An understanding of how facets of a nanocrystal develop is critical for controlling nanocrystal shape and designing novel functional materials. However, the atomic pathways of nanocrystal facet development are mostly unknown because of the lack of direct observation. We report the imaging of platinum nanocube growth in a liquid cell using transmission electron microscopy with high spatial and temporal resolution. The growth rates of all low index facets are similar until the {100} facets stop growth. The continuous growth of the rest facets leads to a nanocube. Our calculation shows that the much lower ligand mobility on the {100} facets is responsible for the arresting of {100} growing facets. These findings shed light on nanocrystal shape-control mechanisms and future design of nanomaterials.

Abstract:Understanding structural details of colloidal nanoparticles is required to bridge our knowledge about their synthesis, growth mechanisms, and physical properties. We introduce a method for determining 3D structures of individual nanoparticles in solution.We combine a graphene liquid cell, high-resolution transmission electron microscopy, a direct electron detector, and an algorithm for single-particle 3D reconstruction originally developed for analysis of biological molecules to produce two near-atomic resolution 3D structures of individual Pt nanocrystals. Since our method derives the 3D structure from images of individual nanoparticles rotating freely in solution, it enables the analysis of heterogeneous populations of potentially unordered nanoparticles that are synthesized in solution, thereby providing a means to understand the structure and stability of defects at the nanoscale. Main Text:Colloidal nanoparticles are clusters of hundreds to thousands of inorganic atoms typically surrounded by organic ligands that stabilize them in solution. The atomic arrangement of colloidal nanoparticles determines their chemical and physical properties, which are distinct from bulk materials and can be exploited for many applications in biological imaging, renewable energy, catalysis, and more. The 3D atomic arrangement on the surface and in the core of a nanocrystal influences the electronic structure, which affects how the nanocrystal functions in catalysis or how it interacts with other components at the atomic scale (1). Introduction of atomic dopants, surface adatoms, defects, and grain boundaries alters the chemical properties of nanocrystals (2). Ensembles of synthesized nanocrystals in solution are structurally inhomogeneous due to the stochastic nature of nanocrystal nucleation and growth (3,4). Therefore, a method for determination of the 3D atomic arrangement of individual unique nanoparticles in solution is needed. 3Electron tomography is routinely used for 3D analysis of materials (5-9). This method cannot be applied to individual particles in a liquid because it relies on acquisition of images of a single object at many different tilt angles over 2 to 5 hours, assuming the object is static during the entire acquisition. Single particle cryo-electron microscopy (cryo-EM) is a common method for the determination of 3D structures in biological sciences. The average 3D Coulomb potential map (density) of a protein is reconstructed from tens of thousands of TEM images of randomly oriented copies of the same protein embedded in vitreous ice (10). The unknown 3D projection angles of the images are determined by computational methods (11). Single-particle cryo-EM has succeeded in reconstructing biological molecules with nearly 3 Å resolution (10, 12). A similar approach was recently applied to reconstruct the atomic structure of homogeneous ultrasmall gold clusters (13). However, the single-particle method is not readily applicable to 3D reconstruction of colloidal nanoparticles due to their intrinsic struc...

electron microscopy ͉ transcription ͉ protein structure ͉ yeast ͉ Schizosaccharomyces pombe T he Mediator complex acts as an interface between genespecific regulatory proteins and the basal RNA polymerase II (pol II) transcription machinery (1). Mediator functions as a key regulator of pol II-dependent genes in Saccharomyces cerevisiae (2), and depletion of human Mediator from nuclear extracts abolishes transcription by pol II (3). The C-terminal domain of pol II (CTD) has an important role for the Mediator function (4, 5), and no fewer than nine SRB genes, encoding for Mediator subunits, were originally identified in a screen for mutants that suppress the cold-sensitive phenotype of a CTD truncation mutant (6). In S. cerevisiae and Schizosaccharomyces pombe, the Mediator complex interacts directly with the unphosphorylated CTD and forms a holoenzyme (5). Based on shape analysis of the low-resolution projection maps, the S. cerevisiae Mediator structure has been divided into three compact and visually distinguishable modules: head, middle, and tail domains of approximately equal mass (7).The subunit composition of S. cerevisiae Mediator has been studied in detail, and 21 proteins are bona fide members of the core Mediator complex (1,8,9). In addition, a subgroup of Srb proteins, Med12͞Srb8, Med13͞Srb9, Cdk8 (cyclin-dependent kinase 8)͞Srb10, and CycC͞Srb11, forms a specific module (the Cdk8 module) that is variably present in Mediator preparations (10, 11). The smaller, core Mediator (S Mediator) lacking the Cdk8 module has a stimulatory effect on basal transcription in vitro (5, 12). The larger form of Mediator (L Mediator), containing the Cdk8 module, instead represses basal transcription in vitro, and genetic analysis also indicates that the Cdk8 module is involved in the negative regulation of genes in vivo (13).The Cdk8 module influences pol II interactions with Mediator, and only S Mediator can interact with pol II and form a holoenzyme complex (11). The molecular mechanism by which the Cdk8 module negatively regulates pol II interactions has not been clarified, but it has been hypothesized that the negative effect of the Cdk8 module on eukaryotic transcription is caused by Cdk8-dependent phosphorylation of CTD. The hyperphosphorylated form of CTD would bind less tightly to Mediator, which may result in dissociation of pol II from the holoenzyme complex (14, 15).Here, we use the S. pombe system to investigate the molecular basis for the distinct functional properties of S and L Mediator. We find that the Cdk8 module binds to the pol II-binding cleft of Mediator, where it sterically blocks interactions with the polymerase. In contrast to earlier assumptions, the Cdk8 kinase activity is dispensable for negative regulation of pol II interactions with Mediator. It should be noted that the structure and function of Mediator appears conserved in fungi and metazoan cells, and to simplify comparisons with other experimental systems, throughout this study we use the recently proposed unifying Mediator nomenclature ...

