Supporting Online Material. Nuclear Pore Complex Structure and Dynamics. revealed by Cryoelectron Tomography

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1 1 Supporting Online Material Nuclear Pore Complex Structure and Dynamics revealed by Cryoelectron Tomography Martin Beck, Friedrich Förster, Mary Ecke, Jürgen M. Plitzko, Frauke Melchior, Günther Gerisch, Wolfgang Baumeister* and Ohad Medalia* Materials and methods Preparation of the nucleus-enriched fraction Dictyostelium discoideum AX2 cells of an exponentially growing suspension culture were washed with 17 mm phosphate buffer (adjusted to ph 7.5 with KOH) and resuspended in 50 mm Tris ph 7.6, 25 mm KCl, 5mM MgCl 2, 50 mm sucrose, 1 mm DTT and protease inhibitor cocktail (Roche) to a final density of approximately 10 8 cells/ml. The cells were lysed by passing them through a 5 µm polycarbonate filter (Whatman, Nuclepore). Nuclei were spun down (5 min at 500g) and washed in the same buffer to obtain an enriched fraction. All steps were carried out on ice. For nuclear import assays the enriched nuclei were resuspended in cytosolic supernatant, which was prepared by spinning the lysate for 5 min at 20,000 g. Nuclear import assay A volume of 30 µl total cell lysate or a nucleus-enriched fraction supplemented with cytosolic supernatant were used for the transport reactions. Transport mixtures with a total volume of 32 µl contained 50 µg/ml FITC-BSA-NLS and an energy regenerating system (1.5 mm ATP, 5 mm creatine phosphate, 35 U/ml creatine phosphate kinase). Energy depletion was achieved

2 2 by pre-incubation of the lysate with apyrase (100 U/mL) and omitting the components of the energy regenerating system. NLS-dependence was shown using a high concentration of FITC- BSA (up to 375 µg/ml) without NLS. Dependence on soluble cytosolic proteins was shown by resuspension of nucleus-enriched pellets in buffer instead of cytosolic supernatant prior to the transport assay. The samples were incubated for 15 min on ice in the dark and then observed with a Zeiss Axioplan 2 light microscope. Quantification of light microscopy images was done by determining the mean fluorescence of individual nuclei with correction for background fluorescence. To confirm active nuclear import as an energy- and time dependent process the assay was carried out in the presence of various amounts of GMP-PNP (Fig. S1A) and for different time intervals (Fig. S1B). Cryo-electron tomography Enriched suspensions of nuclei were pipetted onto lacy carbon grids (Plano) and rapidly frozen in liquid ethane as described by (1). The data acquisition was done as described previously (2). In brief, the specimens were transferred into a Phillips CM 300 FEG microscope equipped with a Gatan post column GIF 2002 energy filter. Tilt series were collected, typically covering an angular range from -63 to 63, sampled with 1.5 tilt increments and at 15 µm underfocus. Therefore, the theoretical resolution limit as set by the contrast transfer function was 5.5 nm. The pixel size was 0.82 nm at the specimen level. Reconstructions were calculated by weighted backprojection (3). All surface rendered views were created with the Amira software package (TGS). An anisotropic denoising algorithm (4) was used to improve the visualisation shown in (Fig. 1E).

3 3 Image processing 3D alignment of NPCs Individual NPCs were located manually in the tomograms and the corresponding subvolumes were extracted for further processing. An iterative averaging algorithm, similar to approaches used in the field of single particle analysis (5) was used for structure determination. The angular and spatial alignment was performed by an improved procedure based on the method described by (6). The cross correlation function used for alignment accounts for the missing wedge in Fourier space (Förster et al, in preparation). Membrane-bound complexes represent highly anisotropic structures and consequently, their information content varies considerably as a function of spatial direction. Therefore, the cross-correlation function was normalized accordingly (7). Exclusion of particles possessing a low signal-to-noise-ratio (SNR) was done by thresholding based on the intensity of the obtained cross-correlation coefficients. Since the nuclear envelope surrounding the NPCs is highly flexible, the individual particles were masked close to the perimeter of the NPC prior to alignment. During the iterative process the position of the mask was adjusted due to the shifts determined. The orientations of the individual complexes were described by the Eulerian angles ϕ, ψ, and θ. Initially, ψ and θ were approximated by the orientation of the particles on the surface of the nucleus, the shape of which was assumed ellipsoid. An initial model for the NPC was generated using a random distribution of the polar angle ϕ. Eight-fold rotational symmetry along ϕ was detected and therefore imposed during the refinement process in order to improve the SNR. All computations were performed with data cubes of 64x64x64 voxels, each voxel corresponding to 35.2 nm 3 at the specimen level. Finally, five iteration cycles were carried out using 128x128x128 voxels, corresponding to 4.4 nm 3 each. In order to determine the resolution of the averaged structure, the aligned particles were divided into 2 stacks and averaged separately. Fourier-ring correlation (8) of both structures decreases to less than 0.5 below 9 nm (using the 2-σ-criterion the resolution is 6.5 nm, Fig. S3A). Because of the denser

