Journal of Structural Biology

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Functional insights from high resolution structures of mouse protein arginine methyltransferase 6

Wed, 09/02/2015 - 00:52
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.





Categories: Journal Articles

Selective colors reflection from stratified aragonite calcium carbonate plates of mollusk shells

Wed, 09/02/2015 - 00:52
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.





Categories: Journal Articles

3D reconstruction of SEM images by use of optical photogrammetry software

Wed, 09/02/2015 - 00:52
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.





Categories: Journal Articles

Crystal structure of designed PX domain from cytokine-independent survival kinase and implications on evolution-based protein engineering

Wed, 09/02/2015 - 00:52
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.





Categories: Journal Articles

Improving the visualization of cryo-EM density reconstructions

Wed, 09/02/2015 - 00:52
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.





Categories: Journal Articles

Molecular mechanism for the action of the anti-CD44 monoclonal antibody MEM-85

Wed, 09/02/2015 - 00:52
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.





Categories: Journal Articles

Label-free microscopy and stress responses reveal the functional organization of Pseudodiaptomus marinus copepod myofibrils

Wed, 09/02/2015 - 00:52
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.





Categories: Journal Articles

Structure of EspB, a secreted substrate of the ESX-1 secretion system of Mycobacterium tuberculosis

Wed, 09/02/2015 - 00:52
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.





Categories: Journal Articles

Directly reconstructing principal components of heterogeneous particles from cryo-EM images

Wed, 09/02/2015 - 00:52
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.





Categories: Journal Articles

A focused ion beam milling and lift-out approach for site-specific preparation of frozen-hydrated lamellas from multicellular organisms

Wed, 09/02/2015 - 00:52
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.





Categories: Journal Articles