A posteriori correction of camera characteristics from large image data sets

Large datasets are emerging in many fields of image processing including: electron microscopy, light microscopy, medical X-ray imaging, astronomy, etc. Novel computer-controlled instrumentation facilitates the collection of very large datasets containing thousands of individual digital images. In single-particle cryogenic electron microscopy (“cryo-EM”), for example, large datasets are required for achieving quasi-atomic resolution structures of biological complexes. Based on the collected data alone, large datasets allow us to precisely determine the statistical properties of the imaging sensor on a pixel-by-pixel basis, independent of any “a priori” normalization routinely applied to the raw image data during collection (“flat field correction”). Our straightforward “a posteriori” correction yields clean linear images as can be verified by Fourier Ring Correlation (FRC), illustrating the statistical independence of the corrected images over all spatial frequencies. The image sensor characteristics can also be measured continuously and used for correcting upcoming images

Applying an Empirical Hydropathic Forcefield in Refinement May Improve Low-Resolution Protein X-Ray Crystal Structures

BACKGROUND: The quality of X-ray crystallographic models for biomacromolecules refined from data obtained at high-resolution is assured by the data itself. However, at low-resolution, >3.0 Å, additional information is supplied by a forcefield coupled with an associated refinement protocol. These resulting structures are often of lower quality and thus unsuitable for downstream activities like structure-based drug discovery. METHODOLOGY: An X-ray crystallography refinement protocol that enhances standard methodology by incorporating energy terms from the HINT (Hydropathic INTeractions) empirical forcefield is described. This protocol was tested by refining synthetic low-resolution structural data derived from 25 diverse high-resolution structures, and referencing the resulting models to these structures. The models were also evaluated with global structural quality metrics, e.g., Ramachandran score and MolProbity clashscore. Three additional structures, for which only low-resolution data are available, were also re-refined with this methodology. RESULTS: The enhanced refinement protocol is most beneficial for reflection data at resolutions of 3.0 Å or worse. At the low-resolution limit, ≥4.0 Å, the new protocol generated models with Cα positions that have RMSDs that are 0.18 Å more similar to the reference high-resolution structure, Ramachandran scores improved by 13%, and clashscores improved by 51%, all in comparison to models generated with the standard refinement protocol. The hydropathic forcefield terms are at least as effective as Coulombic electrostatic terms in maintaining polar interaction networks, and significantly more effective in maintaining hydrophobic networks, as synthetic resolution is decremented. Even at resolutions ≥4.0 Å, these latter networks are generally native-like, as measured with a hydropathic interactions scoring tool

Synthesis and Biological Evaluation of 2-Methyl-4,5-Disubstituted Oxazoles as a Novel Class of Highly Potent Antitubulin Agents

Antimitotic agents that interfere with microtubule formation are one of the major classes of cytotoxic drugs for cancer treatment. Multiple 2-methyl-4-(3′,4′,5′-trimethoxyphenyl)-5-substituted oxazoles and their related 4-substituted-5-(3′,4′,5′-trimethoxyphenyl) regioisomeric derivatives designed as cis-constrained combretastatin A-4 (CA-4) analogues were synthesized and evaluated for their antiproliferative activity in vitro against a panel of cancer cell lines and, for selected highly active compounds, interaction with tubulin, cell cycle effects and in vivo potency. Both these series of compounds were characterized by the presence of a common 3′,4′,5′-trimethoxyphenyl ring at either the C-4 or C-5 position of the 2-methyloxazole ring. Compounds 4g and 4i, bearing a m-fluoro-p-methoxyphenyl or p-ethoxyphenyl moiety at the 5-position of 2-methyloxazole nucleus, respectively, exhibited the greatest antiproliferative activity, with IC50 values of 0.35-4.6 nM (4g) and 0.5–20.2 nM (4i), which are similar to those obtained with CA-4. These compounds bound to the colchicine site of tubulin and inhibited tubulin polymerization at submicromolar concentrations. Furthermore, 4i strongly induced apoptosis that follows the mitochondrial pathway. In vivo, 4i in a mouse syngeneic model demonstrated high antitumor activity which significantly reduced the tumor mass at doses ten times lower than that required for CA-4P, suggesting that 4i warrants further evaluation as a potential anticancer drug

Design and Synthesis of Potent in Vitro and in Vivo Anticancer Agents Based on 1-(3′,4′,5′-Trimethoxyphenyl)-2-Aryl-1H-Imidazole

