3-D Structure of Negatively Stained Chromatin Fibers

Abstract

In the eukaryotic nucleus, DNA is packaged into nucleosomes, and the nucleo- some chain folded into '30nm' chromatin fibers. A number of different model structures have been proposed for the arrangement of nucleosome within the fiber1 and most of these are based on helical symmetry. Indications of helical order have been observed over short regions of fiber but the structure is too disordered to allow reconstruction by Fourier methods. We have used tomography, an approach developed for asymmetric specimens, to prepare 3D reconstructions of chromatin fibers.Nuclei from Necturus maculosus erythrocytes were briefly digested with micrococcal nuclease, and the released chromatin fibers fixed with 0.1% gluta- raldehyde, and stained with 1.5% sodium phosphotungstate pH 7.0 containing 0.015. glucose. Tilt series were taken at 5° intervals from +70° to -70° using a Philips 420 EM at 100KV and 30,000x magnification. Straight regions of fiber showing indications of internal order were digitized with a pixel size of l.lnm, reconstruction carried out as described, and the final 3D data sets filtered to a resolution of 3.5nm.