Divide-and-Conquer Crystallographic Approach Towards an Atomic Structure of Intermediate Filaments Sergei V. Strelkov 1 , Harald Herrmann 2 , Norbert Geisler 3 , Ariel Lustig 1 Sergei Ivaninskii 1 , Ralf Zimbelmann 2 , Peter Burkhard 1 and Ueli Aebi 1 * 1 Maurice E. Mu È ller Institute for Structural Biology Biozentrum, University of Basel, Klingelbergstrasse 70 CH-4056 Basel, Switzerland 2 Division of Cell Biology German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg Germany 3 Division of Biochemistry and Cell Biology, Max Planck Institute for Biophysical Chemistry, Am Faberg 11 D-37070 Go Èttingen, Germany Intermediate ®laments (IFs) represent an essential component of the cytoskeleton in higher eukaryotic cells. The elementary building block of the IF architecture is an elongated dimer with its dominant central part being a parallel double-stranded a-helical coiled coil. Filament formation proceeds via a speci®c multi-step association of the dimers into the unit- length ®laments, which subsequently anneal longitudinally and ®nally radially compact into mature ®laments. To tackle the challenge of a crys- tallographic structure determination, we have produced and character- ised 17 overlapping soluble fragments of human IF protein vimentin. For six fragments ranging in length between 39 and 84 amino acid residues, conditions yielding macroscopic crystals could be established and X-ray diffraction data were collected to the highest resolution limit between 1.4 and 3 A Ê . We expect that solving the crystal structures of these and further fragments will eventually allow us to patch together a molecular model for the full-length vimentin dimer. This divide-and-conquer approach will be subsequently extended to determining the crystal structures of a number of complexes formed by appropriate vimentin fragments, and will eventually allow us to establish the three- dimensional architecture of complete ®laments at atomic resolution. # 2001 Academic Press Keywords: intermediate ®lament; vimentin; protein assembly; crystallisation; atomic structure *Corresponding author Introduction Together with actin ®laments and microtubules, intermediate ®laments (IF) are part of a complex ®lament network, called the cytoskeleton, which is present in all eukaryotic cells. 1,2 The diverse family of cytoplasmatic IF proteins contains four major homology classes, with individual IF proteins mostly being cell-speci®c and tissue-speci®c. The elementary building block of all IFs is an elongated dimer of two parallel polypeptide chains, which may be identical, as in the case of desmin and vimentin IFs, or have related sequences, as in cytokeratin IFs. The IF protein dimers exhibit a dis- tinct tripartite structure consisting of an approxi- mately 45 nm long central rod domain, and N and C-terminal domains. 3,4 The rod domain is a parallel double-stranded a-helical coiled coil containing about 310 amino acid residues per chain. 5 The coiled coil consists of four segments, denoted 1A, 1B, 2A and 2B, which are distinguished by a seven-residue sequence repeat pattern (the heptad repeat; for a recent review, see 6 ). The segments are sequentially connected by short linkers L1, L12 and L2 (Figure 1). The head and tail domains lack the heptad repeat pattern and vary considerably in length and sequence among different IF proteins. In particular, the only charged residues within the N-terminal head domain of human vimentin are 11 arginine residues, so that it carries a strong positive charge at physiological conditions. Association of dimers during ®lament assembly involves several modes of lateral and longitudinal interaction. 7±9 Several higher-order assembly inter- mediates, such as tetramers, hexamers and octa- mers, could be detected in vitro. The lateral association of dimers in type III intermediate ®la- ments is evidently restricted to the stage of a dis- tinct multimer named unit-length ®lament (ULF); E-mail address of the corresponding author: Ueli.Aebi@unibas.ch Abbreviations used: IF, intermediate ®lament; ULF, unit-length ®lament; wt, wild-type; MPD, 2-methyl-2,4- pentanediol; PEG, polyethyleneglycol; TEM, transmission electron microscopy. doi:10.1006/jmbi.2001.4442 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 306, 773±781 0022-2836/01/040773±9 $35.00/0 # 2001 Academic Press