Publication date: August 2015
Source:Journal of Structural Biology, Volume 191, Issue 2

Author(s): Christin Dittmann, Hong-Mei Han, Markus Grabenbauer, Michael Laue

Bacterial spores of the genera Bacillus and Clostridium are extremely resistant against desiccation, heat and radiation and involved in the spread and pathogenicity of health relevant species such as Bacillus anthracis (anthrax) or Clostridium botulinum. While the resistance of spores is very well documented, underlying mechanisms are not fully understood. In this study we show, by cryo-electron microscopy of vitreous sections and particular resin thin section electron microscopy, that dormant Bacillus spores possess highly ordered crystalline core structures, which contain the DNA, but only if small acid soluble proteins (SASPs) are present. We found those core structures in spores of all Bacillus species investigated, including spores of anthrax. Similar core structures were detected in Geobacillus and Clostridium species which suggest that highly ordered, at least partially crystalline core regions represent a general feature of bacterial endospores. The crystalline core structures disintegrate in a period during spore germination, when resistance against most stresses is lost. Our results suggest that the DNA is tightly packed into a crystalline nucleoid by binding SASPs, which stabilizes DNA fibrils and protects them against modification. Thus, the crystalline nucleoid seems to be the structural and functional correlate for the remarkable stability of the DNA in bacterial endospores.

Publication date: August 2015
Source:Journal of Structural Biology, Volume 191, Issue 2

Author(s): Peter Werner, Horst Blumtritt, Igor Zlotnikov, Andreas Graff, Yannicke Dauphin, Peter Fratzl

We report on a structural analysis of several basal spicules of the deep-sea silica sponge Monorhaphis chuni by electron microscope techniques supported by a precise focused ion beam (FIB) target preparation. To get a deeper understanding of the spicules length growth, we concentrated our investigation onto the apical segments of two selected spicules with apparently different growth states and studied in detail permanent and temporary growth structures in the central compact silica axial cylinder (AC) as well as the structure of the organic axial filament (AF) in its center. The new findings concern the following morphology features: (i) at the tip we could identify thin silica layers, which overgrow as a tongue-like feature the front face of the AC and completely fuse during the subsequent growth state. This basically differs from the radial growth of the surrounding lamellar zone of the spicules made of alternating silica lamellae and organic interlayers. (ii) A newly detected disturbed cylindrical zone in the central region of the AC (diameter about 30μm) contains vertical and horizontal cavities, channels and agglomerates, which can be interpreted as permanent leftover of a formerly open axial channel, later filled by silica. (iii) The AF consists of a three-dimensional crystal-like arrangement of organic molecules and amorphous silica surrounding these molecules. Similar to an inorganic crystal, this encased protein crystal is typified by crystallographic directions, lattice planes and surface steps. The 〈001〉 growth direction is especially favored, thereby scaffolding the axial cylinders growth and consequently the spicules’ morphology.

Publication date: August 2015
Source:Journal of Structural Biology, Volume 191, Issue 2

Author(s): Luc Bonnefond, Johann Stojko, Justine Mailliot, Nathalie Troffer-Charlier, Vincent Cura, Jean-Marie Wurtz, Sarah Cianférani, Jean Cavarelli

PRMT6 is a protein arginine methyltransferase involved in transcriptional regulation, human immunodeficiency virus pathogenesis, DNA base excision repair, and cell cycle progression. Like other PRMTs, PRMT6 is overexpressed in several cancer types and is therefore considered as a potential anti-cancer drug target. In the present study, we described six crystal structures of PRMT6 from Mus musculus, solved and refined at 1.34Å for the highest resolution structure. The crystal structures revealed that the folding of the helix αX is required to stabilize a productive active site before methylation of the bound peptide can occur. In the absence of cofactor, metal cations can be found in the catalytic pocket at the expected position of the guanidinium moiety of the target arginine substrate. Using mass spectrometry under native conditions, we show that PRMT6 dimer binds two cofactor and a single H4 peptide molecules. Finally, we characterized a new site of in vitro automethylation of mouse PRMT6 at position 7.

