Identification of Proteins Targeted by the Thioredoxin Superfamily in Plasmodium falciparum

The malarial parasite Plasmodium falciparum possesses a functional thioredoxin and glutathione system comprising the dithiol-containing redox proteins thioredoxin (Trx) and glutaredoxin (Grx), as well as plasmoredoxin (Plrx), which is exclusively found in Plasmodium species. All three proteins belong to the thioredoxin superfamily and share a conserved Cys-X-X-Cys motif at the active site. Only a few of their target proteins, which are likely to be involved in redox reactions, are currently known. The aim of the present study was to extend our knowledge of the Trx-, Grx-, and Plrx-interactome in Plasmodium. Based on the reaction mechanism, we generated active site mutants of Trx and Grx lacking the resolving cysteine residue. These mutants were bound to affinity columns to trap target proteins from P. falciparum cell extracts after formation of intermolecular disulfide bonds. Covalently linked proteins were eluted with dithiothreitol and analyzed by mass spectrometry. For Trx and Grx, we were able to isolate 17 putatively redox-regulated proteins each. Furthermore, the approach was successfully established for Plrx, leading to the identification of 21 potential target proteins. In addition to confirming known interaction partners, we captured potential target proteins involved in various processes including protein biosynthesis, energy metabolism, and signal transduction. The identification of three enzymes involved in S-adenosylmethionine (SAM) metabolism furthermore suggests that redox control is required to balance the metabolic fluxes of SAM between methyl-group transfer reactions and polyamine synthesis. To substantiate our data, the binding of the redoxins to S-adenosyl-L-homocysteine hydrolase and ornithine aminotransferase (OAT) were verified using BIAcore surface plasmon resonance. In enzymatic assays, Trx was furthermore shown to enhance the activity of OAT. Our approach led to the discovery of several putatively redox-regulated proteins, thereby contributing to our understanding of the redox interactome in malarial parasites

Overview and status of the 0.5NA EUV microfield exposure tool at Berkeley Lab

A 0.5-NA extreme ultraviolet micro-field exposure tool has been installed and commissioned at beamline 12.0.1.4 of the Advanced Light Source synchrotron facility at Lawrence Berkeley National Laboratory. Commissioning has demonstrated a patterning resolution of 13 nm half-pitch with annular 0.35-0.55 illumination; a patterning resolution of 8 nm half-pitch with annular 0.1-0.2 illumination; critical dimension (CD) uniformity of 0.7 nm 1σ on 16 nm nominal CD across 80% of the 200 um x 30 um aberration corrected field of view; aerial image vibration relative to the wafer of 0.75 nn RMS and focus control and focus stepping better than 15 nm

PROS: An IRAF based system for analysis of x ray data

PROS is an IRAF based software package for the reduction and analysis of x-ray data. The use of a standard, portable, integrated environment provides for both multi-frequency and multi-mission analysis. The analysis of x-ray data differs from optical analysis due to the nature of the x-ray data and its acquisition during constantly varying conditions. The scarcity of data, the low signal-to-noise ratio and the large gaps in exposure time make data screening and masking an important part of the analysis. PROS was developed to support the analysis of data from the ROSAT and Einstein missions but many of the tasks have been used on data from other missions. IRAF/PROS provides a complete end-to-end system for x-ray data analysis: (1) a set of tools for importing and exporting data via FITS format -- in particular, IRAF provides a specialized event-list format, QPOE, that is compatible with its IMAGE (2-D array) format; (2) a powerful set of IRAF system capabilities for both temporal and spatial event filtering; (3) full set of imaging and graphics tasks; (4) specialized packages for scientific analysis such as spatial, spectral and timing analysis -- these consist of both general and mission specific tasks; and (5) complete system support including ftp and magnetic tape releases, electronic and conventional mail hotline support, electronic mail distribution of solutions to frequently asked questions and current known bugs. We will discuss the philosophy, architecture and development environment used by PROS to generate a portable, multimission software environment. PROS is available on all platforms that support IRAF, including Sun/Unix, VAX/VMS, HP, and Decstations. It is available on request at no charge

Verification of the PROS timing analysis package

ROSAT observations of known pulsars are used to verify the functions of timing programs. The Crab Pulsar and PSR 0540-69, with 33 and 50 millisecond periods, are used to examine the fast Fourier transform and the epoch-folding task used to search for periodic signals. These fast pulsars provide a more vigorous test of the system than those with periods of a few seconds

