The in situ three-dimensional organization of chromatin at the nucleosome and oligonucleosome levels is unknown. Here we use cryo-electron tomography to determine the in situ structures of HeLa nucleosomes, which have canonical core structures and asymmetric, flexible linker DNA. Subtomogram remapping suggests that sequential nucleosomes in heterochromatin follow irregular paths at the oligonucleosome level. This basic principle of higher-order repressive chromatin folding is compatible with the conformational variability of the two linker DNAs at the single-nucleosome level.

Chromosomes condense during mitosis in most eukaryotes. This transformation involves rearrangements at the nucleosome level and has consequences for transcription. Here, we use cryo-electron tomography (cryo-ET) to determine the 3D arrangement of nuclear macromolecular complexes, including nucleosomes, in frozenhydrated Schizosaccharomyces pombe cells. Using 3D classification analysis, we did not find evidence that nucleosomes resembling the crystal structure are abundant. This observation and those from other groups support the notion that a subset of fission yeast nucleosomes may be partially unwrapped in vivo. In both interphase and mitotic cells, there is also no evidence of monolithic structures the size of Hi-C domains. The chromatin is mingled with two features: pockets, which are positions free of macromolecular complexes; and "megacomplexes," which are multimegadalton globular complexes like preribosomes. Mitotic chromatin is more crowded than interphase chromatin in subtle ways. Nearestneighbor distance analyses show that mitotic chromatin is more compacted at the oligonucleosome than the dinucleosome level. Like interphase, mitotic chromosomes contain megacomplexes and pockets. This uneven chromosome condensation helps explain a longstanding enigma of mitosis: a subset of genes is up-regulated. chromatin | condensation | cryo-ET | fission yeast C hromatin structure influences key nuclear activities such as transcription, DNA repair, and replication (1). The fundamental unit of chromatin is the nucleosome, which consists of ∼147 bp of DNA wrapped around a histone octamer (2). In mammalian cells, 2-35 nucleosomes pack into irregular "clutches" (3). More than 500 sequential nucleosomes (calculated from the nucleosome repeat length) are thought to interact as topologically associating domains (4). Likewise, in the fission yeast Schizosaccharomyces pombe, some 300-7,000 nucleosomes are thought to associate as compact globular chromatin bodies called domains (5-7). Furthermore, superresolution imaging of S. pombe revealed that the chromatin is not uniformly distributed in vivo (8). These studies suggest that chromatin higher-order structure arises from physical interactions within large groups of nucleosomes. In mitotic cells, chromosomes condense into discrete structures that can be resolved in a light microscope. The factors involved in condensation have been well characterized (9), and a number of models have been considered for the largescale organization of chromatin domains (10). However, the molecular details of chromatin reorganization are still unknown. Knowledge of how chromosomes condense in 3D at the molecular level is needed to explain the nearly global transcriptional repression that happens to the mitotic cells of most eukaryotes (11). Such a model could also explain how a subset of fission yeast genes escape this mitotic repression and get up-regulated (12)(13)(14). Some insights on in vivo chromatin organization were made possible by new methods, including chromatin-conformation capture (Hi-...

VPS13 is a eukaryotic lipid transport protein localized at membrane contact sites. Previous studies suggested that it may transfer lipids between adjacent bilayers by a bridge-like mechanism. Direct evidence for this hypothesis from a full-length structure and from electron microscopy (EM) studies in situ is still missing, however. Here, we have capitalized on AlphaFold predictions to complement the structural information already available about VPS13 and to generate a full-length model of human VPS13C, the Parkinson’s disease–linked VPS13 paralog localized at contacts between the endoplasmic reticulum (ER) and endo/lysosomes. Such a model predicts an ∼30-nm rod with a hydrophobic groove that extends throughout its length. We further investigated whether such a structure can be observed in situ at ER–endo/lysosome contacts. To this aim, we combined genetic approaches with cryo-focused ion beam (cryo-FIB) milling and cryo–electron tomography (cryo-ET) to examine HeLa cells overexpressing this protein (either full length or with an internal truncation) along with VAP, its anchoring binding partner at the ER. Using these methods, we identified rod-like densities that span the space separating the two adjacent membranes and that match the predicted structures of either full-length VPS13C or its shorter truncated mutant, thus providing in situ evidence for a bridge model of VPS13 in lipid transport.

The 30-nm fiber is commonly formed by oligonucleosome arrays in vitro but rarely found inside cells. To determine how chromatin higher-order structure is controlled, we used electron cryotomography (cryo-ET) to study the undigested natural chromatin released from two single-celled organisms in which 30-nm fibers have not been observed in vivo: picoplankton and yeast. In the presence of divalent cations, most of the chromatin from both organisms is condensed into a large mass in vitro. Rare irregular 30-nm fibers, some of which include face-to-face nucleosome interactions, do form at the periphery of this mass. In the absence of divalent cations, picoplankton chromatin decondenses into open zigzags. By contrast, yeast chromatin mostly remains condensed, with very few open motifs. Yeast chromatin packing is largely unchanged in the absence of linker histone and mildly decondensed when histones are more acetylated. Natural chromatin is therefore generally nonpermissive of regular motifs, even at the level of oligonucleosomes.