The cryo-EM method can be used to determine the three-dimensional structure of biomacromolecules in near native condition at close to atomic resolution, and has the potential to reveal conformations of dynamic molecular complexes. This article is part of a Special Issue entitled "Biophysical Exploration of Dynamical Ordering of Biomolecular Systems" edited by Dr. Koichi Kato.

Heterochromatin plays important roles in transcriptional silencing and genome maintenance by the formation of condensed chromatin structures, which determine the epigenetic status of eukaryotic cells. The trimethylation of histone H3 lysine 9 (H3K9me3), a target of heterochromatin protein 1 (HP1), is a hallmark of heterochromatin formation. However, the mechanism by which HP1 folds chromatin-containing H3K9me3 into a higher-order structure has not been elucidated. Here we report the three-dimensional structure of the H3K9me3-containing dinucleosomes complexed with human HP1α, HP1β, and HP1γ, determined by cryogenic electron microscopy with a Volta phase plate. In the structures, two H3K9me3 nucleosomes are bridged by a symmetric HP1 dimer. Surprisingly, the linker DNA between the nucleosomes does not directly interact with HP1, thus allowing nucleosome remodeling by the ATP-utilizing chromatin assembly and remodeling factor (ACF). The structure depicts the fundamental architecture of heterochromatin.

Femtosecond time-resolved infrared spectroscopy was used to study the vibrational response of riboflavin in DMSO to photoexcitation at 387 nm. Vibrational cooling in the excited electronic state is observed and characterized by a time constant of 4.0 +/- 0.1 ps. Its characteristic pattern of negative and positive IR difference signals allows the identification and determination of excited-state vibrational frequencies of riboflavin in the spectral region between 1100 and 1740 cm (-1). Density functional theory (B3LYP), Hartree-Fock (HF) and configuration interaction singles (CIS) methods were employed to calculate the vibrational spectra of the electronic ground state and the first singlet excited pipi* state as well as respective electronic energies, structural parameters, electronic dipole moments and intrinsic force constants. The harmonic frequencies of the S 1 excited state calculated by the CIS method are in satisfactory agreement with the observed band positions. There is a clear correspondence between computed ground- and excited-state vibrations. Major changes upon photoexcitation include the loss of the double bond between the C4a and N5 atoms, reflected in a downshift of related vibrations in the spectral region from 1450 to 1720 cm (-1). Furthermore, the vibrational analysis reveals intra- and intermolecular hydrogen bonding of the riboflavin chromophore.

Papillomaviruses, members of a group of dsDNA viruses associated with epithelial growths and tumors, have compact capsids assembled from 72 pentamers of the protein L1. We have determined the structure of bovine papillomavirus by electron cryomicrosopy (cryoEM), at ∼3.6 Å resolution. The density map, obtained from single-particle analysis of ∼4,000 particle images, shows the trace of the L1 polypeptide chain and reveals how the N-and C-terminal "arms" of a subunit (extensions from its β-jelly-roll core) associate with a neighboring pentamer. Critical contacts come from the C-terminal arm, which loops out from the core of the subunit, forms contacts (including a disulfide) with two subunits in a neighboring pentamer, and reinserts into the pentamer from which it emanates. This trace corrects one feature of an earlier model. We discuss implications of the structure for virion assembly and for pathways of infectious viral entry. We suggest that it should be possible to obtain image reconstructions of comparable resolution from cryoEM images of asymmetric particles. From the work on papillomavirus described here, we estimate that such a reconstruction will require about 1.5 million images to achieve the same number of averaged asymmetric units; structural variability will increase this number substantially. virus assembly | virus structure | icosahedral symmetry

Ebola virus causes haemorrhagic fever with a high fatality rate in humans and non-human primates. It belongs to the family Filoviridae in the order Mononegavirales, which are viruses that contain linear, non-segmented, negative-sense, single-stranded genomic RNA 1,2. The enveloped, filamentous virion contains the nucleocapsid, consisting of the helical nucleoprotein-RNA complex, VP24, VP30, VP35 and viral polymerase 1,3. The nucleoprotein-RNA complex acts as a scaffold for nucleocapsid formation and as a template for RNA replication and transcription by condensing RNA into the virion 4,5. RNA binding and nucleoprotein oligomerization are synergistic and do not readily occur independently 6. Although recent cryo-electron tomography studies have revealed the overall architecture of the nucleocapsid core 4,5 , there has been no high-resolution reconstruction of the nucleocapsid. Here we report the structure of a recombinant Ebola virus nucleoprotein-RNA complex expressed in mammalian cells without chemical fixation, at near-atomic resolution using single-particle cryoelectron microscopy. Our structure reveals how the Ebola virus nucleocapsid core encapsidates its viral genome, its sequenceindependent coordination with RNA by nucleoprotein, and the dynamic transition between the RNA-free and RNA-bound states. It provides direct structural evidence for the role of the N terminus of nucleoprotein in subunit oligomerization, and for the hydrophobic and electrostatic interactions that lead to the formation of the helical assembly. The structure is validated as representative of the native biological assembly of the nucleocapsid core by consistent dimensions and symmetry with the full virion 5. The atomic model provides a detailed mechanistic basis for understanding nucleocapsid assembly and highlights key structural features that may serve as targets for anti-viral drug development. We expressed and purified C-terminally truncated Zaire ebolavirus nucleoprotein (NP), containing residues 1−450 (NP 1−450) in a human cell line (Fig. 1a), in which it sequestered cellular RNA and assembled into a rigid helix indistinguishable from the viral nucleocapsid core 3-5,7. The sample was imaged without fixation using cryo-electron microscopy (cryo-EM), and its structure was determined by single-particle analysis (SPA) (Fig. 1b, Extended Data Fig. 1a-e, Extended Data Table 1). The complex forms a left-handed helical tube with outer and inner diameters of 295 and 175 Å, respectively, and features a characteristic zipper-like arrangement of parallel C-terminal α-helices (Fig. 1b-d). Its diameter and helical symmetry correspond closely to those of the recently reported Ebola virion reconstructed by cryo-electron tomography (cryo-ET) 4,5. Our cryo-EM map enabled generation of an atomic model, including clearly resolved RNA nucleotides (Fig. 1e, Extended Data Figs. 1d, 1f, 2). The transition from the RNA-free to the RNA-bound state requires conformational changes in both RNA and NP. Encapsidation of RNA by NP has been addressed by two lead...