4 4 environment in the nucleoplasm, different thresholds have been applied for the surface rendering of the nuclear basket and the core structure, consisting of the three rings, cytoplasmic filaments and the CP/T (see Fig. 2, 3, nuclear basket in brown, core in blue). The grey value of a subregion in the averaged structure corresponding to nucleoplasm was chosen as threshold level for the intensity of the nuclear basket, while the grey value of a subregion corresponding to solvent (cytoplasmic side) was chosen as threshold level for the intensity of the core structure. Database information The electron density map of the structure has been uploaded to the European Electron Microscopy Database (EMBL-ESI, accession number EMD-1097). Quantitative analysis of the CP/T Due to the missing wedge problem, the classification of particles derived from tomographic volumes by multivariate statistical analysis (9) was not successful. With NPCs active in transport, the highest variability is expected inside the central channel and indeed individual NPCs showed strong variations in this feature of the structure (Fig. S2). Determination of the centres of gravity and the occupied volume served as a classification criterion independent of the missing wedge. CP/Ts were extracted in silico by application of an ellipsoidal mask fitting into the central channel. The grey value of a subregion in the averaged structure corresponding to the solvent was chosen as threshold level for the intensity. The occupied voxels have been summed, and local centres of gravity were determined. The largest occupied volume that we detected (1180 voxels) is consistent with a sphere-shaped complex with a diameter of ~ 40 nm; however, reliable estimates regarding the size of cargo complexes cannot be deduced from such data. The cargo interacts with nucleoporins lining the central channel, and there could be more than one, bound cargo complex. The distribution of the

5 5 occupied volumes is broader than a normal distribution (Fig. 3A), which would indicate that the CP/T in different NPCs comprises essentially the same components. The observed mass is thus likely to represent an average of different structures, rather than a constitutive substructure within the central channel of the NPC, supporting the notion that the CP/T comprises, in part, cargo complexes arrested during translocation. In contrast to the occupied volumes, the distribution of the centers of gravity along the nucleocytoplasmic axis shows two distinct peaks (Fig. 3B). A double Gaussian function can be fitted to this distribution, indicating that two preferred positions of mass within the central channel exist, and that these are likely to correspond to different NPC states. Hence, the distribution of the centers of gravity along the nucleocytoplasmic axis provides a means for an objective classification of individual NPCs. The local minimum between the two peaks served as a criterion for separating the particles into two stacks (position marked white in Fig. S2B, right). The two classes consisted of 115 (LR-class, represented by ~9200 individual images) and 82 (CFclass, represented by ~6500 individual images) particles and have been averaged as described above. The spatial orientation of the particles within each class covered the entire angular spectrum (Fig. S3B). The resolutions of both structures according to the Fourier-ringcorrelation-0.5-criterion were about 10.5 nm (Fig. S3). Structures generated with an equal number of randomly chosen particles had significantly lower resolutions. Surface rendering of both classes was done as described above (see 3D alignment of NPCs).

6 6 Supporting figures Fig. S1. Active nuclear import into isolated Dictyostelium nuclei. (A) Dependence of nuclear import on a non-cleavable GTP analogue. Import reactions have been carried out with various amounts of GMP-PNP, which had an inhibitory effect on nuclear import. (B) Time course of nuclear import. Import reactions were carried out over the periods of time indicated.

7 7 Fig. S2. Visualisation of a nuclear envelope and variations of the CP/T in individual NPCs. (A) Gallery of NPCs sliced perpendicular to the nucleocytoplasmic axis at the lumenal spoke ring plane in comparison to the averaged structure (right). The CP/Ts are connected to the rings by internal filaments (arrows). (B) Gallery of NPCs sliced parallel to the nucleocytoplasmic axis in comparison to the averaged structure (right). The CP/Ts vary in size and shape and occupy diverse positions.

8 8 Fig. S3. Resolution determination and particle orientation. (A) Resolution determination by Fourier ring correlation (FRC). The FRC of the 2 stacks of aligned particles decreased to less than 0.5 below 9 nm (green dotted line). Using the 2-σ criterion (red line), the resolution was 6.5 nm. (B) Individual NPCs of the two classes cover the entire spectra of angular orientation. Plot of the Eulerian angles Phi and Psi against each other (CF-class in blue, LR-class in red). (C) Same as (A) but for the CF-class. The resolution was 10.5 nm. (D) Same as (A) but for the LR-class. The resolution was 10.5 nm.

9 9 Supporting references and notes 1. J. Dubochet et al., Q Rev Biophys 21, (May, 1988). 2. O. Medalia et al., Science 298, (Nov 8, 2002). 3. R. Hegerl, J Struct Biol 116, 30-4 (Oct, 1996). 4. A. S. Frangakis, R. Hegerl, J Struct Biol 135, (Sep, 2001). 5. M. Van Heel, Ultramicroscopy 21, (1987). 6. J. Walz et al., J Struct Biol 120, (Dec, 1997). 7. A. S. Frangakis et al., Proc Natl Acad Sci U S A 99, (Oct 29, 2002). 8. W. O. Saxton, W. Baumeister, J Microsc 127 (Pt 2), (Aug, 1982). 9. J. Frank, M. van Heel, J Mol Biol 161, (Oct 15, 1982).