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Architecture of the Xenopus nuclear pore complex revealed by three-dimensional cryo-electron microscopy.

The nuclear pore complex spans the nuclear envelope and functions as a macromolecular transporter in the ATP-dependent process of nucleocytoplasmic transport. In this report, we present three dimensional (3D) structures for both membrane-associated and detergent- extracted Xenopus NPCs, imaged in frozen buffers by cryo-electron microscopy. A comparison of the differing configurations present in the 3D maps suggests that the spokes may possess an intrinsic conformational flexibility. When combined with recent data from a 3D map of negatively stained NPCs (Hinshaw, J. E., B. O. Carragher, and R. A. Milligan. 1992. Cell. 69:1133-1141), these observations suggest a minimal domain model for the spoke-ring complex which may account for the observed plasticity of this assembly. Moreover, lumenal domains in adjacent spokes are interconnected by radial arm dimers, forming a lumenal ring that may be responsible for anchoring the NPC within the nuclear envelope pore. Importantly, the NPC transporter is visualized as a centrally tapered cylinder that spans the entire width of the NPC, in a direction normal to the nuclear envelope. The central positioning, tripartite structure, and hollow nature of the transporter suggests that it may form a macromolecular transport channel, with a globular gating domain at each end. Finally, the packing of the transporter within the spokes creates a set of eight internal channels that may be responsible, in part, for the diffusion of ions and small molecules across the nuclear envelope.

aged in frozen buffers by cryo-electron microscopy. A comparison of the differing configurations present in the 3D maps suggests that the spokes may possess an intrinsic conformational flexibility. When combined with recent data from a 3D map of negatively stained NPCs (Hinshaw, J. E., B. O. Carragher, and R. A. Milligan. 1992. Cell. 69:1133-1141), these observations suggest a minimal domain model for the spoke-ring complex which may account for the ob-served plasticity of this assembly. Moreover, lumenal domains in adjacent spokes are interconnected by radial arm dimers, forming a lumenal ring that may be responsible for anchoring the NPC within the nuclear envelope pore. Importantly, the NPC transporter is visualized as a centrally tapered cylinder that spans the entire width of the NPC, in a direction normal to the nuclear envelope. The central positioning, tripartite structure, and hollow nature of the transporter suggests that it may form a macromolecular transport channel, with a globular gating domain at each end. Finally, the packing of the transporter within the spokes creates a set of eight internal channels that may be responsible, in part, for the diffusion of ions and small molecules across the nuclear envelope. T hE nuclear pore complex (NPC) ~ resides within a pore formed by the fusion of the inner and outer nuclear membranes and is tethered to the nuclear lamina. Therefore, the NPC is ideally situated to mediate the bidirectional exchange of small molecules and catalyze the active transport of proteins, cellular and viral RNPs (15,25,29,48). Accumulating evidence suggests that nucleocytoplasmic transport is a singular reaction in that: (a) transport is bidirectional yet vectorial for a particular substrate at a given instant; (b) transport occurs through the center of the NPC as visualized by thin sections of Balbiani ring mRNPs transiting the NPC (61); (c) an unusually broad range of substrate types and sizes are transported; (d) substrates need not be deformed in passage (21); (e) the import of many karyophilic proteins is dependent on a positively charged nuclear localization signal (NLS), that is bipartite in nucleoplasmin and SWI5 (43,56); and (f) import of karyophilic proteins requires at least two cytosolic factors (1,44,45) and involves specific binding to O-linked N-acetyl glucosamine containing nucleoporins (58). 1. Abbreviations used in this paper: CP, cytoplasmic particles; CR/NR, cytoplasmic ring/nucleoplasmic ring; CTF, contrast transfer function; ISR, inner spoke ring; NLS, nuclear localization signal; NPC, nuclear pore complex; RA, radial arm; 3D, three dimensional.
Substrates transported by the NPC are chemically diverse and include: proteins, mRNPs, snRNPs, pre-ribosomal subunits, and tRNAs. Recent data suggests that they use a common transport machine located within the NPC, as both import and export reactions for these substrates demonstrate ATP-dependent saturation kinetics (9,14,30,41,46,65) and manifest similar inhibition profiles (13,14,19,24,41). Interestingly, protein import and the export of nucleic acids appears to occur through common NPCs (17). In addition, the transport of proteins and mRNPs are topologically similar, as both processes use a central channel within the NPC that constrains substrates to a maximum diameter of 200-250/~ during translocation (28,61,23,40). The identity and structure of the macromolecular transport machine located centrally within the NPC has not been established.
The NPC is a cylindrical assembly with octagonal symmetry and a mass of '~125 Mda (18,52). Disassembly studies (52,62) and quantitative image analyses of en face and side-on projections have suggested that the NPC is comprised of a set of thin rings which sandwich a more massive spoke assembly (3,52,62). Projections of frozen-hydrated specimen have revealed pairs of radial arms that interconnect adjacent spokes and a central ringlike transporter (3), that may be capable of forming a gated transport channel for substrates (4,8). The precise symmetry of the NPC proper may vary depending on structural changes induced during enucleation and sample preparation (3). The NPC may he either eightfold symmetric about an axis perpendicular to the nuclear envelope (3,18,28,52) or "o822 symmetric with an approximate twofold axis relating the oppositely facing halves of the spoke-ring complex (3,4,42,62). Recently, Hinshaw and co-workers (34) have completed a single particle three-dimensional (3D) reconstruction from detergent-extracted (dform) and detached Xenopus NPCs with 'o822 symmetry. As visualized in negative stain, the minimal building block of the spoke-ring complex is composed of five domains including two domains within each of the averaged thin rings and three domains in each half-spoke. Peripheral assemblies, such as cytoplasmic particles, filaments (28,53,54,55,59,62), and nucleoplasmic cages/baskets (28,36,54,55) were not visualized in these detached specimens.
In this report, we present data from independent 3D reconstructions of Xenopus NPCs tethered to the nuclear lamina in the presence or absence of the nuclear envelope. The maps provide insights on issues concerning: (a) the minimal domain structure of the spoke-ring complex and its observed conformational flexibility; (b) the possible role of the NPC transporter in nucleocytoplasmic transport; (c) the positions of internal channels that may function as diffusion pores; and (d) interactions between the NPC and the encircling nuclear envelope.