Activation of complement C5 generates the potent anaphylatoxin C5a and leads to pathogen lysis, inflammation and cell damage. The therapeutic potential of C5 inhibition has been demonstrated by eculizumab, one of the world's most expensive drugs. However, the mechanism of C5 activation by C5 convertases remains elusive, thus limiting development of therapeutics. Here we identify and characterize a new protein family of tick-derived C5 inhibitors. Structures of C5 in complex with the new inhibitors, the phase I and phase II inhibitor OmCI, or an eculizumab Fab reveal three distinct binding sites on C5 that all prevent activation of C5. The positions of the inhibitor-binding sites and the ability of all three C5-inhibitor complexes to competitively inhibit the C5 convertase conflict with earlier steric-inhibition models, thus suggesting that a priming event is needed for activation.

Precise three-dimensional (3D) atomic structure determination of individual nanocrystals is a prerequisite for understanding and predicting their physical properties. Nanocrystals from the same synthesis batch display what are often presumed to be small but possibly important differences in size, lattice distortions, and defects, which can only be understood by structural characterization with high spatial 3D resolution. We solved the structures of individual colloidal platinum nanocrystals by developing atomic-resolution 3D liquid-cell electron microscopy to reveal critical intrinsic heterogeneity of ligand-protected platinum nanocrystals in solution, including structural degeneracies, lattice parameter deviations, internal defects, and strain. These differences in structure lead to substantial contributions to free energies, consequential enough that they must be considered in any discussion of fundamental nanocrystal properties or applications.

Summary The protein density and arrangement of subunits of a complete, 31-protein, RNA polymerase II (pol II) transcription pre-initiation complex (PIC) were determined by cryo-electron microscopy and a combination of chemical cross-linking and mass spectrometry. The PIC showed a marked division in two parts, one containing all the general transcription factors (GTFs), and the other pol II. Promoter DNA was associated only with the GTFs, suspended above the pol II cleft and not in contact with pol II. This structural principle of the PIC underlies its conversion to a transcriptionally active state; the PIC is poised for the formation of a transcription bubble and descent of the DNA into the pol II cleft.

Intense femtosecond x-ray pulses from free-electron laser sources allow the imaging of individual particles in a single shot. Early experiments at the Linac Coherent Light Source (LCLS) have led to rapid progress in the field and, so far, coherent diffractive images have been recorded from biological specimens, aerosols, and quantum systems with a few-tens-of-nanometers resolution. In March 2014, LCLS held a workshop to discuss the scientific and technical challenges for reaching the ultimate goal of atomic resolution with single-shot coherent diffractive imaging. This paper summarizes the workshop findings and presents the roadmap toward reaching atomic resolution, 3D imaging at free-electron laser sources.

The membrane attack complex (MAC)/perforin-like protein complement component 9 (C9) is the major component of the MAC, a multi-protein complex that forms pores in the membrane of target pathogens. In contrast to homologous proteins such as perforin and the cholesterol-dependent cytolysins (CDCs), all of which require the membrane for oligomerisation, C9 assembles directly onto the nascent MAC from solution. However, the molecular mechanism of MAC assembly remains to be understood. Here we present the 8 Å cryo-EM structure of a soluble form of the poly-C9 component of the MAC. These data reveal a 22-fold symmetrical arrangement of C9 molecules that yield an 88-strand pore-forming β-barrel. The N-terminal thrombospondin-1 (TSP1) domain forms an unexpectedly extensive part of the oligomerisation interface, thus likely facilitating solution-based assembly. These TSP1 interactions may also explain how additional C9 subunits can be recruited to the growing MAC subsequent to membrane insertion.