Publication date: August 2015
Source:Journal of Structural Biology, Volume 191, Issue 2

Author(s): Chutiparn Lertvachirapaiboon, Tewarak Parnklang, Prompong Pienpinijtham, Kanet Wongravee, Chuchaat Thammacharoen, Sanong Ekgasit

An interaction between the incident light and the structural architecture within the shell of Asian green mussel (Perna viridis) induces observable pearlescent colors. In this paper, we investigate the influence of the structural architecture on the expressed colors. After a removal of the organic binder, small flakes from crushed shells show vivid rainbow reflection under an optical microscope. An individual flake expresses vivid color under a bright-field illumination while become transparent under a dark-field illumination. The expressed colors of the aragonite flakes are directly associated with its structural architecture. The flakes with aragonite thickness of 256, 310, and 353nm, respectively, appear blue, green, and red under an optical microscope. The spectral simulation corroborates the experimentally observed optical effects as the flakes with thicker aragonite layers selectively reflected color with longer wavelengths. Flakes with multiple aragonite thicknesses expressed multi-color as the upper aragonite layers allow reflected colors from the lower layers to be observed.

Publication date: August 2015
Source:Journal of Structural Biology, Volume 191, Issue 2

Author(s): Mona Eulitz, Gebhard Reiss

Reconstruction of the three-dimensional (3D) surface of an object to be examined is widely used for structure analysis in science and many biological questions require information about their true 3D structure. For Scanning Electron Microscopy (SEM) there has been no efficient non-destructive solution for reconstruction of the surface morphology to date. The well-known method of recording stereo pair images generates a 3D stereoscope reconstruction of a section, but not of the complete sample surface. We present a simple and non-destructive method of 3D surface reconstruction from SEM samples based on the principles of optical close range photogrammetry. In optical close range photogrammetry a series of overlapping photos is used to generate a 3D model of the surface of an object. We adapted this method to the special SEM requirements. Instead of moving a detector around the object, the object itself was rotated. A series of overlapping photos was stitched and converted into a 3D model using the software commonly used for optical photogrammetry. A rabbit kidney glomerulus was used to demonstrate the workflow of this adaption. The reconstruction produced a realistic and high-resolution 3D mesh model of the glomerular surface. The study showed that SEM micrographs are suitable for 3D reconstruction by optical photogrammetry. This new approach is a simple and useful method of 3D surface reconstruction and suitable for various applications in research and teaching.

Publication date: August 2015
Source:Journal of Structural Biology, Volume 191, Issue 2

Author(s): David Shultis, Gregory Dodge, Yang Zhang

The Phox homology domain (PX domain) is a phosphoinositide-binding structural domain that is critical in mediating protein and cell membrane association and has been found in more than 100 eukaryotic proteins. The abundance of PX domains in nature offers an opportunity to redesign the protein using EvoDesign, a computational approach to design new sequences based on structure profiles of multiple evolutionarily related proteins. In this study, we report the X-ray crystallographic structure of a designed PX domain from the cytokine-independent survival kinase (CISK), which has been implicated as functioning in parallel with PKB/Akt in cell survival and insulin responses. Detailed data analysis of the designed CISK-PX protein demonstrates positive impacts of knowledge-based secondary structure and solvation predictions and structure-based sequence profiles on the efficiency of the evolutionary-based protein design method. The structure of the designed CISK-PX domain is close to the wild-type (1.54Å in Cα RMSD), which was accurately predicted by I-TASSER based fragment assembly simulations (1.32Å in Cα RMSD). This study represents the first successfully designed conditional peripheral membrane protein fold and has important implications in the examination and experimental validation of the evolution-based protein design approaches.

Publication date: August 2015
Source:Journal of Structural Biology, Volume 191, Issue 2

Author(s): M. Spiegel, A.K. Duraisamy, G.F. Schröder

Cryo-electron microscopy yields 3D density maps of macromolecules from single-particle images, tomograms, or 2D crystals. An optimal visualization of the density map is important for its proper interpretation. We have developed a method to improve the visualization of density maps by using general statistical information about proteins for the sharpening process. In particular, the packing density of atoms is highly similar between different proteins, which allows for building a pseudo-atomic model to approximate the true mass distribution. From this model the radial structure factor and density value histogram are estimated and applied as constraints to the 3D reconstruction in reciprocal- and real-space, respectively. Interestingly, similar improvements are obtained when using the correct radial structure factor and density value histogram from a crystal structure. Thus, the estimated pseudo-atomic model yields a sufficiently accurate mass distribution to optimally sharpen a density map.

