The compaction level of arrays of nucleosomes may be understood in terms of the balance between the selfrepulsion of DNA (principally linker DNA) and countering factors including the ionic strength and composition of the medium, the highly basic N termini of the core histones, and linker histones. However, the structural principles that come into play during the transition from a loose chain of nucleosomes to a compact 30-nm chromatin fiber have been difficult to establish, and the arrangement of nucleosomes and linker DNA in condensed chromatin fibers has never been fully resolved. Based on images of the solution conformation of native chromatin and fully defined chromatin arrays obtained by electron cryomicroscopy, we report a linker histone-dependent architectural motif beyond the level of the nucleosome core particle that takes the form of a stem-like organization of the entering and exiting linker DNA segments. DNA completes Ϸ1.7 turns on the histone octamer in the presence and absence of linker histone. When linker histone is present, the two linker DNA segments become juxtaposed Ϸ8 nm from the nucleosome center and remain apposed for 3-5 nm before diverging. We propose that this stem motif directs the arrangement of nucleosomes and linker DNA within the chromatin fiber, establishing a unique threedimensional zigzag folding pattern that is conserved during compaction. Such an arrangement with peripherally arranged nucleosomes and internal linker DNA segments is fully consistent with observations in intact nuclei and also allows dramatic changes in compaction level to occur without a concomitant change in topology.

The "30-nm" chromatin fibers, as observed in eukaryotic nuclei, are considered a discrete level in a hierarchy of DNA folding. At present, there is considerable debate as to how the nudeosomes and linker DNA are organized within chromatin fibers, and a number of models have been proposed, many of which are based on helical symmetry and imply specific contacts between nucleosomes. However, when observed in nuclei or after isolation, chromatin fibers show considerable structural irregularity. In the present study, chromatin folding is considered solely in terms of the known properties of the nudeosome-linker unit, taking into account the relative rotation between consecutive nucleosomes that results from the helical twist of DNA. Model building based on this premise, and with a constant length of linker DNA between consecutive nucleosomes, results in a family of fiber-and ribbon-like structure. When the linker length between nucleosomes is aflowed to vary, as occurs in nature, fibers showing the types of irregulaity observed in nuclei and in isolated chromatin are created. The potential application of the model in determining the three-dimensional organization of chromatin in which nucleosome positions are known is discussed.The genome of most eukaryotes is complexed with proteins to form chromatin (1). Under low salt conditions in vitro, the complex assumes its simplest conformation and is seen as a beaded chain of 11-nm (diameter) nucleosomes (e.g., ref.2). As the ionic strength of the medium is raised, the nucleosomal chain condenses, eventually forming a compact fiber 30-40 nm in diameter (1-3). These compact fibers observed in isolated chromatin in vitro are presumed to be related to similar structures seen in thin sections of certain types of nuclei such as nucleated erythrocytes (e.g., refs. 4 and 5). X-ray scattering studies of whole cells and nuclei often show 30-to 45-nm reflections that have been interpreted as arising from the center-to-center spacing of compact chromatin fibers (6). These results suggest that the compact chromatin fiber constitutes a distinct level of chromatin organization, especially for transcriptionally inactive chromatin. In this context, the architecture of the fiber would implicitly define the substrate for the regulatory events that lead to chromatin unfolding, a prerequisite for transcription.Attempts to deduce the structure of compact chromatin fibers have resulted in a number of proposals that include both symmetrical and nonsymmetrical arrangements of nucleosomes (reviewed in ref. 7). There is evidence suggesting some degree of symmetry in fiber structure: an intermediate conformation between the nucleosome chain and the compact chromatin fiber is a "zig-zag ribbon" (2) that can display considerable regularity (8), and isolated fibers prepared for electron microscopy contain regions with a limited amount of internal order (2, 3, 9-11). All proposed model structuresThe publication costs of this article were defrayed in part by page charge payment. This article must ...

Abstract. The three dimensional (3D) structure of chromatin fibers in sections of nuclei has been determined using electron tomography. Low temperature embedding and nucleic acid-specific staining allowed individual nucleosomes to be clearly seen, and the tomographic data collection parameters provided a reconstruction resolution of 2.5 nm. Chromatin fibers have complex 3D trajectories, with smoothly bending regions interspersed with abrupt changes in direction, and U turns. Nucleosomes are located predominantly at the fiber periphery, and linker DNA tends to project toward the fiber interior. Within the fibers, a unifying structural motif is a two nucleosome-wide ribbon that is variably bent and twisted, and in which there is little face-to-face contact between nucleosomes. It is suggested that this asymmetric 3D zig-zag of nucleosomes and linker DNA represents a basic principle of chromatin folding that is determined by the properties of the nucleosome-linker unit. This concept of chromatin fiber architecture is contrasted with helical models in which specific nucleosome-nucleosome contacts play a major role in generating a symmetrical higher order structure. The transcriptional control implications of a more open and irregular chromatin structure are discussed.

Past attempts to detect tropomyosin in electron micrograph images of frozen-hydrated troponin-regulated thin filaments under relaxing conditions have not been successful. This raised the possibility that tropomyosin may be disordered on filaments in the off-state, a possibility at odds with the steric blocking model of muscle regulation. By using cryoelectron microscopy and helical image reconstruction we have now resolved the location of tropomyosin in both relaxing and activating conditions. In the off-state, tropomyosin adopts a position on the outer domain of actin with a binding site virtually identical to that determined previously by negative staining, although at a radius of 3.8 nm, slightly higher than found in stained filaments. Molecular fitting to the atomic model of F-actin shows that tropomyosin is localized over sites on actin subdomain 1 required for myosin binding. Restricting access to these sites would inhibit the myosin-cross-bridge cycle, and hence contraction. Under high Ca(2+) activating conditions, tropomyosin moved azimuthally, away from its blocking position to the same site on the inner domain of actin previously determined by negative staining, also at 3.8 nm radius. These results provide strong support for operation of the steric mechanism of muscle regulation under near-native solution conditions and also validate the use of negative staining in investigations of muscle thin filament structure.