Specimen Preparation and Image Processing
The maintenance of amphibians, isolation of oocytes, and preparation of frozen-hydrated nuclear envelope spreads for cryo-electron microscopy were carried out as described previously (3). Two microscopes were used in the data collection, a Philips EM400T at the Deptartment of Cell Biology, Stanford University Medical School (Stanford, CA) and an EM400 in the Structural Studies Division at the Medical Research Council Laboratory of Molecular Biology. Both microscopes were modified with objective lens current meters by Dr. C. Toyoshima (Department of Biological Sciences, Tokyo Institute of Technology, Tokyo, Japan) which were used to adjust the zero and tilted specimen pair z-heights, such that a consistent objective lens current was obtained, thereby ensuring a uniform magnification. Micrographs were scanned on a flatbed densitometer manufactured by Joyce, Loebl and Co. Ltd. (Gateshead, England), that was extensively modified at the LMB. The effective step sizes on the images were 24.6 and 26.7/~ depending on the initial magnification. 3D image processing used SECRET, a 3D reconstruction method for single particles based on a conical tilt geometry (50,51), implemented within the SPIDER software package (26). A suite of roughly 100 SPIDER procedures and sub-procedures were written and integrated into a single automated 2D/3D program set, ALIGN___EM, based in part on a previous set of programs written by M. Radermacher (New York State Department of Health, Albany, NY) (RCDOALL). ALIGN_..EM runs five deep within a batch job and provides a highly structured environment for data and document files during the analysis of large datasets. The current version has over 40 steps in a sequential menu driven format that covers 2D/3D alignments and the preparation of computed 3D maps for subsequent analysis and presentation. Specific modifications were made in the procedures used to align tilted particles, to accommodate the low-contrast images of frozen-hydrated specimens. In particular, larger areas of 256 × 256 were windowed out around the zero and tilted particles and used only in the alignment of the untilted/tilted image pairs, to boost the signal of the cross-correlation peak. Images in 128 × 128 format were used routinely for all other calculations. In addition, all tilted particles used in the 3D reconstructions had the first minima of their contrast transfer functions (CTFs) outside the nominal resolution range. Cylindrical averages of the tilted and aligned particles were used to judge the accuracy of alignment along with the convergence of the step sizes assigned to each particle during refinement. Further improvemerits in the tilt centration parameters were achieved by iterative refinement of each tilted image against a projection of an unweighted 3D reconstruction, calculated from the entire dataset (Radermacher, 1992). In this approach the tilted image under refinement (with negative contrast) was removed from the trial 3D reconstruction by back projection, before a centration alignment between the original tilted image (with positive con-tras0 and the equivalent projection calculated from the 3D volume. Corrected xy shifts were then used to reinsert the tilted image into the 3D volume by back projection and the process continued for all images in the dataset through two complete cycles. This refinement resulted in small but significant changes in the xy shifts obtained from cross-correlational alignments of the zero/tilted image pairs. Datasets with tilt angles of 34 ° (mform) and 42 ° (dform) were used to minimize problems from nearest neighbor overlap in the tilted specimens; however this resulted in rather large missing cones. Therefore, a real space/Fourier space method (POCs/solvent flattening; 12) was employed that uses volume constraints to iteratively extend the molecular transform into the cone of missing information. This approach was incorporated into SPIDER by M. Radermacher and carded out as follows. A weighted 3D reconstruction of the complete dataset was used to generate a 3D mask of the NPC using a combination of thresholding and low pass filtering. The mask was then applied to the 3D volume in real space and the 3D Fourier transform calculated. The resulting molecular transform in the missing cone was left untouched between cycles, but the calculated Fourier in the measured area of the molecular transform was replaced with the original molecular transform (from the unmasked object) and the resulting hybrid back Fourier transformed. This procedure was carded out for two to three cycles before defining a smaller mask and repeating the process. The final molecular volume of the mask was roughly 1.5 times the calculated molecular volume of the NPC based on STEM mass measurements (52). This cutoff was used to compensate for the unknown mass and undetermined occupancy of the cytoplasmic particles and material "trapped" within the transporter (4). Solvent flattening produced 3D maps whose peak distribution and relative intensities were unaffected in the xy plane sections; however, the modified maps demonstrated a higher contrast relative to background and a net shrinkage along the z-axis of 12-16%, resulting in height/diameter ratios that agree with side-on views (3). The series of 3D masks were used to refine half-datasets in parallel and the resulting 3D maps used to calculate phase residuals (27). Phases within the missing cone were random at the beginning of POCs refinement, but converged to give residuals similar to those in the original measured regions of the molecular transform (,,o100]~). Final 3D volumes were merged from the half-datasets and low pass filtered at 80/~ resolution to compensate for doubling the amount of data. The POCs method worked well because the original NPCs are not appreciably flattened in the frozen-hydrated state. Moreover, an excellent fit of common features was obtained when the central sections in each map were scaled together, but regions within the scaled maps corresponding to the thin rings in the mform were 6-10% smaller in diameter than their counterparts from the dform. Multicolor surface rendering techniques were used to highlight specific regions within the 3D maps using an algorithm written by M. Radermacber to create a composite surface for display on WEB, a Motif display program written by A. Leith (New York State Department of Health, Albany, NY). Cutoffs were chosen to illustrate reproducible features and generally fell at the boundaries of steep density gradients. Unknown parameters such as the absolute occupancy of cytoplasmic particles and material caught in transit within the transporter, coupled with uncertainties arising from the loss of the lowest resolution frequencies of the CTF, prevented us from placing the surface maps on an absolute volume scale commensurate with the measured mass (52).

Results
We have used cryo-electron microscopy of frozen-hydrated specimens to visualize the architecture of the NPC in 3D. This approach minimizes perturbations that can occur during negative staining and drying of large specimens. These methods have produced both pseudosymmetric ('o822) and asymmetric (C8) averages of the NPC associated with either the nuclear envelope or the nuclear lamina (3,4,7). We have used the Single Exposure random Conical tilt Reconstruction Technique (SECRET; 50, 51) which uses a pair of micrographs, one tilted and one untilted, to obtain the orientation angles necessary to calculate 3D maps from tilted projections. Maps of membrane-associated (mform) and detergent-  Necturus NPCs associated with a detergent-extracted nucleus isolated from an animal with unhealthy oocytes. Note the weak inner spoke ring (ISR) formed by the slewed inner spoke domains. Density within the spokes is somewhat asymmetric. Bar, 500 ./~. extracted (dform) NPCs from Xenopus were obtained by this method and are described in the following sections. At this stage, the resolution is inadequate to precisely identify subunit boundaries within the molecular envelopes of the maps. However, a detailed side-by-side comparison allows a qualitative description of the minimal number of domains which comprise the framework of the spoke-ring complex. This analysis confirms the 3D map of Hinshaw and coworkers (34) and extends our understanding of the conformationally flexible spoke-ring assembly and its interactions with the central NPC transporter.

NPCs Associated with the Nuclear Envelope
Representative areas of a micrograph pair recorded at 0 ° and 34 ° from a spread Xenopus nuclear envelope are shown in Fig. 1, a and b. The mform NPCs used for the 3D reconstruction were circular with a distinct eightfold symmetry and demonstrated a ring of density centered at a radius of "~420-430/~ that traverses the centers of individual spokes in projection (3,4,36,62). The specimens are unstained; hence, protein dominates the scattering and is dark. The tilt axis (34 °) is located parallel to the white arrow in Fig. 1 b, along the major axis of the elliptical NPCs in the tilted image. Although a few NPCs retain their ringlike central transporters (white arrowheads Fig. 1 a), the majority have lost them during specimen preparation. In addition, large particles located between the NPCs (indicated by black arrowheads) may correspond to either detached cytoplasmic particles observed in the 3D maps or possibly to membraneassociated ribosomes (62).