Publication date: August 2015
Source:Journal of Structural Biology, Volume 191, Issue 2

Author(s): Jana Škerlová, Vlastimil Král, Michael Kachala, Milan Fábry, Ladislav Bumba, Dmitri I. Svergun, Zdeněk Tošner, Václav Veverka, Pavlína Řezáčová

The hyaluronate receptor CD44 plays role in cell adhesion and migration and is involved in tumor metastasis. The extracellular domain of CD44 comprises the hyaluronate-binding domain (HABD) and the membrane-proximal stem region; the short intracellular portion interacts with adaptor proteins and triggers signaling pathways. Binding of hyaluronate to CD44 HABD induces an allosteric conformational change, which results in CD44 shedding. A poorly characterized epitope in human CD44 HABD is recognized by the murine monoclonal antibody MEM-85, which cross-blocks hyaluronate binding to CD44 and also induces CD44 shedding. MEM-85 is of therapeutic interest, as it inhibits growth of lung cancer cells in murine models. In this work, we employed a combination of biophysical methods to determine the MEM-85 binding epitope in CD44 HABD and to provide detailed insight into the mechanism of MEM-85 action. In particular, we constructed a single-chain variable fragment (scFv) of MEM-85 as a tool for detailed characterization of the CD44 HABD–antibody complex and identified residues within CD44 HABD involved in the interaction with scFv MEM-85 by NMR spectroscopy and mutational analysis. In addition, we built a rigid body model of the CD44 HABD–scFv MEM-85 complex using a low-resolution structure obtained by small-angle X-ray scattering. The MEM-85 epitope is situated in the C-terminal part of CD44 HABD, rather than the hyaluronate-binding groove, and the binding of MEM-85 induces a structural reorganization similar to that induced by hyaluronate. Therefore, the mechanism of MEM-85 cross-blocking of hyaluronate binding is likely of an allosteric, relay-like nature.

Publication date: August 2015
Source:Journal of Structural Biology, Volume 191, Issue 2

Author(s): Ali Ibrahim, Charles Henri Hage, Anissa Souissi, Aymeric Leray, Laurent Héliot, Sami Souissi, Bernard Vandenbunder

Pseudodiaptomus marinus copepods are small crustaceans living in estuarine areas endowed with exceptional swimming and adaptative performances. Since the external cuticle acts as an impermeable barrier for most dyes and molecular tools for labeling copepod proteins with fluorescent tags are not available, imaging cellular organelles in these organisms requires label free microscopy. Complementary nonlinear microscopy techniques have been used to investigate the structure and the response of their myofibrils to abrupt changes of temperature or/and salinity. In contrast with previous observations in vertebrates and invertebrates, the flavin autofluorescence which is a signature of mitochondria activity and the Coherent Anti-Stokes Raman Scattering (CARS) pattern assigned to T-tubules overlapped along myofibrils with the second harmonic generation (SHG) striated pattern generated by myosin tails in sarcomeric A bands. Temperature jumps from 18 to 4°C or salinity jumps from 30 to 15psu mostly affected flavin autofluorescence. Severe salinity jumps from 30 to 0psu dismantled myofibril organization with major changes both in the SHG and CARS patterns. After a double stress (from 18°C/30psu to 4°C/0psu) condensed and distended regions appeared within single myofibrils, with flavin autofluorescence bands located between sarcomeric A bands. These results shed light on the interactions between the different functional compartments which provide fast acting excitation–contraction coupling and adequate power supply in copepods muscles.

Publication date: August 2015
Source:Journal of Structural Biology, Volume 191, Issue 2

Author(s): Natalia Korotkova, Jérémie Piton, Jonathan M. Wagner, Stefanie Boy-Röttger, Aleksandre Japaridze, Timothy J. Evans, Stewart T. Cole, Florence Pojer, Konstantin V. Korotkov

Mycobacterium tuberculosis secretes multiple virulence factors during infection via the general Sec and Tat pathways, and via specialized ESX secretion systems, also referred to as type VII secretion systems. The ESX-1 secretion system is an important virulence determinant because deletion of ESX-1 leads to attenuation of M. tuberculosis. ESX-1 secreted protein B (EspB) contains putative PE (Pro-Glu) and PPE (Pro-Pro-Glu) domains, and a C-terminal domain, which is processed by MycP1 protease during secretion. We determined the crystal structure of PE–PPE domains of EspB, which represents an all-helical, elongated molecule closely resembling the structure of the PE25–PPE41 heterodimer despite limited sequence similarity. Also, we determined the structure of full-length EspB, which does not have interpretable electron density for the C-terminal domain confirming that it is largely disordered. Comparative analysis of EspB in cell lysate and culture filtrates of M. tuberculosis revealed that mature secreted EspB forms oligomers. Electron microscopy analysis showed that the N-terminal fragment of EspB forms donut-shaped particles. These data provide a rationale for the future investigation of EspB’s role in M. tuberculosis pathogenesis.

