. The holding potential was between –80 and +60 mV. Each point corresponds to the mean ± S.D. (n ≥ 3 for immunopure InsP3R and n ≥ 5 for InsP3RGE). The conductance was determined as the slope of the current-voltage relationship. Recordings from bilayers were carried out in standard solutions containing of 2 µM InsP3, 10 µM ryanodine, and 0.5 mM ATP as described under "Experimental Procedures."

 

The unitary conductance of the immunopurified receptor was compared with InsP3R channels obtained from CHAPS-solubilized cerebellar microsomes (InsP3RGE) (Fig. 1C). The conductance for the immunopurified channel was 222.2 ± 12.4 picosiemens, and the conductance for the gradient enriched was 275.8 ± 12.73 picosiemens. These results are similar to those obtained for the native and recombinant type 1 and 2 InsP3 receptors and suggest that the function of purified InsP3R1 is preserved through our purification steps (10, 24, 25).

Visualization of InsP3R1 and Image Analysis—The purified InsP3R1 channel was vitrified in Buffer A containing 0.15% Triton X-100. Since no additional modulator was included during the cryospecimen preparation, the channel is considered to be in its closed conformation. Because of the low signal-to-noise ratio in the raw images, characteristic of membrane proteins on a continuous carbon substrate, the detailed features of the channel particles can be visually recognized only in the class averages after alignment/classification (Fig. 2). Each of these images represents the CTF-corrected average of several particles in nearly identical orientation. The image analysis revealed a sufficiently random distribution of orientations of the InsP3R1 particles in the data set, which were used for the three-dimensional reconstruction. Some characteristic views of the InsP3R1 clearly exhibit 4-fold symmetry (Fig. 2) and have dimensions of ~250 x 250 Å, which are consistent with previously reported results from negative stain electron microscopy (5, 6, 26). Moreover the 4-fold symmetry of the channel was further confirmed by the 4-fold appearance in some of the eigenimages calculated from multivariate statistical analysis using IMAGIC (15, 19). Thus, the final reconstruction was performed with imposed C4 symmetry.



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FIG. 2.
Image analysis of electron images of ice-embedded InsP3R1. Shown are examples of characteristic views used to reconstruct the three-dimensional structure of the InsP3R1 (top row) and corresponding reprojections from the three-dimensional reconstruction (bottom row). The scale bar is 420 Å.

 

Overall Features of Three-dimensional Structure—The 30-Å-resolution three-dimensional reconstruction of the InsP3R1 exhibits two major morphological regions on opposite sides of the channel (Fig. 3). These consist of a pinwheel-shaped region of about 250 Å in diameter comprised of four curved radial spokes (Fig. 3A) and an opposing square-shaped region of 130 Å in side length exhibiting a central mass depression of ~35 Å in diameter (Fig. 3C). At this resolution, we cannot be certain of the exact molecular boundary of individual subunits. However, we could describe one-quarter of the channel, which is equivalent to a single channel subunit (Mr ~ 313,000) in terms of multiple structural domains (A, B, C, D, and transmembrane (TM)) based on their morphological appearance and density connectivity. Each spoke consists of two domains, A and B, which are roughly equal in size (~100 x 60 x 55 Å). The spoke is connected to a central core region at the 4-fold axis of the structure by a bridge density, domain C. The central core region has a crown shape on one end and a funnel-shaped cavity on the opposite end (Fig. 3D). The TM domain has a bean-like shape of roughly 50 x 75 x 120 Å and is connected to domain C by a 40-Å-long column density, domain D (Fig. 3, B and D).



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FIG. 3.
Surface representation of the three-dimensional reconstruction of InsP3R1. The three-dimensional reconstruction is shown in three different views: A, view from cytoplasm facing the cytoplasmic region; B, side view with the 4-fold axis in the plane of the image with the cytoplasmic region up and the TM region down; C, view from the ER lumen facing the TM region; D, side view of three-dimensional reconstruction cut open through 4-fold axis in the direction indicated with the dashed line in A. Two opposite ends of the central core region, the crown-shaped subdomain and the funnel-shaped cavity, are indicated with an arrow and an asterisk, respectively. The reconstruction is contoured at a molecular volume corresponding to a channel molecular mass of ~1.3 MDa, assuming a protein density of 1.35 g/cm3.

