Electron crystallography is arguably the only electron cryomicroscopy (cryo EM) technique able to deliver atomic resolution data (better then 3 Å) for membrane proteins embedded in a membrane. The progress in hardware improvements and sample preparation for diffraction analysis resulted in a number of recent examples where increasingly higher resolutions were achieved. Other chapters in this book detail the improvements in hardware and delve into the intricate art of sample preparation for microscopy and electron diffraction data collection and processing. In this chapter, we describe in detail the protocols for molecular replacement for electron diffraction studies. The use of a search model for phasing electron diffraction data essentially eliminates the need of acquiring image data rendering it immune to aberrations from drift and charging effects that effectively lower the attainable resolution.

The distribution of chromatin-associated proteins plays a key role in directing nuclear function. Previously, we developed an image-based method to quantify the nuclear distributions of proteins and showed that these distributions depended on the phenotype of human mammary epithelial cells. Here we describe a method that creates a hierarchical tree of the given cell phenotypes and calculates the statistical significance between them, based on the clustering analysis of nuclear protein distributions.

Genes include cis-regulatory regions that contain transcriptional enhancers. Recent reports have shown that developmental genes often possess multiple discrete enhancer modules that drive transcription in similar spatio-temporal patterns: primary enhancers located near the basal promoter and secondary, or ’shadow’, enhancers located at more remote positions. It has been proposed that the seemingly redundant activity of primary and secondary enhancers contributes to phenotypic robustness. We tested this hypothesis by generating a deficiency that removes two newly discovered enhancers of shavenbaby (svb, a transcript of the ovo locus), a gene encoding a transcription factor that directs development of Drosophila larval trichomes. At optimal temperatures for embryonic development, this deficiency causes minor defects in trichome patterning. In embryos that develop at both low and high extreme temperatures, however, absence of these secondary enhancers leads to extensive loss of trichomes. These temperature-dependent defects can be rescued by a transgene carrying a secondary enhancer driving transcription of the svb cDNA. Finally, removal of one copy of wingless, a gene required for normal trichome patterning, causes a similar loss of trichomes only in flies lacking the secondary enhancers. These results support the hypothesis that secondary enhancers contribute to phenotypic robustness in the face of environmental and genetic variability.

An HTS screening campaign identified a series of low molecular weight phenols that showed excellent selectivity (>100-fold) for HDAC1/HDAC2 over other Class I and Class II HDACs. Evolution and optimization of this HTS hit series provided HDAC1-selective (SHI-1) compounds with excellent anti-proliferative activity and improved physical properties. Dose-dependent efficacy in a mouse HCT116 xenograft model was demonstrated with a phenylglycine SHI-1 analog.

The antennae of male silk moths are extremely sensitive to the female sex pheromone such that a male moth can find a female up to 4.5 km away. This remarkable sensitivity is due to both the morphological and biochemical design of these antennae. Along the branches of the plumose antennae are the sensilla trichodea, each consisting of a hollow cuticular hair containing two unbranched dendrites bathed in a fluid, the receptor lymph ,3. The dendrites and receptor lymph are isolated from the haemolymph by a barrier of epidermal cells which secreted the cuticular hair. Pheromone molecules are thought to diffuse down 100 A-wide pore tubules through the cuticular wall and across the receptor lymph space to receptors located in the dendritic membrane. To prevent the accumulation of residual stimulant and hence sensory adaptation, the pheromone molecules are subsequently inactivated in an apparent two-step process of rapid ’early inactivation’ followed by much slower enzymatic degradation. The biochemistry involved in this sequence of events is largely unknown. We report here the identification of three proteins which interact with the pheromone of the wild silk moth Antheraea polyphemus: a pheromone-binding protein and a pheromone-degrading esterase, both uniquely located in the pheromone-sensitive sensilla; and a second esterase common to all cuticular tissues except the sensilla.

Proteomic studies have identified thousands of eukaryotic phosphorylation sites (phosphosites), but few are functionally characterized. Nishi et al., in this issue of Structure, characterize phosphosites at protein-protein interfaces and estimate the effect of their phosphorylation on interaction affinity, by combining proteomics data with protein structures.

{At distal dendritic locations, the threshold for action potential generation is higher and the amplitude of back-propagating spikes is decreased. To study whether these characteristics depend upon Na+ channels, their voltage-dependent properties at proximal and distal dendritic locations were compared in CA1 hippocampal neurons. Distal Na+ channels activated at more hyperpolarized voltages than proximal (half-activation voltages were -20.4 +/- 2.4 mV vs. -12.0 +/- 1.7 mV for distal and proximal patches, respectively

Photoactivatable pharmacological agents have revolutionized neuroscience, but the palette of available compounds is limited. We describe a general method for caging tertiary amines by using a stable quaternary ammonium linkage that elicits a red shift in the activation wavelength. We prepared a photoactivatable nicotine (PA-Nic), uncageable via one- or two-photon excitation, that is useful to study nicotinic acetylcholine receptors (nAChRs) in different experimental preparations and spatiotemporal scales.

Key to understanding a protein’s biological function is the accurate determination of its spatial distribution inside a cell. Although fluorescent protein markers allow the targeting of specific proteins with molecular precision, much of this information is lost when the resultant fusion proteins are imaged with conventional, diffraction-limited optics. In response, several imaging modalities that are capable of resolution below the diffraction limit (approximately 200 nm) have emerged. Here, both single- and dual-color superresolution imaging of biological structures using photoactivated localization microscopy (PALM) are described. The examples discussed focus on adhesion complexes: dense, protein-filled assemblies that form at the interface between cells and their substrata. A particular emphasis is placed on the instrumentation and photoactivatable fluorescent protein (PA-FP) tags necessary to achieve PALM images at approximately 20 nm resolution in 5 to 30 min in fixed cells.

Commentary: A paper spearheaded by Hari which gives a thorough description of the methods and hardware needed to successfully practice PALM, including cover slip preparation, cell transfection and fixation, drift correction with fiducials, characterization of on/off contrast ratios for different photoactivted fluorescent proteins, identifying PALM-suitable cells, and mechanical and optical components of a PALM system.

Superresolution fluorescence microscopy permits the study of biological processes at scales small enough to visualize fine subcellular structures that are unresolvable by traditional diffraction-limited light microscopy. Many superresolution techniques, including those applicable to live cell imaging, utilize genetically encoded photocontrollable fluorescent proteins. The fluorescence of these proteins can be controlled by light of specific wavelengths. In this review, we discuss the biochemical and photophysical properties of photocontrollable fluorescent proteins that are relevant to their use in superresolution microscopy. We then describe the recently developed photoactivatable, photoswitchable, and reversibly photoswitchable fluorescent proteins, and we detail their particular usefulness in single-molecule localization-based and nonlinear ensemble-based superresolution techniques. Finally, we discuss recent applications of photocontrollable proteins in superresolution imaging, as well as how these applications help to clarify properties of intracellular structures and processes that are relevant to cell and developmental biology, neuroscience, cancer biology and biomedicine.