Averaged projection maps resulting from computer-based alignments of untilted and tilted (34 °) NPCs are shown in Fig. 2, a and d. Contrast has been reversed relative to the micrographs; protein is now white. The averaged map calculated from the untilted NPCs is similar to maps obtained previously from Xenopus (3). For example, the eight spokes and The Journal of Cell Biology, Volume 122, 1993 Figure 3. Diagram of a model cylinder with the x-, y-and z-axes indicated. The slices described in the text are shown including an xy slice cut perpendicular to the z-axis (the dark plane) and a z-slice which corresponds to a cross-section cut parallel to and containing the z-axis. As an example, a z slice is shown cut along an arbitrary radial axis denoted by the angle theta. The z slices described in the text were chosen as follows. The spoke z slice was chosen to bisect individual spokes along their approximate twofold axis. Radial ann z slices were obtained by rotating the plane of the cross-section by 22.5 ° clockwise, thereby bisecting the radial arms and the inner spoke ring. their attachments to the radial arms are visible. In addition' the spokes are bisected by a ring of density that may arise from the border of the fused inner and outer nuclear membranes. The transporter is absent from the 2D map; instead a central cavity is present with a diameter of ,x,440/~ ( Fig.  2 a and see Fig. 4). A cylindrically averaged map was calculated from the tilted and aligned NPCs (Fig. 2 d). A cylindrical average is obtained because all possible rotational orientations, relative to the central eightfold axis, are present in the corresponding untilted NPCs. Hence, after tilting to 34 ° and averaging over 196 NPCs a rotationally "blurred" image is obtained without subunit details as viewed from above. Importantly, the cylindrical average provides direct evidence that the cytoplasmic and nucleoplasmic rings are present within the specimens. Moreover, the membrane border and spoke subunits appear to be split into concentric elliptical rings that "encircle" the central cavity. After the tilted NPCs were aligned to a common origin, a 3D map was calculated by back projection. The cytoplasmic and nucleoplasmic sides were then identified as follows. First, features within the map could be correlated directly with their counterparts in the 3D map of the dform. This form possesses a distinct handedness in projection that has been used previously to assign a unique viewing direction (3). Second, the cytoplasmic ring in the maps was "denser" due to the presence of the cytoplasmic particles. This observation is in agreement with previous observations (33,52,55,62).
After refinement, differing views of domains within the 3D maps were obtained by taking appropriate xy slices or z slices (cross-sections) of the 3D volume. The orientation of xy slices cut perpendicular to the z-axis is shown in Fig. 3, within an idealized cylindrical volume. In Fig. 3, the z-axis would correspond to the central eightfold symmetry axis of the NPC. A set of xy-slices for the mform is shown in Fig.  4 (a-f), and the spacings of these slices are shown in a side view of a surface model to orient the reader (Fig. 4 g). The 3D map demonstrates an approximate twofold axis of symmetry within the central section (arrows 1 and 2 in Fig. 4 d) that is maintained within the spokes. Symmetry axis 1 is centered on each spoke while symmetry axis 2 is rotated 22.5 ° clockwise from axis 1 and bisects the radial arms. In total, both approximate twofold axes are repeated eight times around the cylindrical NPC. The approximate twofold symmetry of the spokes is shown by comparing xy-slices in Fig.  4, c and f, which are related by a 180 ° rotation about the center of the spokes. However, the thin rings are different at this resolution (80-100A) with the cytoplasmic ring having eight paired domains (labeled 1 and 2) and an inwards pointing aspect (see asterisk in Fig. 4 b), while the nucleoplasmic ring has eight contiguous single domains and is less intense (compare Fig. 4, b and e). Furthermore, the departure from perfect twofold symmetry is most marked at the cytoplasmic ring which is decorated with the remnants of cytoplasmic particles or collapsed filaments (see Fig. 4 a). The centers of the cytoplasmic particles are offset counterclockwise by ,'o17 ° from the putative spoke twofold axis (symmetry axis "1" in Fig. 4 d).
Inspection of the central xy slice in Fig. 4 d reveals three density peaks within each spoke including: the inner domain (IS), a central domain (CS), and the lumenal domain (LS). Furthermore, certain spoke domains join together to form larger concentric structures. For example, the inner spoke domains are interconnected to form an inner spoke ring (ISR) and the lumenal domains penetrate into the nuclear lumen where they are linked by radial arm dimers (RA), forming a lumenal ring. Additional features are visible in z-slices, as defined in Fig. 3, that correspond to planar cross-sections cut parallel to the z-axis along the arrows labeled 1 and 2 in Fig. 4 d. In Fig. 5 a, the z-slice bisects a spoke showing the vertical and radial density variations within oppositely facing spokes. Interestingly, the inner spoke domains make their strongest contacts to the rodlike density that contains the central domain at the upper and lower extremities, rather than at the midline. Other contributions to the vertical rodshaped density result from the juxtaposition of the outer vertical domains (Vo) and the central domain with the nuclear envelope border. The lumenal spoke domain protrudes from the spoke surface into the lumen of the nuclear envelope. A second cross-section cut 22.5 ° away and bisecting the radial arms, shows portions of five domains including the ISR, the cytoplasmic and nucleoplasmic rings (CR/NR), the cytoplasmic particles (CP), and the radial arms (RA). A weaker band of density (indented in the middle) runs vertically along the outer surface of the inner spoke ring and, at this resolution, connects the cytoplasmic and nncleoplasmic thin rings. This density may be attributed in part to the border of the nuclear envelope (M). In Fig. 4 d, the central domains (CS) appear to traverse this ring of density. However, structural rearrangements within the inner spoke ring may also contribute to the "membrane" density at this radius.
Color-coded surface views of the 3D map are presented in Akey and Radermacher Architecture of the Xenopus Nuclear Pore Complex  Note that the inner spoke domain is split into cytoplasmic and nucleoplasmic domains (small black arrows on le~) that join at the midline of the spokes and interact with the inner vertical domains and the central domain. (d) A z slice which bisects the radial arms of the dform is shown. Note that the membrane density is missing and the inner spoke ring is more rounded. Fig. 6. End-on and oblique views (at 45°), as viewed from the cytoplasmic side, emphasize the connectivity of the lumenal ring (Fig. 6, a and b). Within the lumenal ring, the radial arms attach diagonally in an alternating fashion to the top and bottom of adjacent lumenal spoke domains. The spoke-ring complex is colored a medium blue while the lumenal ring is darker blue. The cut-away surfaces presented in Fig. 6, c and d are shown at a higher cutoff to minimize contributions from the nuclear envelope. In these views, the individual spokes and a dimeric splitting within the inner spoke ring are visualized.