 

Comparisons with Other Structures—The overall appearance of our three-dimensional reconstruction is consistent with the map of the InsP3R1 based on the negatively stained InsP3R1 (8). The pronounced differences are observed in the arrangement of the pinwheel region. This region in the negatively stained microscopy map is rather square-shaped with dimensions of ~180 x 180 x 110 Å and exhibits very weak connecting densities between the peripheral density corresponding to our A domain and the central region. Unlike our three-dimensional map, the column domain D connecting the pinwheel structure with the TM domain is missing. These discrepancies may be introduced by different specimen preparative methods.

Another 24-Å-resolution map of the InsP3R1 in its closed state was reported based on electron cryomicroscopy and single-particle reconstruction (7). This three-dimensional map is strikingly different from our three-dimensional reconstruction. While this map also contains a 4-fold symmetry, it appears to consist of a central core with very little peripheral mass in the pinwheel region. Since this reconstruction was also performed using EMAN (without EMAN's CTF correction) with some initial analysis in IMAGIC, it seems unlikely that this is a software problem but probably represents a fundamental difference in the raw data. The receptor purification strategy used in this study uses single-step chromatography over an immunoaffinity matrix with Triton X-100 while Jiang et al. (7) used CHAPS. Additionally da Fonseca (8) used essentially the same strategy as Jiang et al. (7) with the only major difference being the use of Triton X-100 and Surfact-Amps X-100. It is possible that the apparent discrepancies between the structures are a consequence of the different detergents. Additionally our protein isolation was performed at pH 7.4, whereas the other preparations (7, 8) were made at alkaline pH. These pH changes may induce additional conformational transitions of the channel. Consistent with this notion, it has been demonstrated that a sharp increase in InsP3 binding to cerebellar membranes occurs at alkaline pH (27). Another more remote possibility for the observed differences in the structures is the use of receptor from Bos tarus, while the other structures are derived from rat cerebella. However, this is highly unlikely since the rat and bovine receptors have a 92% amino acid identity. Clearly more experimentation will be required to determine the cause of this discrepancy.

Localization of InsP3-binding Domain—The N terminus of the InsP3R1 monomer has been established to contain the InsP3-binding site (residues 226–578), which provides specific and high affinity ligand binding independent of tetramer formation. The x-ray structure of the InsP3-binding core of mouse InsP3R1 in complex with InsP3 was recently reported at 2.2-Å resolution (21). To assign mass density for this domain within our three-dimensional structure of InsP3R1, we docked the crystal structure blurred to 30-Å resolution into the three-dimensional map of InsP3R1 using FOLDHUNTER software (22). The shown fit represents the highest scoring match and places the InsP3-binding region in the B domain within the pinwheel structure (Fig. 4). The next several highest scores were in virtually identical locations and overall orientations. Since the next match in a substantially different location was such a poor fit visually that it was considered unreasonable, we consider this fit to be definitive within the resolution limit of the current data.



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FIG. 4.
Assignment of electron densities for InsP3-binding core domain. A, fitting of 30-Å electron density map of the InsP3-binding domain (blue mesh) within the B domains of the cytoplasmic region of InsP3R1 tetramer shown in its top view. The x-ray structure of the InsP3-binding domain is shown as a ribbon diagram with overlapping electron density map. B, side view of the region extracted from the InsP3R1 tetramer as indicated in A with the dashed lines. C, zoom of the region indicated with the dashed lines in A. The white star marks the C terminus of the x-ray structure. The scale bar in A and B is 50 Å and in C is 25 Å.

 

Our localization of the InsP3-binding domain is consistent with its recent mapping using heparin-gold labeling and negative stain electron microscopy (26). In their study, the heparin-binding domain was localized to the peripheral region of the InsP3R1 at a distance over 50 Å away from the central region of the channel, which is equivalent to domain B in our structure. However, our localization is different from that of another electron microscopy study, which suggested the InsP3-binding domain is close to the 4-fold axis (8). Their conclusion was inferred from an earlier ligand binding study using dimers of InsP3 linked by polyethylene glycol molecules of varying lengths (10–80 Å) (28) that suggested that the InsP3-binding sites are separated by no more than 20 Å. This estimate is inconsistent with our map where the InsP3-binding sites assigned to the B domains are spaced at least 100 Å apart. A possible explanation for this discrepancy is that the radioligand binding studies were performed at pH 8.3 (28), which is optimal for binding of InsP3, while our study and the heparin-binding site mapping study (26) used a pH of 7.4. Conformational transitions may take place in the channel upon changing the pH as suggested previously (27).