Publication date: August 2015
Source:Journal of Structural Biology, Volume 191, Issue 2

Author(s): Hemant D. Tagare, Alp Kucukelbir, Fred J. Sigworth, Hongwei Wang, Murali Rao

Structural heterogeneity of particles can be investigated by their three-dimensional principal components. This paper addresses the question of whether, and with what algorithm, the three-dimensional principal components can be directly recovered from cryo-EM images. The first part of the paper extends the Fourier slice theorem to covariance functions showing that the three-dimensional covariance, and hence the principal components, of a heterogeneous particle can indeed be recovered from two-dimensional cryo-EM images. The second part of the paper proposes a practical algorithm for reconstructing the principal components directly from cryo-EM images without the intermediate step of calculating covariances. This algorithm is based on maximizing the posterior likelihood using the Expectation–Maximization algorithm. The last part of the paper applies this algorithm to simulated data and to two real cryo-EM data sets: a data set of the 70S ribosome with and without Elongation Factor-G (EF-G), and a data set of the influenza virus RNA dependent RNA Polymerase (RdRP). The first principal component of the 70S ribosome data set reveals the expected conformational changes of the ribosome as the EF-G binds and unbinds. The first principal component of the RdRP data set reveals a conformational change in the two dimers of the RdRP.

Publication date: Available online 26 July 2015
Source:Journal of Structural Biology

Author(s): Julia Mahamid, Ruud Schampers, Hans Persoon, Anthony A. Hyman, Wolfgang Baumeister, Jürgen M. Plitzko

Cryo-electron tomography provides 3D views of cellular architecture with molecular resolution. A principal limitation of cryo-transmission electron microscopy performed on cells or tissues is the accessible specimen thickness. Recently it has been shown that cryo-focused ion beam milling of plunge-frozen eukaryotic cells can produce homogeneously thin, distortion free lamellas for cryo-electron tomography. Multicellular organisms and tissue cannot be properly vitrified and thinned using this technique because they are considerably thicker. High pressure freezing is therefore necessary to provide optimal preservation. Here, we describe a workflow for preparing lamellas from Caenorhabditis elegans worms using cryo-FIB applied to high pressure frozen samples. We employ cryo-planing followed by correlative cryo-fluorescence microscopy to navigate this large multicellular volume and to localize specific targets within. To produce vitreous lamellas amenable to cryo-TEM observations at these targeted locations, we have developed a dedicated lift-out procedure at cryogenic temperature.

Publication date: Available online 1 September 2015
Source:Journal of Molecular Biology

Author(s): Viktor Haellman, Martin Fussenegger

Higher multi-cellular organisms have evolved sophisticated intra- and inter-cellular biological networks that enable cell growth and survival to fulfil an organism’s needs. Although such networks allow the assembly of complex tissues and even provide healing and protective capabilities, malfunctioning cells can have severe consequences for an organism’s survival. In humans, such events can result in severe disorders and diseases, including metabolic and immunological disorders [1, 2], as well as cancer [3]. Dominating the therapeutic frontier for these potentially lethal disorders, cell and gene therapies aim to relieve or eliminate patient suffering by restoring the function of damaged, diseased, and aging cells and tissues via the introduction of healthy cells or alternative genes. However, despite recent success, these efforts have yet to achieve sufficient therapeutic effects, and further work is needed to ensure the safe and precise control of transgene expression and cellular processes. In this review, we describe the biological tools and devices that are at the forefront of synthetic biology and discuss their potential to advance the specificity, efficiency, and safety of the current generation of cell and gene therapies, including how they can be used to confer curative effects that far surpass those of conventional therapeutics. We also highlight the current therapeutic delivery tools and the current limitations that hamper their use in human applications.
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Publication date: Available online 31 August 2015
Source:Journal of Molecular Biology

Author(s): Alan X. Ji, Anh Chu, Tine Kragh Nielsen, Samir Benlekbir, John L. Rubinstein, Gilbert G. Privé

