X-ray free electron lasers are used in measuring diffraction patterns from nanocrystals in the 'diffract-before-destroy' mode by outrunning radiation damage. The finite-sized nanocrystals provide an opportunity to recover intensity between Bragg spots by removing the modulating function that depends on crystal shape, i.e. the transform of the crystal shape. This shape-transform dividing-out scheme for solving the phase problem has been tested using simulated examples with cubic crystals. It provides a phasing method which does not require atomic resolution data, chemical modification to the sample, or modelling based on the protein databases. It is common to find multiple structural units (e.g. molecules, in symmetry-related positions) within a single unit cell, therefore incomplete unit cells (e.g. one additional molecule) can be observed at surface layers of crystals. In this work, the effects of such incomplete unit cells on the 'dividing-out' phasing algorithm are investigated using 2D crystals within the projection approximation. It is found that the incomplete unit cells do not hinder the recovery of the scattering pattern from a single unit cell (after dividing out the shape transforms from data merged from many nanocrystals of different sizes), assuming that certain unit-cell types are preferred. The results also suggest that the dynamic range of the data is a critical issue to be resolved in order to apply the shape transform method practically.
X-ray free-electron lasers provide novel opportunities to conduct single particle analysis on nanoscale particles. Coherent diffractive imaging experiments were performed at the Linac Coherent Light Source (LCLS), SLAC National Laboratory, exposing single inorganic core-shell nanoparticles to femtosecond hard-X-ray pulses. Each facetted nanoparticle consisted of a crystalline gold core and a differently shaped palladium shell. Scattered intensities were observed up to about 7 nm resolution. Analysis of the scattering patterns revealed the size distribution of the samples, which is consistent with that obtained from direct real-space imaging by electron microscopy. Scattering patterns resulting from single particles were selected and compiled into a dataset which can be valuable for algorithm developments in single particle scattering research.
Single particle diffractive imaging data from Rice Dwarf Virus (RDV) were recorded using the Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS). RDV was chosen as it is a well-characterized model system, useful for proof-of-principle experiments, system optimization and algorithm development. RDV, an icosahedral virus of about 70 nm in diameter, was aerosolized and injected into the approximately 0.1 μm diameter focused hard X-ray beam at the CXI instrument of LCLS. Diffraction patterns from RDV with signal to 5.9 Ångström were recorded. The diffraction data are available through the Coherent X-ray Imaging Data Bank (CXIDB) as a resource for algorithm development, the contents of which are described here.
Viral protein U (Vpu) is a type-III integral membrane protein encoded by Human Immunodeficiency Virus-1 (HIV- 1). It is expressed in infected host cells and plays several roles in viral progeny escape from infected cells, including down-regulation of CD4 receptors. But key structure/function questions remain regarding the mechanisms by which the Vpu protein contributes to HIV-1 pathogenesis. Here we describe expression of Vpu in bacteria, its purification and characterization. We report the successful expression of PelB-Vpu in Escherichia coli using the leader peptide pectate lyase B (PelB) from Erwinia carotovora. The protein was detergent extractable and could be isolated in a very pure form. We demonstrate that the PelB signal peptide successfully targets Vpu to the cell membranes and inserts it as a type I membrane protein. PelB-Vpu was biophysically characterized by circular dichroism and dynamic light scattering experiments and was shown to be an excellent candidate for elucidating structural models.
One of the identified health risk areas for human spaceflight is infectious disease, particularly involving environmental microorganisms already found on the International Space Station (ISS). In particular, bacteria belonging to the Burkholderia cepacia complex (Bcc) which can cause human disease in those who are immunocompromised, have been identified in the ISS water supply. This present study characterized the effect of spaceflight analog culture conditions on Bcc to certain physiological stresses (acid and thermal as well as intracellular survival in U927 human macrophage cells). The NASA-designed Rotating Wall Vessel (RWV) bioreactor was used as the spaceflight analogue culture system in these studies to grow Bcc bacterial cells under Low Shear Modeled Microgravity (LSMMG) conditions. Results show that LSMMG culture increased the resistance of Bcc to both acid and thermal stressors, but did not alter phagocytic uptake in 2-D monolayers of human monocytes.