Putative Assignment of Sequence to Domains within Three-dimensional Map—Thus, having assigned the InsP3-binding core to the B domain, it seems reasonable to suggest that the pinwheel region of the channel is exposed to the cytoplasm, while the opposite square-shaped region includes the transmembrane channel domain and faces into the endoplasmic reticulum. This assignment concurs with the topological model of the InsP3R deduced from biochemical, electrophysiological, and molecular experiments (14), suggesting that about 89% of the channel polypeptide is located in the cytoplasm, while the remaining, smaller portion (11%) represents the transmembrane channel domain. The square-shaped region of our three-dimensional reconstruction includes about 20% of the total channel volume, thus the remaining portion, that is about 80% of the total volume, accounts for the pinwheel structure and the column domains.

Our fitted model shows that the C terminus (residue 602) of the InsP3-binding fragment that covers the InsP3-binding core (residues 226–578) is located close to the A domain (Fig. 4C). This putative position suggests that the A domain may include part of the sequence following the InsP3-binding domain, which has been referred to as the modulatory/coupling domain (residues 579–2275). The modulatory domain has been reported to contain binding sites for InsP3R1 channel modulators including Ca2+, calmodulin, FKBP12 (12-kDa FK506-binding protein), and ATP and modulatory phosphorylation sites. We propose that the internal coupling domain resides within the spoke and the central core regions. These regions probably would undergo significant conformational changes to modulate the channel gating. In support of this hypothesis, it was previously shown that a cytoplasmic N-terminal region including the ligand-binding and a portion of the coupling/regulatory domain (residues 1–1803) of the recombinant type 1 receptor indeed undergoes a large conformational change upon the binding of InsP3 (11). It was reported recently that high Ca2+ concentrations regulate the structural rearrangement of the peripheral regions of the InsP3R1 (26) and possibly InsP3-mediated gating.

Furthermore our fitting suggests that the N-terminal sequence (residues 1–223) of the InsP3R1 monomer resides in the domain C and/or the column domain D. Therefore the column domain D probably contains the C-terminal sequence (residues 2590–2749) and provides a connection of the ligand-binding region to the channel pore-forming region. It is known that the transmembrane sequences reside between residues 2276 and 2589. Therefore, the TM domain would be partially made up with this sequence. Our assignment places the N and C termini of the InsP3R1 monomer close to each other, which is consistent with cross-linking and co-immunoprecipitation studies (29).

Thus, we have presented the three-dimensional map for domain organization of the InsP3R1. In this model, the InsP3-binding region is positioned in one of the domains within the cytoplasmic region that is far from the transmembrane domain of InsP3R1. This localization of the ligand-binding region implies a highly allosteric mechanism for regulation of channel gating, which would require long range conformational changes in the cytoplasmic region of InsP3R1 to trigger the channel opening. Further structural studies at higher resolution combined with specific labeling techniques as well as recombinant receptor mutants will refine the proposed model for the three-dimensional arrangement of the InsP3R1 channel.


    FOOTNOTES
 
* This research was supported by National Institutes of Health Grants MH53367 (to G. A. M.) and P41RR02250 (to W. C.) and by a grant from the Muscular Dystrophy Association and American Heart Association Grant 9730258N (to I. I. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Molecular Physiology and Biophysics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Tel.: 713-798-6989; E-mail: irinas{at}bcm.tmc.edu.

1 The abbreviations used are: InsP3R, inositol 1,4,5-trisphosphate receptor; InsP3, inositol 1,4,5-trisphosphate; InsP3R1, type 1 InsP3R; ER, endoplasmic reticulum; CTF, contrast transfer function; T1C, type 1 C-terminal; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; TM, transmembrane. Back


