The effect of secondary electrons on radiolysis as observed by in liquid TEM: The role of window material and electrical bias

The effect of window material on electron beam induced phenomena in liquid phase electron microscopy (LPEM) is an interesting yet under-explored subject. We have studied the differences of electron beam induced gold nanoparticle (AuNP) growth subject to three encapsulation materials: Silicon Nitride (Si3N4), carbon and formvar. We find Si3N4 liquid cells (LCs) to result in significantly higher AuNP growth yield as compared to LCs employing the other two materials. In all cases, an electrical bias of the entire LC structures significantly affected particle growth. We demonstrate an inverse correlation of the AuNP growth rate with secondary electron (SE) emission from the windows. We attribute these differences at least in part to variations in SE emission dynamics, which is seen as a combination of material and bias dependent SE escape flux (SEEF) and SE return flux (SERF). Furthermore, our model predictions qualitatively match electrochemistry expectations

Epstein–Barr virus LMP2A imposes sensitivity to apoptosis

In cell lines, the Epstein–Barr virus (EBV)-encoded protein latent membrane protein 2A (LMP2A) protects B-cells from apoptosis by blocking B-cell receptor (BCR) signalling. However, EBV-infected B-cells in vivo are extremely different from cell lines. This study used a murine transgenic model in which B-cells express LMP2A and a BCR specific for hen egg lysozyme to determine whether LMP2A protects resting and antigen-activated B-cells from apoptosis. LMP2A allows BCR signal transduction and induces constitutive activation of NF-κB to increase Bcl-2 levels that afford LMP2A-mediated protection from apoptosis in the absence or presence of antigen. In contrast, low levels of NF-κB inhibitor only affected Bcl-2 and Bcl-xL levels and increased apoptosis in LMP2A-negative B-cells after BCR cross-linking. These data suggest that LMP2A uniquely makes resting B-cells sensitive to NF-κB inhibition and apoptosis and suggest that NF-κB may be a novel target to eradicate latently EBV-infected B-cells

The first cytoplasmic loop of the mannitol permease from Escherichia coli is accessible for sulfhydryl reagents from the periplasmic side of the membrane

The mannitol permease (EIIMtl) from Escherichia coli couples mannitol transport to phosphorylation of the substrate. Renewed topology prediction of the membrane-embedded C domain suggested that EIIMtl contains more membrane-embedded segments than the six proposed previously on the basis of a PhoA fusion study. Cysteine accessibility was used to confirm this notion. Since cysteine 384 in the cytoplasmic B domain is crucial for the phosphorylation activity of EIIMtl, all cysteine mutants contained this activity-linked cysteine residue in addition to those introduced for probing the membrane topology of the protein. To distinguish between the activity-linked cysteine and the probed cysteine, either trypsin was used to specifically digest the two cytoplasmic domains (A and B), thereby removing Cys384, or Cys384 was protected by phosphorylation from alkylation by N-ethylmaleimide (NEM). Our data show that upon phosphorylation EIIMtl undergoes major conformational changes, whereby residues in the putative first cytoplasmic loop become accessible to NEM. Other residues in this loop were accessible to NEM in intact cells and inside-out membrane vesicles, but cysteine residues at these positions only reacted with the membrane-impermeable sulfhydryl reagent from the periplasmic side of the protein. These and other results suggest that the predicted loop between TM2 and TM3 may fold back into the membrane and form part of the translocation path

Serial protein crystallography in an electron microscope

Serial X-ray crystallography at free-electron lasers allows to solve biomolecular structures from sub-micron-sized crystals. However, beam time at these facilities is scarce, and involved sample delivery techniques are required. On the other hand, rotation electron diffraction (MicroED) has shown great potential as an alternative means for protein nano-crystallography. Here, we present a method for serial electron diffraction of protein nanocrystals combining the benefits of both approaches. In a scanning transmission electron microscope, crystals randomly dispersed on a sample grid are automatically mapped, and a diffraction pattern at fixed orientation is recorded from each at a high acquisition rate. Dose fractionation ensures minimal radiation damage effects. We demonstrate the method by solving the structure of granulovirus occlusion bodies and lysozyme to resolutions of 1.55 Å and 1.80 Å, respectively. Our method promises to provide rapid structure determination for many classes of materials with minimal sample consumption, using readily available instrumentation