Imaging single cells in a beam of live cyanobacteria with an X-ray laser

Citation: van der Schot, G., Svenda, M., Maia, F., Hantke, M., DePonte, D. P., Seibert, M. M., . . . Ekeberg, T. (2015). Imaging single cells in a beam of live cyanobacteria with an X-ray laser. Nature Communications, 6, 9. doi:10.1038/ncomms6704There exists a conspicuous gap of knowledge about the organization of life at mesoscopic levels. Ultra-fast coherent diffractive imaging with X-ray free-electron lasers can probe structures at the relevant length scales and may reach sub-nanometer resolution on micron-sized living cells. Here we show that we can introduce a beam of aerosolised cyanobacteria into the focus of the Linac Coherent Light Source and record diffraction patterns from individual living cells at very low noise levels and at high hit ratios. We obtain two-dimensional projection images directly from the diffraction patterns, and present the results as synthetic X-ray Nomarski images calculated from the complex-valued reconstructions. We further demonstrate that it is possible to record diffraction data to nanometer resolution on live cells with X-ray lasers. Extension to sub-nanometer resolution is within reach, although improvements in pulse parameters and X-ray area detectors will be necessary to unlock this potential.Additional Authors: Almeida, N. F.;Odic, D.;Hasse, D.;Carlsson, G. H.;Larsson, D. S. D.;Barty, A.;Martin, A. V.;Schorb, S.;Bostedt, C.;Bozek, J. D.;Rolles, D.;Rudenko, A.;Epp, S.;Foucar, L.;Rudek, B.;Hartmann, R.;Kimmel, N.;Holl, P.;Englert, L.;Loh, N. T. D.;Chapman, H. N.;Andersson, I.;Hajdu, J.;Ekeberg, T

Matched Filters for Source Detection in the Poissonian Noise Regime

A procedure is described for estimating an optimum kernel for the detection by convolution of signals among Poissonian noise. The technique is applied to the detection of x-ray point sources in XMM-Newton data, and is shown to yield an improvement in detection sensitivity of up to 60% over the sliding-box method used in the creation of the 1XMM catalog

Three-Dimensional Reconstruction of the Giant Mimivirus Particle with an X-Ray Free-Electron Laser

Citation: Ekeberg, T., Svenda, M., Abergel, C., Maia, F., Seltzer, V., Claverie, J. M., . . . Hajdu, J. (2015). Three-Dimensional Reconstruction of the Giant Mimivirus Particle with an X-Ray Free-Electron Laser. Physical Review Letters, 114(9), 6. doi:10.1103/PhysRevLett.114.098102We present a proof-of-concept three-dimensional reconstruction of the giant mimivirus particle from experimentally measured diffraction patterns from an x-ray free-electron laser. Three-dimensional imaging requires the assembly of many two-dimensional patterns into an internally consistent Fourier volume. Since each particle is randomly oriented when exposed to the x-ray pulse, relative orientations have to be retrieved from the diffraction data alone. We achieve this with a modified version of the expand, maximize and compress algorithm and validate our result using new methods.Additional Authors: Andersson, I.;Loh, N. D.;Martin, A. V.;Chapman, H.;Bostedt, C.;Bozek, J. D.;Ferguson, K. R.;Krzywinski, J.;Epp, S. W.;Rolles, D.;Rudenko, A.;Hartmann, R.;Kimmel, N.;Hajdu, J

Chloroplasts lacking class I glutaredoxins are functional but show a delayed recovery of protein cysteinyl redox state after oxidative challenge

Redox status of protein cysteinyl residues is mediated via glutathione (GSH)/glutaredoxin (GRX) and thioredoxin (TRX)-dependent redox cascades. An oxidative challenge can induce post-translational protein modifications on thiols, such as protein S-glutathionylation. Class I GRX are small thiol-disulfide oxidoreductases that reversibly catalyse S-glutathionylation and protein disulfide formation. TRX and GSH/GRX redox systems can provide partial backup for each other in several subcellular compartments, but not in the plastid stroma where TRX/light-dependent redox regulation of primary metabolism takes place. While the stromal TRX system has been studied at detail, the role of class I GRX on plastid redox processes is still unknown. We generate knockout lines of GRXC5 as the only chloroplast class I GRX of the moss Physcomitrium patens. While we find that PpGRXC5 has high activities in GSH-dependent oxidoreductase assays using hydroxyethyl disulfide or redox-sensitive GFP2 as substrates in vitro, Δgrxc5 plants show no detectable growth defect or stress sensitivity, in contrast to mutants with a less negative stromal EGSH (Δgr1). Using stroma-targeted roGFP2, we show increased protein Cys steady state oxidation and decreased reduction rates after oxidative challenge in Δgrxc5 plants in vivo, indicating kinetic uncoupling of the protein Cys redox state from EGSH. Compared to wildtype, protein Cys disulfide formation rates and S-glutathionylation levels after H2O2 treatment remained unchanged. Lack of class I GRX function in the stroma did not result in impaired carbon fixation. Our observations suggest specific roles for GRXC5 in the efficient transfer of electrons from GSH to target protein Cys as well as negligible cross-talk with metabolic regulation via the TRX system. We propose a model for stromal class I GRX function in efficient catalysis of protein dithiol/disulfide equilibria upon redox steady state alterations affecting stromal EGSH and highlight the importance of identifying in vivo target proteins of GRXC5

Gas Dynamic Virtual Nozzle for Generation of Microscopic Droplet Streams

As shown by Ganan-Calvo and co-workers, a free liquid jet can be compressed in iameter through gas-dynamic forces exerted by a co-flowing gas, obviating the need for a solid nozzle to form a microscopic liquid jet and thereby alleviating the clogging problems that plague conventional droplet sources of small diameter. We describe in this paper a novel form of droplet beam source based on this principle. The source is miniature, robust, dependable, easily fabricated, and eminently suitable for delivery of microscopic liquid droplets, including hydrated biological samples, into vacuum for analysis using vacuum instrumentation. Monodisperse, single file droplet streams are generated by triggering the device with a piezoelectric actuator. The device is essentially immune to clogging