NPCs Associated with the Nuclear Lamina after Detergent Extraction
Nuclear envelope ghosts were obtained by TX-100 extraction of nuclei, extruded into low salt buffers, and subsequently spread onto grids for rapid freezing. Under these conditions NPCs remain attached to the disordered lamina (but see references 2, 3). Areas of a zero tilt/42 ° tilt pair are shown in Fig. 1, c and d. The position of the tilt axis is indicated by the white arrow and the lamina is visible as a diffuse net-work of fine strands located between the NPCs (black arrowheads labeled "L" in Fig. 1 c). In general, the NPCs demonstrate a high occupancy of central ring-like transporters (white arrowheads in Fig. 1 c). Averages from 284 dform NPCs after alignment are shown in Fig. 2 b (untilted) and e (cylindrical average). The spoke-ring complex is more asymmetric than observed in the best images from Necturus (3) and displays the lower point group symmetry C8. The radial arms are visible between the spokes at high radius but are characteristically more diffuse than in maps of mform NPCs (3). In addition, the NPC transporter is visualized in projection as an averaged"plug" that may encompass a number of different transport-related configurations (C. W. Akey, unpublished data; see references 4,8). Interestingly, the transporter is visible in the tilted cylindrical average as a horizontal band of density within the inner bright elliptical ring, and the cytoplasmic and nucleoplasmic thin rings are resolved (Fig. 2 e). Numerous attempts were made to obtain symmetrical (,,o822) specimens from Xenopus without success. A projection map from a second dform specimen is shown in Fig. 2 c. The similarity of the two averages suggests that this configuration is reproducible and similar maps have The nucleoplasmic thin ring is also indicated (NR). (c) A cut-away surface view of the mform NPC is shown with the cytoplasmic surface upwards. This view is shown at a higher cutoff to eliminate weaker density between adjacent spokes that is contributed in part, by the nuclear envelope. The positions of the cytoplasmic and nucleoplasmic thin rings are indicated as is the lumenal spoke domain. (d) The same cut-away surface is shown rotated 45 ° towards the viewer to show the gaps between adjacent spokes which delineate the cytoplasmic ring and the dimeric splitting that occurs in the inner spoke ring.
been obtained from negatively stained NPCs (11,52). For comparison, a map of dform NPCs is shown in Fig. 2 f (n = 284) in which the pore complexes were obtained from older Necturus oocytes. Note that the preservation of twofold symmetry within the spokes and inner spoke ring is inter-mediate in character between the Xenopus maps and the best maps from Necturus (3).
Representative xy slices taken from the 3D volume of the dform NPC, equivalent to those presented from the 3D map of the reform NPC, are shown in Fig. 7. The in-plane sym-  metry of the central slice (Fig. 7 d) is more complicated than in the mform NPC. Instead of one set of approximate twofold axes there are two sets of "local" approximate twofold symmetry axes rotationally offset by ,o12-15 °. The first set is indicated by the white arrows labeled "1" and "2" in Fig. 7 d and reflects the approximate twofold symmetry of the lumenal domains and radial arms. The position of the corresponding "spoke" twofold symmetry axis in the second set (offset by 45* for clarity) is shown by the dashed arrow. This approximate twofold axis bisects the central and inner spoke domains. The local rotational distortion that gives rise to the two sets of offset symmetry axes is less marked for the inner spoke domains located above and below the central xy slice (Fig. 8 a). Nearly equivalent slices located above and below the midplane of the spoke complex show the spokes as "pinwheels" with opposite handedness, as expected for a structure with twofold symmetry (Fig. 7, c and f). However, the correspondence in detail between the slices is only approximate. In addition, both the cytoplasmic and nucleoplasmic thin rings have eight paired domains although the density in the nucleoplasmic rings is weaker (compare Fig. 7, b and e). Both the positions and shapes of peaks within the cytoplasmic rings of the mform and dform NPCs are similar, including the small inward pointing regions. Unexpectedly, the cytoplasmic particles are better preserved in the dform NPC (Fig. 7 a), due possibly to a higher occupancy after specimen preparation. Cylindrical features at the centers of the xy slices are described in the next section on the NPC transporter.
There are large and intriguing differences present in the 3D maps of the mform and dform NPCs. In comparing cross-sections from the two maps (Fig. 5, a and c), it appears that the lumenal domains extend radially to a comparable distance. Other spoke domains are present in differing orientations relative to their neighboring domains. For example, the outer rod of vertical density in the mform NPC (Fig. 5  a) is kinked in the dform NPC (Fig. 5 c) rather than straight. Moreover, two vertical domains (Vo) appear to link the lumenal domain to the cytoplasmic and nucleoplasmic rings. The outer vertical domains also may form lateral contacts with the inner vertical domains (Vi). The "twofold" related inner vertical domains connect the inner spoke domain to the cytoplasmic and nucleoplasmic rings, respectively, although the lower connection is more disordered in the map of the dform. The structural role of the central domain may be pivotal, as a radial displacement of this domain may possibly have occurred in response to relative movements of the lumenal and outer vertical domains (Fig. 7 d). It seems likely that mechanical distortions may have induced the observed rotational displacement of this domain from the spoke twofold axis. In addition, the inner spoke domain is a dimer of two smaller domains that are connected to the central domain by a pair of strand-like connections at the midplane of the map (dotted line in Fig. 7 d). These strandlike connections may move outwards in the presence of the nuclear envelope and contribute to the ring of density observed at a radius of 420--430/~ in the mform NPC.
A cross-section located 22.5 ° clockwise from Fig. 5 c, bisects the radial arms as shown in Fig. 5 d. A number of features in common with the mform NPC are visible including the cytoplasmic particles, the cytoplasmic and nucleoplasmic rings, the radial arms and the inner spoke ring. However, the inner spoke ring has become more rounded and the vertical density located between the spokes in the 3D map of the mform NPC is absent. This latter density is abolished after detergent extraction; hence, it may originate in part from the nuclear envelope pore in which the NPC resides. The inner spoke ring plays a critical role in maintaining the integrity of the NPC, yet is rather variable when comparing projection maps of symmetric and asymmetric dform NPCs (this work; and references 3, 34) with maps of mform NPCs (3,4). This variability may reflect differing interactions of the inner spoke ring with the encircling nuclear envelope, as the result of detergent extraction or specimen preparation. In addition, the thin rings in the scaled 3D map of the reform NPC are 6-10% smaller in diameter than in the dform NPC. This suggests that the thin rings may be compressed radially as the result of an inward-directed pressure associated with the nuclear envelope.
Domain connectivity within a wedge-shaped spoke is shown in a rotation series of four surface maps in Fig. 8, with the rotation axis aligned vertically. The path of strongest connectivity may run as follows: the cytoplasmic portion of the inner spoke domain connects to the ridgelike inner vertical domain, which interacts with both the cytoplasmic thin ring and the outer vertical domain. The outer vertical domain contacts the lumenal domain which is linked to adjacent spokes by radial arm dimers forming a lumenal ring. The connectivity sequence described for the cytoplasmic half of the spoke is continued around the nucleoplasmic half as shown in Fig. 8, c and d. The asterisk in Fig. 8 b marks the location of the central domain which is intimately associated with the inner spoke domain and rotationaily offset from the spoke midline. The surface morphologies of the spoke ring assembly presented in this report and the 3D map described by Hinshaw and co-workers (34) are similar but not identical. Importantly, comparisons between maps of distinct but related forms have allowed us to make a qualitative assessment of domain connectivity and potential flexibility, after compensating for disorder within the nucleoplasmic halves of our 3D maps.