    ACKNOWLEDGMENTS
 
We thank M. L. Baker and M. T. Dougherty for help with visualization, M. R. Baker and O. Thomas for help with digitizing electron micrographs, and S. L. Hamilton for helpful discussions.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. Galvan, D. L., Borrego-Diaz, E., Perez, P. J., and Mignery, G. A. (1999) J. Biol. Chem. 274, 29483–29492[Abstract/Free Full Text]
  2. Galvan, D. L., and Mignery, G. A. (2002) J. Biol. Chem. 277, 48248–48260[Abstract/Free Full Text]
  3. Joseph, S. K., Boehning, D., Pierson, S., and Nicchitta, C. V. (1997) J. Biol. Chem. 272, 1579–1588[Abstract/Free Full Text]
  4. Ramos-Franco, J., Galvan, D., Mignery, G. A., and Fill, M. (1999) J. Gen. Physiol. 114, 243–250[Abstract/Free Full Text]
  5. Chadwick, C. C., Saito, A., and Fleischer, S. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2132–2136[Abstract]
  6. Maeda, N., Niinobe, M., and Mikoshiba, K. (1990) EMBO J. 9, 61–67[Abstract]
  7. Jiang, Q. X., Thrower, E. C., Chester, D. W., Ehrlich, B. E., and Sigworth, F. J. (2002) EMBO J. 21, 3575–3581[Abstract/Free Full Text]
  8. Da Fonseca, P. C., Morris, S. A., Nerou, E. P., Taylor, C. W., and Morris, E. P. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 3936–3941[Abstract/Free Full Text]
  9. Mignery, G. A., Newton, C. L., Archer, B. T., III, and Sudhof, T. C. (1990) J. Biol. Chem. 265, 12679–12685[Abstract/Free Full Text]
  10. Ramos-Franco, J., Caenepeel, S., Fill, M., and Mignery, G. (1998) Biophys. J. 75, 2783–2793[Abstract/Free Full Text]
  11. Mignery, G. A., and Sudhof, T. C. (1990) EMBO J. 9, 3893–3898[Abstract]
  12. Perez, P. J., Ramos-Franco, J., Fill, M., and Mignery, G. A. (1997) J. Biol. Chem. 272, 23961–23969[Abstract/Free Full Text]
  13. Bers, D. M., Patton, C. W., and Nuccitelli, R. (1994) Methods Cell Biol. 40, 3–29[Medline] [Order article via Infotrieve]
  14. Baudet, S., Hove-Madsen, L., and Bers, D. M. (1994) Methods Cell Biol. 40, 93–113[Medline] [Order article via Infotrieve]
  15. Serysheva, I. I., Orlova, E. V., Chiu, W., Sherman, M. B., Hamilton, S. L., and van Heel, M. (1995) Nat. Struct. Biol. 2, 18–24[Medline] [Order article via Infotrieve]
  16. Serysheva, I. I., Ludtke, S. J., Baker, M. R., Chiu, W., and Hamilton, S. L. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 10370–10375[Abstract/Free Full Text]
  17. Ludtke, S. J., Baldwin, P. R., and Chiu, W. (1999) J. Struct. Biol. 128, 82–97[CrossRef][Medline] [Order article via Infotrieve]
  18. Ludtke, S. J., Jakana, J., Song, J., Chuang, D. T., and Chiu, W. (2001) J. Mol. Biol. 314, 241–250[CrossRef]
  19. van Heel, M., Harauz, G., and Orlova, E. V. (1996) J. Struct. Biol. 116, 17–24[CrossRef][Medline] [Order article via Infotrieve]
  20. Baker, M. L., Serysheva, I. I., Sencer, S., Wu, Y., Ludtke, S. J., Jiang, W., Hamilton, S. L., and Chiu, W. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 12155–12160[Abstract/Free Full Text]
  21. Bosanac, I., Alattia, J. R., Mal, T. K., Chan, J., Talarico, S., Tong, F. K., Tong, K. I., Yoshikawa, F., Furuichi, T., Iwai, M., Michikawa, T., Mikoshiba, K., and Ikura, M. (2002) Nature 420, 696–700[CrossRef][Medline] [Order article via Infotrieve]
  22. Jiang, W., Baker, M. L., Ludtke, S. J., and Chiu, W. (2001) J. Mol. Biol. 308, 1033–1044[CrossRef][Medline] [Order article via Infotrieve]
  23. Huang, C. C., Couch, G. S., Pettersen, E. F., and Ferrin, T. E. (1996) Pac. Symp. Biocomput. 1, 724
  24. Ramos-Franco, J., Fill, M., and Mignery, G. A. (1998) Biophys. J. 75, 834–839[Abstract/Free Full Text]
  25. Ramos-Franco, J., Bare, D., Caenepeel, S., Nani, A., Fill, M., and Mignery, G. (2000) Biophys. J. 79, 1388–1399[Abstract/Free Full Text]
  26. Hamada, K., Miyata, T., Mayanagi, K., Hirota, J., and Mikoshiba, K. (2002) J. Biol. Chem. 277, 21115–21118[Abstract/Free Full Text]
  27. Worley, P. F., Baraban, J. M., Supattapone, S., Wilson, V. S., and Snyder, S. H. (1987) J. Biol. Chem. 262, 12132–12136[Abstract/Free Full Text]
  28. Riley, A. M., Morris, S. A., Nerou, E. P., Correa, V., Potter, B. V., and Taylor, C. W. (2002) J. Biol. Chem. 277, 40290–40295[Abstract/Free Full Text]
  29. Boehning, D., and Joseph, S. K. (2000) EMBO J. 19, 5450–5459[Abstract/Free Full Text]