A novel series of tubulin polymerization inhibitors, based on the 1-(3',4',5'-trimethoxyphenyl)-2-aryl-1H-imidazole scaffold and designed as cis-restricted combretastatin A-4 analogues, was synthesized with the goal of evaluating the effects of various patterns of substitution on the phenyl at the 2-position of the imidazole ring on biological activity. A chloro and ethoxy group at the meta- and para-positions, respectively, produced the most active compound in the series (4o), with IC50 values of 0.4-3.8 nM against a panel of seven cancer cell lines. Except in HL-60 cells, 4o had greater antiproliferative than CA-4, indicating that the 3'-chloro-4'-ethoxyphenyl moiety was a good surrogate for the CA-4 B-ring. Experiments carried out in a mouse syngenic model demonstrated high antitumor activity of 4o, which significantly reduced the tumor mass at a dose thirty times lower than that required for CA-4P, which was used as a reference compound. Altogether, our findings suggest that 4o is a promising anticancer drug candidate that warrants further preclinical evaluation

C-terminal Tail of β-Tubulin and its Role in the Alterations of Dynein Binding Mode

Dynein is a cytoskeletal molecular motor protein that moves along the microtubule (MT) and transports various cellular cargos during its movement. Using standard Molecular Dynamics (MD) simulation, Principle Component Analysis (PCA), and Normal Mode Analysis (NMA) methods, this investigation studied large-scale movements and local interactions of dynein’s Microtubule Binding Domain (MTBD) when bound to tubulin heterodimer subunits. Examination of the interactions between the MTBD segments, and their adjustments in terms of intra- and intermolecular distances at the interfacial area with tubulin heterodimer, particularly at α-H16, β-H18 and β-tubulin C-terminal tail (CTT), was the main focus of this study. The specific intramolecular interactions, electrostatic forces and the salt-bridge residue pairs were shown to be the dominating factors in orchestrating movements of the MTBD and MT interfacial segments in the dynein’s low-high affinity binding modes. Important interactions included β-Glu447 and β-Glu449 (CTT) with Arg3469 (MTBD-H6), Lys3472 (MTBD-H6-H7 loop) and Lys3479 (MTBD-H7); β-Glu449 with Lys3384 (MTBD-H8), Lys3386 and His3387 (MTBD-H1). The structural and precise position, orientation, and functional effects of the CTTs on the MT-MTBD, within reasonable cut-off distance for non-bonding interactions and under physiological conditions, are unavailable from the previous studies. The absence of the residues in the highly flexible MT-CTTs in the experimentally solved structures is perhaps in some cases due to insufficient data from density maps, but these segments are crucial in protein binding. The presented work contributes to the information useful for the MT-MTBD structure refinement

X-ray absorption by macromolecular crystals: the effects of wavelength and crystal composition on absorbed dose

Radiation damage restricts the useful lifetime for macromolecular crystals in the X-ray beam, even at cryotemperatures. With the development of structural genomics pipelines, it will be essential to incorporate projected crystal lifetime information into the automated data collection software routines. As a first step towards this goal, a computer program, RADDOSE, is presented which is designed for use by crystallographers in optimizing the amount of data that can be obtained from a particular cryo-cooled crystal at synchrotron beamlines. The program uses the composition of the crystal and buffer constituents, as well as the beam energy, flux and dimensions, to compute the absorption coefficients and hence the theoretical time taken to reach an absorbed dose of 2 × 10 7 Gy, the so-called 'Henderson limit'. At this dose, the intensity of the diffraction pattern is predicted to be halved. A 'diffraction-dose efficiency' quantity is introduced, for the convenient comparison of absorbed dose per diffracted photon for different crystals. Four example cases are considered, and the implications for anomalous data collection are discussed in the light of the results from RADDOSE

Differential specific radiation damage in the Cu-II-bound and Pd-II-bound forms of an alpha-helical foldamer: a case study of crystallographic phasing by RIP and SAD

The high photon flux at third-generation synchrotron sources can inflict significant primary radiation damage upon macromolecular crystals, even when the crystals are cryocooled. However, specific radiation-induced structural changes can be exploited for de novo phasing by an approach known as radiation damage-induced phasing (RIP). Here, RIP and single-wavelength anomalous dispersion (SAD) phasing were alternatively used to derive experimental phases to 1.2 Å resolution for crystals of an -helical 18-residue peptide, MINTS, which was derived from the neurotoxin apamin and the palladium-bound structure of which is now reported. Helix formation is induced by the binding of palladium (or copper) to two histidines spaced four residues apart, while two disulfide bonds tether the N-terminal helix to the C-terminal loop-like part of the peptide. Either RIP or SAD phasing of the palladium-bound and copper-bound forms of MINTS, which crystallized in different space groups, resulted in density maps of superb quality. Surprisingly, RIP phasing of the metal-bound complex structures of MINTS was a consequence of differential radiation damage, resting primarily on the reduction of the disulfide bonds in Pd-MINTS and on depletion of the metal sites in Cu-MINTS. Its miniprotein-like characteristics, versatile metal-binding properties and ease of crystallization suggest MINTS to be a convenient test specimen for methods development in crystallographic phasing based on either synchrotron or in-house X-ray diffraction data