Color coded surface maps of the dform NPC structure are presented in Fig. 9 (a-f). The color coding is as follows: the lumenal ring is dark blue, the spoke-ring complex is medium blue and the transporter is pink. As with the mform NPC, the radial arm dimers connect lumenal domains in adjacent spokes to form a lumenal ring of nucleoporins ( Fig.  9, a and b). Furthermore, the lumenal domain does not protrude from the spoke as strongly but rather is more closely in contact with the outer vertical domains located above and below it (Fig. 9, b and c). Eight large particles are associated with the cytoplasmic ring. Their outermost portions mediate attachment to the cytoplasmic thin ring (Fig. 9 c) while the innermost regions are linked by a weak ring of density ( Fig.  9, a and b). This ring was not observed previously but may represent cytoplasmic filaments that have collapsed onto the NPC as the aqueous buffer layer thins during specimen blotting.
A surface map of the dform NPC in which the transporter and half of the spoke-ring complex have been computationally removed is shown in Fig. 9 d. In this view the alternating sizes of the domains which comprise the inner spoke ring are visible as are the cytoplasmic and nucleoplasmic thin rings. The inner spoke domain is tilted 'o7 ° clockwise from the vertical axis, towards the cytoplasmic surface and is comprised of two sub-domains related by an approximate twofold axis. The inner and outer vertical domains connect the thin rings to the inner spoke ring and outer portions of the spokes, respectively. These domains are emphasized by the small hollow feature that radially separates the lumenal domain from the inner spoke ring/central domain (Fig. 9, d-f).
Moreover, the observed rotational displacement of the central domain within the spoke may have accentuated the size of the hollow. This feature was not visualized in negatively stained NPCs, possibly because specimen compression as-sociated with drying ('o35-50%) may have closed this small hollow (see reference 34).

The NPC Transporter: A Tripartite Cylindrical Assembly
The NPC transporter was not preserved in specimen used to calculate the 3D map of the mform NPC; therefore, this map has a large central channel. However, the central transporter is present at a very high occupancy (~75-80%) in the dform NPCs. A corresponding ring of density is observed at the centers of the xy slices within the 3D map (Fig. 7, b-e), and depending on the level within the map this ringlike feature is either hollow or appears to be partially occluded by "endogenous" material. A different perspective is obtained by viewing the cross-sections shown in Fig. 5, c and d. In these z-slices, the NPC transporter is centrally tapered giving the impression of a tripartite structure constructed with very approximate twofold symmetry. The transporter appears to be hollow but additional density is present in the nucleoplasmic half of the map. This axial material may represent an averaged view of substrates caught "in transit" within the transporter (4,8), coupled with residual noise peaks. The trapping of substrates during enucleation into buffers without ATP may occur rather readily (3,8), as transport through the NPC is probably a highly cooperative process that requires soluble factors and ATP (1,44,46).
The cytoplasmic and nucleoplasmic surfaces of the transporter appear solid; a central channel entrance is not present. It cannot be ascertained if this is an intrinsic feature of the transporter or alternatively, may reflect the average super positioning of substrates docked at entrances to the central transport channel (4,8). The external tapering of the transporter c~linder is mirrored internally. The channel diameter is ~90A wide at the waist and expands out to ll0A in the cytoplasmic half and 160/~ in the nucleoplasmic half of the transporter. To some extent, these differences may reflect the presence of the axial material in the nucleoplasmic half of the transporter. The walls of the transporter are thinnest at the central taper, "o75-90~ These estimates will change with the collection of improved data on NPCs with defined configurations, as partial occupancy of substrates within the transporter may cause the walls to appear thicker after rotational averaging. Interestingly, the small diameter of the putative transport channel at the central waist would suggest that this region, located between possible gating assemblies at either end, may be flexible and undergo considerable expansion during substrate translocation (17,61).
In surface views of the NPC, the transporter appears as a central protruding cylinder with a fiat cytoplasmic surface ( Fig. 9, a-c). The complete transporter is revealed as a tripartite hourglass-shaped cylinder with an axial dimension of'o625~ in views in which half of the spoke-ring assembly has been computationaUy cut away (Fig. 9, e and f). The central waist of the transporter is "o320/~ in diameter while the putative gating assemblies located at either end have maximal diameters of "o420/~. The transporter in this structure is a global average of at least three to four different configurations that may be transport related (unpublished data); hence, additional data and classification analysis will be needed to resolve individual domains or subunits within the assembly. Connections between the transporter and the inner spoke ring form a set of eight inter-connected channels (Fig.  9, a-c). The general shape of these channels can be seen in Fig. 9 e, which is shown at a higher density cutoff such that the connections between the transporter and the spoke-ring assembly are eliminated. These internal channels were observed previously in projection, in class averages of mform NPCs with central transporters (4). The channel crosssections are roughly the correct size (80-90/~) to allow diffusion of ions, small molecules, and small nonnuclear proteins across the pore complex (49). Finally, the NPCtransporter is vertically displaced by roughly 50A towards the cytoplasmic surface of the NPC, away from the center of the spoke assembly (Fig. 9, e and f). This displacement may have occurred as the cumulative result of mechanical stresses during isolation; similar forces may have induced the observed asymmetry between the top and bottom halves of the spokes (see Figs. 8 and 9 e).
We maintain a high degree of confidence in the global structure of the NPC-transporter. First, 3D maps computed from half and quarter datasets are similar. Second, the size of the two globular end domains within the transporter, coupled with the approximate twofold symmetry, suggests that each transp,,o,,rter may be capable of splitting into "halftransporters' equivalent to the ,,o360/~ diameter pluglike particles obtained during disassembly studies (62). Third, the 3D map suggests that the central, disordered cylindrical rod of density observed previously in an initial 3D map (62) may be equivalent to the transporter. Fourth, Np-gold (23) and mRNPs (28,40,61) share a similar topology during transport, as both substrates are restricted to a central channel that spans the entire NPC. These data are explainable in terms of the NPC transporter which may provide a macromolecular channel that traverses the entire NPC.

Discussion
A detailed molecular analysis of the structure and function of the NPC has proven difficult due in part to the size, complexity and low abundance of this intracellular leviathan (39). However, our knowledge has increased in parallel with the development of techniques to visualize and quantitatively analyze the pore complex, in combination with newer methodologies which are now allowing the biochemical and genetic identification of constituent nucleoporins. Furthermore, the approach of combining cryo-electron microscopy and X-ray diffraction analyses with biochemical and genetic information is proving to be a powerful tool in the functional dissection of large macromolecular assemblies (35,60,63). As a first step in understanding the dynamics of NPC conformation and function, we have used cryo-electron microscopy and single particle 3D methods to analyze the structure of NPCs associated with either the nuclear envelope or detergent-extracted nuclear ghosts. To a first approximation, the NPC proper can be described in terms of the spoke-ring complex and the central transporter (but see Fig. 11). In our experience, both assemblies may exhibit differing conformations or configurations in isolated specimen (3,4) and we fully expect that some of these changes may be induced by sample isolation and preparation for EM. However, it is our working hypothesis that both the apparent conformational flexibility of the spoke-ring complex (3) and the observed configurations of the transporter (4, 8) may be relevant in a fundamental way to processes that occur in vivo.

