Vimentin filaments integrate low-complexity domains in a complex helical structure

Intermediate filaments (IFs) are integral components of the cytoskeleton. They provide cells with tissue-specific mechanical properties and are involved in numerous cellular processes. Due to their intricate architecture, a 3D structure of IFs has remained elusive. Here we use cryo-focused ion-beam milling, cryo-electron microscopy and tomography to obtain a 3D structure of vimentin IFs (VIFs). VIFs assemble into a modular, intertwined and flexible helical structure of 40 α-helices in cross-section, organized into five protofibrils. Surprisingly, the intrinsically disordered head domains form a fiber in the lumen of VIFs, while the intrinsically disordered tails form lateral connections between the protofibrils. Our findings demonstrate how protein domains of low sequence complexity can complement well-folded protein domains to construct a biopolymer with striking mechanical strength and stretchability.

(~1.68) the connections of the luminal fiber with the filament tube are visible.At low threshold (~0.18) the protofibrils appear as separated units.The isosurface cross-section visualization of this subtomogram average (Fig. 1d, Extended Data Fig. 1d, and Extended Data Fig. 2e) is a montage between two thresholds to allow for counting of the protofibrils (threshold ~0.72) and visualization of the luminal fiber (threshold ~1.21).Scale bar 10 nm.helical twist and 30 Å to 57 Å for the helical rise, therefore also capturing possible helical assemblies based on 4 or 6 protofibrils.As starting value for the symmetry search an initial twist angle of 72° and an initial helical rise of 37 Å was set (green dot), and this helical symmetry was also imposed on the initial 3D template.In between the helical 3D classifications, those 3D classes converging to the borders of the search interval were iteratively removed.In the final helical 3D classification (the resulting helical symmetry parameter are shown as blue dots in the plot and the corresponding class averages are shown in (c)), the mean helical symmetry was 73.4° for the helical twist angle and 42.1 Å for the helical rise (red dot).This helical symmetry parameter were used as the initial twist and rise values for subsequent local helical symmetry searches during 3D refinement, finally converging to the exact identical helical symmetry parameter as determined before based on power spectrum analysis.(d) The VIF segments underlying the final VIF 3D structure were combined with 2D classification to assess the gain of structural homogeneity during data processing.The image shows a representative subset of 27 out of 100 class averages calculated from the final 236,920 VIF segments.Scale bar is 35 nm.

Supplementary
(b) Visualization of the angular distribution of the final VIF 3D structure (Fig. 2).(c) In order to reach subnanometer resolution it was critical to mask the luminal fiber during 3D refinement.
The blue colored density shows the final VIF average and the grey density the mask applied during the final 3D refinement.The structure of the luminal fiber (yellow density) was obtained from a preceding refinement (resolution ~14 Å) without masking the luminal fiber.For visualization the final VIF structure was combined with the luminal fiber structure (Fig. 2).

Figure 2 .Supplementary Figure 3 .
Cryo-ET of detergent-treated MEFs.(a) Maximum intensity projection of a 3D-SIM image (n=20) of a detergent-treated MEF.The VIF network remains intact following the permeabilization procedure, fixation and staining with vimentin antibody (green).The cell nucleus is stained in red using lamin antibody.Scale bar 10 µm.(b) Slice in x-y-direction with 13.76 Å thickness through a cryo-tomogram of detergent-treated MEFs (n=225).Scale bar 200 nm.(c) The correlation of each class average (n=8) with itself was calculated and the corresponding autocorrelation profiles along the x-axis were plotted (black lines).The red line is the averaged autocorrelation profile over all class averages.The pattern in the averages repeats at a distance of 179 Å ± 40 Å.(d) Model to explain the boundary asymmetry in the class averages.If VIFs are assembled from 5 protofibrils, one side of the VIF would appear brighter in projection (grey rectangle).However, if they are assembled from 4 or 6 Computational assembly of VIFs.(a) VIF segments (615,106 particles with a size of 38 x 38 nm 2 ) were extracted from tomograms of detergent-treated MEFs and combined with 2D classification.The picking distance between the segments was set to 55 Å and the projection thickness to 220 Å [1].The displayed class averages were used for subsequent computational filament assembly.Scale bar 35 nm.(b) The computationally assembled VIFs (ca-VIFs) allow to follow the progression of the filaments with improved signalto-noise ratio over a substantial length (n=5205).The displayed filament box is 353 nm wide.(c) The ca-VIFs are represented by a series of transformed, tailored, and overlapping class averages.The densities are normalized according to the local overlap of the class averages.The image shows the normalization mask that was applied to the ca-VIF shown above.More overlap between the class averages is indicated by brighter regions.(d) Gallery of unbent ca-VIFs (n=5205).Scale bar 100 nm.
(d) Calculating the gold-standard Fourier shell correlation (FSC) curve with the RELION command relion_postprocess, and using the option --ampl_corr, indicates a global resolution of 5.0 Å at FSC = 0.143.(e) Local resolution measurement of the electron density map.(f) The gold-standard FSC curve of the final VIF 3D structure indicates an overall resolution of 7.2 Å at FSC = 0.143.