Structural basis for CRISPR RNA-guided DNA recognition by Cascade

The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and low-resolution structure of Cascade and show how it recognizes double-stranded DNA (dsDNA) targets in a sequence-specific manner. Cascade is a 405-kDa complex comprising five functionally essential CRISPR-associated (Cas) proteins (CasA1B2C6D1E1) and a 61-nucleotide CRISPR RNA (crRNA) with 5′-hydroxyl and 2′,3′-cyclic phosphate termini. The crRNA guides Cascade to dsDNA target sequences by forming base pairs with the complementary DNA strand while displacing the noncomplementary strand to form an R-loop. Cascade recognizes target DNA without consuming ATP, which suggests that continuous invader DNA surveillance takes place without energy investment. The structure of Cascade shows an unusual seahorse shape that undergoes conformational changes when it binds target DNA.

Fibroblast growth factor signalling controls nervous system patterning and pigment cell formation in Ciona intestinalis

During the development of the central nervous system (CNS), combinations of transcription factors and signalling molecules orchestrate patterning, specification and differentiation of neural cell types. In vertebrates, three types of melanin-containing pigment cells, exert a variety of functional roles including visual perception. Here we analysed the mechanisms underlying pigment cell specification within the CNS of a simple chordate, the ascidian Ciona intestinalis. Ciona tadpole larvae exhibit a basic chordate body plan characterized by a small number of neural cells. We employed lineage-specific transcription profiling to characterize the expression of genes downstream of fibroblast growth factor signalling, which govern pigment cell formation. We demonstrate that FGF signalling sequentially imposes a pigment cell identity at the expense of anterior neural fates. We identify FGF-dependent and pigment cell-specific factors, including the small GTPase, Rab32/38 and demonstrated its requirement for the pigmentation of larval sensory organs

Epstein-Barr Virus LMP2A Reduces Hyperactivation Induced by LMP1 to Restore Normal B Cell Phenotype in Transgenic Mice

Epstein-Barr virus (EBV) latently infects most of the human population and is strongly associated with lymphoproliferative disorders. EBV encodes several latency proteins affecting B cell proliferation and survival, including latent membrane protein 2A (LMP2A) and the EBV oncoprotein LMP1. LMP1 and LMP2A signaling mimics CD40 and BCR signaling, respectively, and has been proposed to alter B cell functions including the ability of latently-infected B cells to access and transit the germinal center. In addition, several studies suggested a role for LMP2A modulation of LMP1 signaling in cell lines by alteration of TRAFs, signaling molecules used by LMP1. In this study, we investigated whether LMP1 and LMP2A co-expression in a transgenic mouse model alters B cell maturation and the response to antigen, and whether LMP2A modulates LMP1 function. Naïve LMP1/2A mice had similar lymphocyte populations and antibody production by flow cytometry and ELISA compared to controls. In the response to antigen, LMP2A expression in LMP1/2A animals rescued the impairment in germinal center generation promoted by LMP1. LMP1/2A animals produced high-affinity, class-switched antibody and plasma cells at levels similar to controls. In vitro, LMP1 upregulated activation markers and promoted B cell hyperproliferation, and co-expression of LMP2A restored a wild-type phenotype. By RT-PCR and immunoblot, LMP1 B cells demonstrated TRAF2 levels four-fold higher than non-transgenic controls, and co-expression of LMP2A restored TRAF2 levels to wild-type levels. No difference in TRAF3 levels was detected. While modulation of other TRAF family members remains to be assessed, normalization of the LMP1-induced B cell phenotype through LMP2A modulation of TRAF2 may be a pathway by which LMP2A controls B cell function. These findings identify an advance in the understanding of how Epstein-Barr virus can access the germinal center in vivo, a site critical for both the genesis of immunological memory and of virus-associated tumors

Väst-SOM-undersökningen 2008

Amylomaltases are glycosyl hydrolases belonging to glycoside hydrolase family 77 that are capable of the synthesis of large cyclic glucans and the disproportionation of oligosaccharides. Using protein crystallography, we have generated a flip book movie of the amylomaltase catalytic cycle in atomic detail. The structures include a covalent glycosyl enzyme intermediate and a covalent intermediate in complex with an analogue of a cosubstrate and show how the structures of both enzyme and substrate respond to the changes required by the catalytic cycle as it proceeds. Notably, the catalytic nucleophile changes conformation dramatically during the reaction. Also, Gln- 256 on the 250s loop is involved in orienting the substrate in the + 1 site. The absence of a suitable base in the covalent intermediate structure explains the low hydrolysis activity