Cryo‐electron tomography and small‐angle X‐ray scattering were used to investigate the chromatin folding in metaphase chromosomes. The tomographic 3D reconstructions show that frozen‐hydrated chromatin emanated from chromosomes is planar and forms multilayered plates. The layer thickness was measured accounting for the contrast transfer function fringes at the plate edges, yielding a width of ~ 7.5 nm, which is compatible with the dimensions of a monolayer of nucleosomes slightly tilted with respect to the layer surface. Individual nucleosomes are visible decorating distorted plates, but typical plates are very dense and nucleosomes are not identifiable as individual units, indicating that they are tightly packed. Two layers in contact are ~ 13 nm thick, which is thinner than the sum of two independent layers, suggesting that nucleosomes in the layers interdigitate. X‐ray scattering of whole chromosomes shows a main scattering peak at ~ 6 nm, which can be correlated with the distance between layers and between interdigitating nucleosomes interacting through their faces. These observations support a model where compact chromosomes are composed of many chromatin layers stacked along the chromosome axis.

Early results showed the emanation of chromatin fibers from mitotic chromosomes and nuclei swollen with water. In contrast, under metaphase ionic conditions, it was found that chromatin from mitotic chromosomes is planar and forms multilayered plates. Here, we show that in buffers containing interphase cation concentrations, the chromatin emanated from disrupted nuclei also has a planar morphology. Furthermore, the chromatin fragments produced by micrococcal nuclease digestion of nuclei form the typical beads‐on‐a‐string fibers in the absence of cations, but they self‐assemble into plate‐like structures in buffers containing magnesium. The plates from interphase nuclei do not form the thick multilayered structures observed in metaphase chromosomes, suggesting that they are more exposed to the medium to facilitate DNA replication and gene expression.