Conformational Flexibility and a Minimal Domain Model of the NPC
A comparison of the two 3D structures reported in this work reveals that the spoke-ring complex may be rather flexible and allows a qualitative analysis of domain positions and interactions. Idealized domain models for the m-and dforms ofXenopus NPCs are shown in Fig. 10, redrawn from density maps of z-slices cut along the putative "spoke" (Fig. 10, a and c) and "radial arm" twofold axes (Fig. 10, b and d). Inferred positions of the nuclear envelope and the NPC transporter have been indicated in Fig. 10, a and b. The spoke assembly is twofold symmetric about the midplane of the structure as shown in the best 2D maps (3,4,42,62) and in a recent 3D map of negatively stained NPCs (34). Although there are varying degrees of distortion present in the nucleoplasmic halves of our 3D maps, the major features support this interpretation. Inspection of the two 3D maps suggests that conformational changes involving the spoke domains and thin rings have occurred, possibly as the result of detergentextraction, osmotic shock within the lumen of the nuclear envelope or mechanical stresses during specimen preparation.
As an aid in visualizing the overall design of the NPC, a 3D ribbon diagram of the membrane-associated NPC is shown in Fig. 11. In addition, this diagram includes peripheral assemblies visualized by other methods that form part Figure 9. A gallery of color coded 3D surface views is shown of the detergent-extracted NPC. The spoke-ring assembly including cytoplasmic particles (CP) is medium blue, the lumenal ring is dark blue and the central NPC-transporter is pink. (a) The NPC is shown from the cytoplasmic surface viewed at a glancing angle of 10 °. Note the attachments between the transporter and the surrounding spoke ring assembly and the thin ring of density which links up adjacent cytoplasmic particles. The lumenal spoke, radial arm domains and the cytoplasmic thin ring are labeled. (b) A view of the NPC rotated 45 ° about a vertical axis is shown. The lumenal ring is highlighted in this view. The cytoplasmic particles and the nucleoplasmic thin ring are labeled. (c) A view of the NPC from the cytoplasmic surface is shown in the same orientation as a, but with the top portion of the cytoplasmic particles removed to show the packing of the transporter within the inner spoke ring. Entrances to possible diffusion channels are delineated by the connections between the transporter and the inner spoke ring. (d) A cut-away view of the spoke-ring assembly is presented with the central transporter removed to show the internal surface of the spoke-ring assembly. The cut was made along the approximate spoke twofold axis which bisects two of the wedge-shaped spokes. The lumenal ring and the cytoplasmic particles are indicated. (e) A second cut-away view is shown with the transporter left intact. In this view the cutting plane is oriented along a putative radial arm twofold axis, between adjacent spokes. The packing of the transporter within the wedge-shaped spokes is visible as are opposite surfaces of the spokes (compare with Fig. 8). (f) A cut-away side view of the NPC is shown in the same orientation as d with the transporter left intact. Internal channels located between the hourglass-shaped transporter and the inner spoke ring are revealed. The transporter spans the width of the NPC and the local twofold axis of the transporter is offset from the approximate twofold axis of the spoke-ring assembly by ,,~50 A, towards the cytoplasmic/top surface. The size of the cavity located between the inner spoke/central spoke domains and the lumenal spoke domain has been exaggerated (h). In addition, connections between the transporter and the nueleoplasmic face of the inner spoke domains have been included although they are weak in the original map, due to local disorder. Note the overall change in shape of the inner spoke ring in b and d, which suggests that the nuclear envelope has a significant effect on the conformation of this structural element. of the extended model of the NPC (2,28,33,36,55,57). Individual domains have been reduced in size (or in some instances omitted) for clarity; for example the inner spoke ring has been reduced in height to allow visualization of the central NPC transporter. However, this reduction causes a distortion of the inner vertical domains within the wedgeshaped spokes. Hence, the ribbon diagram should be used to orient the various subassemblies of the NPC, the model cross-sections in Fig. 10 are more accurate in terms of the spatial relationships between spoke domains. Simply, the architecture of the spoke-ring assembly can be described in terms of four colinear rings, that use homologous and heterologous interactions between domains to maintain the integrity of individual spokes while mediating assembly of the eightfold symmetric pore complex. First, the inner spoke ring is formed by domains that bridge the inner domains within adjacent spokes to form an eightfold symmetric annulus. The inner spoke ring appears to be the central organizing element of the spoke-ring complex and may function as an adaptor between the spoke-ring complex and the NPC transporter, to which it is connected (Fig. 11). Second, the cytoplasmic and nucleoplasmic thin rings may stabilize the spoke-ring assembly by making intimate contacts with the inner and outer vertical domains in each of the eight spokes. Third, the anchoring of the spoke assembly within the nuclear envelope may be stabilized by the formation of a lumenal ring, comprised of eight lumenal domains bridged by radial arm dimers (Fig. 11). Importantly, the position of the Figure 11. A 3D representation of the membrane-associated NPC is presented as a ribbon diagram. Structural information from other groups has been incorporated to give an overall impression of our current knowledge of the extended architecture of the NPC (2,28,33,36,55). For clarity, the dimensions of certain features have been minimized and the top cytoplasmic ring (CR) is sliced open to reveal the interior. For example, the inner spoke ring (ISR) is shorter and thinner than in the real structure (see Fig. 9, d-f). These changes result in some artistic adjustments being made in the shape of the inner vertical spoke domains which link up the ISR to the cytoplasmic thin ring. The spokes are wedge shaped with seven domains in the diagram. In actuality the inner spoke domains (pan of the ISR in this diagram), the central and the lumenal domains are dimers, giving a total of 10 domains per spoke. The diagram emphasizes the construction of the NPC from four coUinear ring systems including the cytoplasmic and nucleoplasmic thin rings (CR/ NR), the inner spoke ring (comprised of the inner spoke domains and the material between spokes) and the lumenal ring which links up adjacent spokes within the lumenal space. The lumenal ring is composed of the lumenal domains (LS) and the radial ann dimers (RA). Additional components of the NPC proper are labeled: cytoplasmic particles (CP), the spokes (S), and the tripartite transporter (T). The precise attachment sites of peripheral assemblies are not known. Cytoplasmic components include thin filaments (CF) which attach to the exposed thin ring and the outer nuclear membrane (ONM). On the nucleoplasmic surface, the lamina is apparently anchored into the inner nuclear membrane (INM) by a membrane receptor (RL). An octal~onal cage or basketlike structure (NC) is attached to the outer surface of the nucleoplasmic thin ring. This structure may extend ,,ol,000 A into the nucleoplasm. Moreover, adjacent NPCs may be linked into groups by the nuclear envelope lattice (NEL) which interconnects distal rings (DR) of the cages (33). Finally, thin bridges of material connect each half of the transporter to the inner spoke ring, thereby forming a set of interconnected channels that may share a common vestibule formed by the packing of the centrally tapered transporter within the inner spoke ring.