Cullin3 (Cul3)-based ubiquitin E3 ligase complexes catalyze the transfer of ubiquitin from an E2 enzyme to target substrate proteins. In these assemblies, the C-terminal region of Cul3 binds Rbx1/E2~ubiquitin, while the N-terminal region interacts with various BTB domain proteins that serve as substrate adaptors. Previous crystal structures of the homodimeric BTB proteins KLHL3, KLHL11 and SPOP in complex with the N-terminal domain of Cul3 revealed the features required for Cul3 recognition in these proteins. A second class of BTB-domain containing proteins, the KCTD proteins, are also Cul3 substrate adaptors, but these do not share many of the previously identified determinants for Cul3 binding. We report the pentameric crystal structures of the KCTD1 and KCTD9 BTB domains, and identify plasticity in the KCTD1 rings. We find that the KCTD proteins 5, 6, 9, and 17 bind to Cul3 with high affinity, while the KCTD proteins 1 and 16 do not have detectable binding. Finally, we confirm the 5:5 assembly of KCTD9/Cul3 complexes by electron cryomicroscopy and provide a molecular rationale for BTB-mediated Cul3 binding specificity in the KCTD family.
Graphical abstract

Publication date: 28 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 17

Publication date: 28 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 17

Publication date: 28 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 17

Author(s): Paul C. Driscoll

Publication date: 28 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 17

Author(s): Sujan Devkota, Raji E. Joseph, Lie Min, D. Bruce Fulton, Amy H. Andreotti

Activation of the phospholipase, PLCγ1, is critical for proper T cell signaling following antigen receptor engagement. In T cells, the Tec family kinase, interleukin-2-induced tyrosine kinase (ITK), phosphorylates PLCγ1 at tyrosine 783 (Y783) leading to activation of phospholipase function and subsequent production of the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. In this work, we demonstrate that PLCγ1 can be primed for ITK-mediated phosphorylation on Y783 by a specific region of the adaptor protein, SLP-76. The SLP-76 phosphotyrosine-containing sequence, pY173IDR, does not conform to the canonical recognition motif for an SH2 domain yet binds with significant affinity to the C-terminal SH2 domain of PLCγ1 (SH2C). The SLP-76 pY173 motif competes with the autoinhibited conformation surrounding the SH2C domain of PLCγ1 leading to exposure of the ITK recognition element on the PLCγ1 SH2 domain and release of the target tyrosine, Y783. These data contribute to the evolving model for the molecular events occurring early in the T cell activation process.
Graphical abstract

Publication date: 28 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 17

Author(s): Karen T. Fortmann, Russell D. Lewis, Kim A. Ngo, Riku Fagerlund, Alexander Hoffmann

Whereas ubiquitin-dependent degrons have been characterized in some detail, how proteins may be targeted to ubiquitin-independent proteasomal degradation remains unclear. Here we show that IκBα contains an ubiquitin-independent degron whose activity is portable to heterologous proteins such as the globular protein GFP (green fluorescent protein) via a proteasome-dependent, ubiquitin-independent, non-lysosomal pathway. The ubiquitin-independent degradation signal resides in an 11-amino-acid sequence, which is not only sufficient but also required for IκBα's short half-life. Finally, we show that this degron's activity is regulated by the interaction with NFκB, which controls its solvent exposure, and we demonstrate that this regulation of the degron's activity is critical for IκBα's signaling functions.
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Publication date: 28 August 2015
Source:Journal of Molecular Biology, Volume 427, Issue 17

Author(s): Matthias Spiess, Meriem Echbarthi, Andreas Svanström, Roger Karlsson, Julie Grantham

Chaperonin containing tailless complex polypeptide 1 (CCT) forms a classical chaperonin barrel structure where two rings of subunits surround a central cavity. Each ring consists of eight distinct subunits, creating a complex binding interface that makes CCT unique among the chaperonins. In addition to acting as a multimeric chaperonin, there is increasing evidence indicating that the CCT subunits, when monomeric, possess additional functions. Here we assess the role of the CCT subunits individually, using a GFP (green fluorescent protein) tagging approach to express each of the subunits in their monomeric form in cultured mammalian cells. Over-expression of CCTdelta, but not the other seven CCT subunits, results in the appearance of numerous protrusions at the cell surface. Two point mutations, one in the apical domain and one in the ATP binding pocket of CCTdelta, that abolish protrusion formation have been identified, consistent with the apical domain containing a novel interaction site that is influenced by the ATPase activity in the equatorial domain. Structured illumination microscopy, together with sub-cellular fractionation, reveals that only the wild-type CCTdelta is associated with the plasma membrane, thus connecting spatial organization with surface protrusion formation. Expression of the equivalent subunit in yeast, GFP-Cct4, rescues growth of the temperature-sensitive strain cct4-1 at the non-permissive temperature, indicative of conserved subunit-specific activities for CCTdelta.
Graphical abstract