Summary The insulin receptor (IR) is a dimeric protein that plays a crucial role in controlling glucose homeostasis, regulating lipid, protein and carbohydrate metabolism, and modulating brain neurotransmitter levels1,2. IR dysfunctions have been associated with many diseases, including diabetes, cancer, and Alzheimer’s1,3,4. The primary sequence has been known since the 1980s5, and is composed of an extracellular portion (ectodomain, ECD), a single transmembrane helix and an intracellular tyrosine kinase domain. Insulin binding to the dimeric ECD triggers kinase domain auto-phosphorylation and subsequent activation of downstream signaling molecules. Biochemical and mutagenesis data have identified two putative insulin binding sites (S1 and S2)6. While insulin bound to an ECD fragment containing S1 and the apo ectodomain have been characterized structurally7,8, details of insulin binding to the full receptor and the signal propagation mechanism are still not understood. Here we report single particle cryoEM reconstructions for the 1:2 (4.3 Å) and 1:1 (7.4 Å) IR ECD dimer:Insulin complexes. The symmetric 4.3 Å structure shows two insulin molecules per dimer, each bound between the Leucine-rich sub domain L1 of one monomer and the first fibronectin-like domain (FnIII-1) of the other monomer, and making extensive interactions with the α subunit C-terminal helix (α-CT helix). The 7.4 Å structure has only one similarly bound insulin per receptor dimer. The structures confirm the S1 binding interactions and define the full S2 binding site. These insulin receptor states suggest that recruitment of the α-CT helix upon binding of the first insulin changes the relative sub domain orientations and triggers downstream signal propagation.

The organization of genomic DNA into nucleosomes profoundly affects all DNA-related processes in eukaryotes. The histone chaperone ‘FAcilitates Chromatin Transcription’ (FACT, consisting of subunits SPT16 and SSRP1 1 ) promotes both disassembly and reassembly of nucleosomes during gene transcription, DNA replication, and repair 2 . The mechanism by which FACT causes these opposing outcomes is unknown. Here we report two cryo-EM structures of human FACT in complex with partially assembled ‘sub-nucleosomes’, with supporting biochemical and hydrogen-deuterium exchange (HDX) data. FACT is engaged in extensive interactions with nucleosomal DNA and all histones. The large DNA-binding surface on FACT appears to be protected by the C-terminal domains of both subunits, and this inhibition is released by interaction with H2A-H2B, allowing FACT-H2A-H2B to dock onto a (H3-H4) 2 -DNA complex 3 . SPT16 binds nucleosomal DNA and tethers H2A-H2B through its C-terminal domain by acting as a placeholder for DNA. SSRP1 also contributes to DNA binding, and can assume two conformations, depending on whether a second H2A-H2B dimer is present. Our data suggest a compelling mechanism for how FACT maintains chromatin integrity during polymerase passage, by facilitating H2A-H2B dimer removal, stabilizing intermediate ‘subnucleosomal’ states, and promoting nucleosome reassembly. Our findings reconcile discrepancies regarding the many roles of FACT and underscore the dynamic interactions between histone chaperones and nucleosomes.

Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are not adsorbed to the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment.

Most protein particles prepared in vitreous ice for single particle cryo-electron microscopy are adsorbed to air-water or substrate-water interfaces, potentially causing particles to adopt preferred orientations. Using the Spotiton robot and nanowire grids, we can reduce some of the deleterious effects of the air-water interface by decreasing the dwell time of particles in thin liquid films. We demonstrate this by using single particle cryoEM and cryoET on three biological samples.

Almost every aspect of cryo electron microscopy (cryoEM) has been automated over the last few decades. One of the challenges that remains to be addressed is the robust and reliable preparation of vitrified specimens of suitable ice thickness. We present results from a new device for preparing vitrified samples. The successful use of the device is coupled to a new “self-blotting” grid that we have developed to provide a method for spreading a sample to a thin film without the use of externally applied filter paper. This new approach has the advantage of using small amounts of protein material, resulting in large areas of ice of a well defined thickness containing evenly distributed single particles. We believe that these methods will in the future result in a system for vitrifying grids that is completely automated.

Fidaxomicin is an antibacterial drug in clinical use for treatment of Clostridium difficile diarrhea. The active ingredient of fidaxomicin, lipiarmycin A3 (Lpm), functions by inhibiting bacterial RNA polymerase (RNAP). Here we report a cryo-EM structure of Mycobacterium tuberculosis RNAP holoenzyme in complex with Lpm at 3.5-Å resolution. The structure shows that Lpm binds at the base of the RNAP "clamp." The structure exhibits an open conformation of the RNAP clamp, suggesting that Lpm traps an open-clamp state. Single-molecule fluorescence resonance energy transfer experiments confirm that Lpm traps an open-clamp state and define effects of Lpm on clamp dynamics. We suggest that Lpm inhibits transcription by trapping an open-clamp state, preventing simultaneous interaction with promoter -10 and -35 elements. The results account for the absence of cross-resistance between Lpm and other RNAP inhibitors, account for structure-activity relationships of Lpm derivatives, and enable structure-based design of improved Lpm derivatives.

YiiP is a dimeric antiporter from the cation diffusion facilitator family that uses the proton motive force to transport Zn across bacterial membranes. Previous work defined the atomic structure of an outward-facing conformation, the location of several Zn binding sites, and hydrophobic residues that appear to control access to the transport sites from the cytoplasm. A low-resolution cryo-EM structure revealed changes within the membrane domain that were associated with the alternating access mechanism for transport. In the current work, the resolution of this cryo-EM structure has been extended to 4.1 Å. Comparison with the X-ray structure defines the differences between inward-facing and outward-facing conformations at an atomic level. These differences include rocking and twisting of a four-helix bundle that harbors the Zn transport site and controls its accessibility within each monomer. As previously noted, membrane domains are closely associated in the dimeric structure from cryo-EM but dramatically splayed apart in the X-ray structure. Cysteine crosslinking was used to constrain these membrane domains and to show that this large-scale splaying was not necessary for transport activity. Furthermore, dimer stability was not compromised by mutagenesis of elements in the cytoplasmic domain, suggesting that the extensive interface between membrane domains is a strong determinant of dimerization. As with other secondary transporters, this interface could provide a stable scaffold for movements of the four-helix bundle that confers alternating access of these ions to opposite sides of the membrane.

We present an update describing new features and applications of Spotiton, a novel instrument for vitrifying samples for cryoEM. We have used Spotiton to prepare several test specimens that can be reconstructed using routine single particle analysis to ∼3 Å resolution, indicating that the process has no apparent deleterious effect on the sample integrity. The system is now in routine and continuous use in our lab and has been used to successfully vitrify a wide variety of samples.