central domain within a wedge-shaped spoke may be pivotal, as this domain appears capable of moving ~35--40/~ radially in a rearrangement that may involve a repositioning of the outer vertical and lumenal domains (compare a and c in Fig.  10). Overall, the dimeric inner spoke, central, and lumenal domains each contribute a domain to the vertical inner and outer domains to form half of a wedge-shaped spoke with a total of five domains. Subsequently, each half spoke is paired about a central twofold axis (3,34,42) to generate a complete spoke and eight of these units combine with the cytoplasmic and nucleoplasmic thin rings to form an articulated spoke-ring assembly.
A comparison of the 3D map of Xenopus NPCs reported by Hinshaw and co-workers (34) with our work, reveals that the basic features of the spoke-ring complexes are remarkably similar including a sharing of the same absolute handedness. The 3D maps of both dform NPCs were produced using the random conical tilt reconstruction method (50), with different image processing packages (SPIDER/SUPRIM) and different specimen preparative methods (frozen-hydrated specimens vs. negative-staining). The similarity of the final 3D structures serves to validate both the 3D reconstruction methods and the reliability of the resulting maps. Although rapid freezing can potentially preserve structural order to atomic resolution, specimen dependent factors generally result in a much lower attainable resolution. For example, the nucleoplasmic halves of the 3D maps may be partially disordered resulting from the transduction of mechanical stress along the fibrous lamina and its attachments to the NPC (2,3). Alternatively, changes in spoke-ring conformation may result from hydration changes of the nucleoplasm (swelling), enucleation, and the possible loss of lumenal components.

Cytoplasmic Particles and Peripheral Assemblies
Fingerprinting experiments first demonstrated the existence of asymmetrically shaped particles on the cytoplasmic surface of the NPC with a diameter of ~ 220A (62). Subsequently, Reichelt and co-workers (52) measured particles released from the nuclear envelope with diameters of 230-270A and showed them to have a mass of ~6-6.7 Mda. These particles were associated with the thin rings in some instances; however, their mass appears to preclude them from being ribosomes. Particles are found associated with the surface of the cytoplasmic thin rings in both 3D structures presented in this work. The particles in the map of the dform are better preserved and have dimensions of ,,o 230 x 160/~ when viewed along the eightfold axis from the cytoplasm. The shape of the particles is similar to particle profiles observed in negatively stained specimens obtained by fingerprinting (62), suggesting that these are true particles rather than collapsed filaments (36,55). At a lower density cutoff, a thin ring appears to inter-connect the eight cytoplasmic particles in the dform NPC. This ring may originate from the collapse of thin cytoplasmic filaments during adsorption of the specimen to the carbon film (33,55). Other peripheral assemblies including the nucleoplasmic cages/ baskets (33,36,54,55,57), the nuclear envelope lattice (33) and attachments to the nuclear lamina were not retained during specimen preparation or alternatively, were too strongly disordered to be visualized (see Fig. 11 for the relative positions of these components).

Interactions with the Nuclear Envelope
The NPC resides within a pore in the nuclear envelope formed by the fusion of the inner and outer nuclear membranes. In the current 3D map of the mform NPC, the nuclear envelope appears to conform to the spoke-ring assembly while spanning exposed lateral surfaces of the spokes between the two oppositely facing thin rings. In addition, the ring of density observed in projection maps of_the mform NPCs, occurs at an average radius of 420-430/~ in the 3D map and the central spoke domain traverses this feature. Fi-naUy, the lumenal spoke domains and the radial ann dimers form a ring of nucleoporins on the lumenal side of the nuclear membrane. We suggest that this lumenal ring may play a role in both the assembly and anchoring of the pore complex. Gp-210 a membrane glycoprotein component of the NPC with a single transmembrane domain has been proposed to play a role in anchoring the NPC to the nuclear envelope (32,64). The 3D map of the mform NPC suggests that the large lumenal domain of gp-210 may be localized to either the radial arms or the lumenal domains. In either case, the possibility exists that the lumenal portion of gp-210 may mediate the formation of homodimers, although this species may not be stable when the molecule is removed from the context of the spoke-ring assembly.
Recently, Greber and Gerace (31) have shown that microinjection of total hybridoma mRNA encoding a gp-210 specific IgG (RL27) into tissue culture cells, results in the synthesis and transport of antibody into the lumen of the ER and the nuclear envleope, where it binds gp-210. Remarkably, the nuclear import of micro-injected nucleoplasmin was reduced fourfold and diffusion of a 10-kD dextran was also diminished in these cells. What is the structural basis for this dramatic down regulation of active transport and diffusion from the lumenal side? Depending on the location of the epitope recognized by RL27, the functional effect(s) induced by antibody binding would have to be propagated over a radial distance of * 350-520A, to impinge on the putative diffusion and macromoleeular transport channels. Although of non-physiological origin, the plasticity of the spoke-ring assembly observed in our maps may mimic certain aspects of a possible "conformational signaling" mechanism in which nucleocytoplasmic transport is globally regu-lated by signals originating from the lumenal side of the pore complex. For example, it is conceivable that RL27 binding to the lumenal ring may trigger rearrangements within the outer vertical and lumenal domains, which in turn are transmitted to the inner spoke ring by radial displacement of the central spoke domains. Of course, alternative routes are possible. However, the inner spoke ring may play a critical role in this scenario, as it makes connections to the NPCtransporter that define an internal network of channels. Therefore, it is conceivable that changes within the spokering complex may be transmitted to putative gating assemblies within the central transporter by way of the inner spoke ring. Overall, our observations on the conformational plasticity of the spoke-ring complex in vitro, lend support to the emerging hypothesis that the spokes may transduce signals provided by as yet unidentified lumenal or intracellular effeetors, thereby modulating global transport properties of the NPC under physiological conditions (22,31).

Internal Eh'ffusion Channels
Micro-injection studies with dextrans (49) and polyvinylpyrrolidone-coated gold particles (20) have established that diffusion channels with a limiting diameter of ~90-100/~ may be present in the pore complex, resulting in an estimated transport cutoff for non-nuclear proteins and small molecules of •40 kD. The 3D map of the dform NPC suggests that 8 diffusion channels may be located internally between the inner spoke ring and the central NPC transporter, with 16 entrances (eight from either side) delineated by connections between the transporter and the inner spoke domains (Fig. 10, a and b). The 16 entrances may link up in a common cylindrical vestibule created by the packing of the centrally tapered transporter within the surrounding inner spoke ring. A clearer picture of this internal channel network should emerge with higher resolution data on a defined transporter configuration. Recently, eight possible diffusion channels have been hypothesized by Hinshaw et al. (34) to occur between adjacent spokes at a radius outside the inner spoke ring. Our current 3D map of the reform NPC in vitro does not directly support this proposal. However, it is conceivable that the nuclear membrane may bulge outwards between adjacent spokes in vivo while remaining tethered to the central spoke domains, thereby creating "peripheral" diffusion channels for soluble and membrane proteins between the spokes. Additional experiments are needed to determine the radial position(s) of diffusion channels within the NPC in vivo.

The NPC Transporter and Models of the Translocation Step
Previous work in projection has established that a central ringlike structure is present within the NPC. This assembly has been named the transporter, as it appears to define the macromolecular transport channel for NP-gold conjugates and endogenous substrates (3,4,8). In this work, the 3D map of the dform NPC demonstrates a central cylindrical feature that may represent a "global" average of transporters trapped in a number of transport-related configurations during enucleation. Interestingly, the averaged transporter has a tripartite structure and is comprised of two "globular" assemblies which join face-to-face about an approximate twofold axis to form a cylinder with a tapered center. The NPC transporter is hollow with 75-90/~-thick walls and a total length of 625/k. Studies of the nuclear import of karyophilic proteins suggest that nuclear import occurs within a central channel of the NPC as follows. Initially, microinjected substrates accumulate at the nuclear periphery in a temperature independent fashion, ATP-dependent vectorial translocation then ensues after substrate binding to the NPC (46,47,53). Further mapping studies using nucleoplasmin-gold have suggested that transport involves substrate docking to the central NPC transporter, before a possible gating event within this assembly (8).
A number of models for the translocation step in nucleocytoplasmic transport have been discussed. For instance, it has been suggested that substrates might move along illaments postulated to traverse the NPC aided by an ATPase/ motor molecule (10). Recent data have confirmed observations that filaments are attached to both the cytoplasmic and nucleoplasmic surfaces of the NPC (33,36,53,55,57). However, our maps suggest that there is no physical room for filaments to traverse the NPC itself when the transporter is present; hence, this mode of translocation would appear unlikely. A second model has posited that a substrate bearing container may transit through the center of the spoke assembly, thereby providing protection for mRNPs. This model was recently brought to the forefront with the suggestion that vaults, a large assembly with 822 symmetry, might be involved in transport (37). However, images in which large mRNPs (28,40,61) and Np-gold (23) are caught transiting the entire width of the NPC would argue against the transport container hypothesis. Moreover, a container mechanism appears unnecessary as many mRNPs spend a large fraction of their lifetimes associated with fibrous tracts (reviewed in ref- erence 38). Finally, the dimensions of the vaults and the NPC transporter in frozen-hydrated specimens are dissimilar (vaults: 490 x 275]t; transporter: 625 x 420/~) and their morphologies are rather different.
The recent visualization of octagonally arranged baskets or cages on the nucleoplasmic face of the NPC has sparked interest in the possible roles of these structures in transport (33,36,54,55). However, the strict localization of the cages on the nucleoplasmic side of the NPC would pose a topological problem to possible roles in a bidirectional gating mechanism. In addition, recent micrographs of the nucleoplasmic surface of metal-coated amphibian oocytes have demonstrated the presence of a filamentous lattice that interconnects adjacent cages at their distal rings (33). Perhaps, the cages provide access to the nucleoplasmic face of the NPC transporter for macromolecules within the context of the high density of peripheral chromatin and the thickened lamina present in many cells.
Labeling studies with WGA and MAb414 suggest that the transporter may be eightfold symmetric, as these bivalent probes often label individual transporters in groups of four (8,57). A direct determination of the symmetry of the transporter must await monospecific FAbs for components of the transporter. Recently, a macromolecular lock/double iris hypothesis has been proposed to describe the translocation of substrates catalyzed by the NPC (4). In this model the transporter is proposed to have ,~822 symmetry (reviewed in references 5 and 6). The limited number of NPCs in our dataset coupled with the apparent admixture of differing transporter configurations, results in a globally averaged view of the transporter that does not allow a direct confirmation of the irislike gating mechanism at this stage. However, the tripartite morphology of the NPC transporter when combined with data on substrate translocation through the centers of NPCs (4,8,23), lends support to the hypothesis that this process may involve two oppositely facing gates. In this model, the two globular end domains of the NPC transporter would correspond to possible gating assemblies. It is suggested that these putative gates may open asynchronously to regulate transport along a central channel like a macromolecular lock (4,7). Furthermore, it is tempting to speculate that the translocation mechanism may use induced changes in channel diameter, within the centrally tapered region of the transporter, to communicate the status of oppositely facing gates.

Conclusions
The NPC is comprised of a symmetrical or pseudo-symmetrical spoke-ring complex located in the plane of the nuclear envelope, that frames a central transport machine. In addition, data from other groups indicates that the NPC makes peripheral attachments to cytoplasmic filaments, particles, nucleoplasmic cages/baskets and the lamina to form an extended transport assembly (see Fig. 11; reviewed in references 7 and 55). Our 3D maps suggest that each half spoke is comprised of five domains that appear capable of moving relative to each other, providing an inherent conformational flexibility within the spoke-ring assembly. Therefore, it is hypothesized that the structural plasticity observed in vitro may play a role in the global regulation of transport in vivo, at the level of the NPC. In addition, the framework of the pore complex is firmly anchored within the nuclear envelope as the tip of each spoke penetrates into the nuclear lumen. In this way, a lumenal ring of nucleoporins is formed by the association of adjacent lumenal spoke domains with intervening radial arm dimers. Interestingly, the nuclear envelope appears to conform to the surface of the octagonal spoke complex while leaving the thin rings exposed to their respective cellular compartments (also see reference 33). However, the lumen between nuclear membranes may undergo osmotic swelling during the isolation of macro-nuclei. Hence, it is not known whether the membrane-associated NPC described in this work represents the in vivo conformation or merely a single structure in a range of possible structures (reference 34; and this work). Moreover, the visualization of the central transporter as an elongated centrally tapered cylinder, lends support to the proposed macromolecular lock hypothesis. In this model, it is suggested that substrate passage across the nuclear envelope may require two asynchronous gating events within the context of a central channel (4). High resolution data are now needed on distinct conformations of the spoke-ring complex and the associated transporter to extend our understanding of this unique transport machine. This work has benefited from the suggestions of M. Rout and the support ofN. Unwin. The computational biology was greatly aided by A. Leith who made available upgrades of SPIDER/WEB and D. Atkinson, who set up our computer network. We gratefully acknowledge the advice provided by R. Nolte on color slide and print making. Finally, we thank colleagues in the Department of Cell Biology, Stanford University School of Medicine and the Structural Studies Division, Medical Research Council Laboratory of Molecular Biology, for access to and support of the Philips EM400 series microscopes.
This work was supported by the National Institutes of Health (R01 GM45377 and R01 GM29169).
Received for publication 22 September 1992 and in revised form 6 April 1993.