Journal Cover Journal of Structural Biology
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   Hybrid Journal Hybrid journal (It can contain Open Access articles)
   ISSN (Print) 1047-8477 - ISSN (Online) 1095-8657
   Published by Elsevier Homepage  [3175 journals]
  • Spotiton: New features and applications
    • Authors: Venkata P. Dandey; Hui Wei; Zhening Zhang; Yong Zi Tan; Priyamvada Acharya; Edward T. Eng; William J. Rice; Peter A. Kahn; Clinton S. Potter; Bridget Carragher
      Pages: 161 - 169
      Abstract: Publication date: May 2018
      Source:Journal of Structural Biology, Volume 202, Issue 2
      Author(s): Venkata P. Dandey, Hui Wei, Zhening Zhang, Yong Zi Tan, Priyamvada Acharya, Edward T. Eng, William J. Rice, Peter A. Kahn, Clinton S. Potter, Bridget Carragher
      We present an update describing new features and applications of Spotiton, a novel instrument for vitrifying samples for cryoEM. We have used Spotiton to prepare several test specimens that can be reconstructed using routine single particle analysis to ∼3 Å resolution, indicating that the process has no apparent deleterious effect on the sample integrity. The system is now in routine and continuous use in our lab and has been used to successfully vitrify a wide variety of samples.

      PubDate: 2018-04-18T07:40:01Z
      DOI: 10.1016/j.jsb.2018.01.002
       
  • Automated tracing of helical assemblies from electron cryo-micrographs
    • Authors: Stefan T. Huber; Tanja Kuhm; Carsten Sachse
      Pages: 1 - 12
      Abstract: Publication date: April 2018
      Source:Journal of Structural Biology, Volume 202, Issue 1
      Author(s): Stefan T. Huber, Tanja Kuhm, Carsten Sachse
      Structure determination of helical specimens commonly requires datasets from thousands of micrographs often obtained by automated cryo-EM data acquisition. Interactive tracing of helical assemblies from such a number of micrographs is labor-intense and time-consuming. Here, we introduce an automated tracing tool MicHelixTrace that precisely locates helix traces from micrographs of rigid as well as very flexible helical assemblies with small numbers of false positives. The computer program is fast and has low computational requirements. In addition to helix coordinates required for a subsequent helical reconstruction work-flow, we determine the persistence length of the polymer ensemble. This information provides a useful measure to characterize mechanical properties of helical assemblies and to evaluate the potential for high-resolution structure determination.

      PubDate: 2018-04-18T07:40:01Z
      DOI: 10.1016/j.jsb.2017.11.013
       
  • Direct regulation of p190RhoGEF by activated Rho and Rac GTPases
    • Authors: Olugbenga Dada; Stephen Gutowski; Chad A. Brautigam; Zhe Chen; Paul C. Sternweis
      Pages: 13 - 24
      Abstract: Publication date: April 2018
      Source:Journal of Structural Biology, Volume 202, Issue 1
      Author(s): Olugbenga Dada, Stephen Gutowski, Chad A. Brautigam, Zhe Chen, Paul C. Sternweis
      Rho family GTPases regulate a wide range of cellular processes. This includes cellular dynamics where three subfamilies, Rho, Rac, and Cdc42, are known to regulate cell shape and migration though coordinate action. Activation of Rho proteins largely depends on Rho Guanine nucleotide Exchange Factors (RhoGEFs) through a catalytic Dbl homology (DH) domain linked to a pleckstrin homology (PH) domain that subserves various functions. The PH domains from Lbc RhoGEFs, which specifically activate RhoA, have been shown to bind to activated RhoA. Here, p190RhoGEF is shown to also bind Rac1·GTP. Crystal structures reveal that activated Rac1 and RhoA use their effector-binding surfaces to associate with the same hydrophobic surface on the PH domain. Both activated RhoA and Rac1 can stimulate exchange of nucleotide on RhoA by localization of p190RhoGEF to its substrate, RhoA·GDP, in vitro. The binding of activated RhoA provides a mechanism for positive feedback regulation as previously proposed for the family of Lbc RhoGEFs. In contrast, the novel interaction between activated Rac1 and p190RhoGEF reveals a potential mechanism for cross-talk regulation where Rac can directly effect stimulation of RhoA. The greater capacity of Rac1 to stimulate p190RhoGEF among the Lbc RhoGEFs suggests functional specialization.

      PubDate: 2018-04-18T07:40:01Z
      DOI: 10.1016/j.jsb.2017.11.014
       
  • Correlative imaging reveals physiochemical heterogeneity of
           microcalcifications in human breast carcinomas
    • Authors: Jennie A.M.R. Kunitake; Siyoung Choi; Kayla X. Nguyen; Meredith M. Lee; Frank He; Daniel Sudilovsky; Patrick G. Morris; Maxine S. Jochelson; Clifford A. Hudis; David A. Muller; Peter Fratzl; Claudia Fischbach; Admir Masic; Lara A. Estroff
      Pages: 25 - 34
      Abstract: Publication date: April 2018
      Source:Journal of Structural Biology, Volume 202, Issue 1
      Author(s): Jennie A.M.R. Kunitake, Siyoung Choi, Kayla X. Nguyen, Meredith M. Lee, Frank He, Daniel Sudilovsky, Patrick G. Morris, Maxine S. Jochelson, Clifford A. Hudis, David A. Muller, Peter Fratzl, Claudia Fischbach, Admir Masic, Lara A. Estroff
      Microcalcifications (MCs) are routinely used to detect breast cancer in mammography. Little is known, however, about their materials properties and associated organic matrix, or their correlation to breast cancer prognosis. We combine histopathology, Raman microscopy, and electron microscopy to image MCs within snap-frozen human breast tissue and generate micron-scale resolution correlative maps of crystalline phase, trace metals, particle morphology, and organic matrix chemical signatures within high grade ductal carcinoma in situ (DCIS) and invasive cancer. We reveal the heterogeneity of mineral-matrix pairings, including punctate apatitic particles (<2 µm) with associated trace elements (e.g., F, Na, and unexpectedly Al) distributed within the necrotic cores of DCIS, and both apatite and spheroidal whitlockite particles in invasive cancer within a matrix containing spectroscopic signatures of collagen, non-collagen proteins, cholesterol, carotenoids, and DNA. Among the three DCIS samples, we identify key similarities in MC morphology and distribution, supporting a dystrophic mineralization pathway. This multimodal methodology lays the groundwork for establishing MC heterogeneity in the context of breast cancer biology, and could dramatically improve current prognostic models.
      Graphical abstract image

      PubDate: 2018-04-18T07:40:01Z
      DOI: 10.1016/j.jsb.2017.12.002
       
  • Photosome membranes merge and organize tending towards rhombohedral
           symmetry when light is emitted
    • Authors: Malika Ouldali; Virginie Maury; Gisèle Nicolas; Jean Lepault
      Pages: 35 - 41
      Abstract: Publication date: April 2018
      Source:Journal of Structural Biology, Volume 202, Issue 1
      Author(s): Malika Ouldali, Virginie Maury, Gisèle Nicolas, Jean Lepault
      Polynoid worm elytra emit light when mechanically or electrically stimulated. Specialized cells, the photocytes, contain light emitting machineries, the photosomes. Successive stimulations induce light intensity variations and show a coupling within and between photosomes. Here, we describe, using electron tomography of cryo-substituted elytra and freeze-fracturing, the structural transition associated to light emission: undulating tubules come closer, organize and their number forming photosomes increases. Two repeating undulating tubules in opposite phase compose the photosome. Undulations are located on three hexagonal layers that regularly repeat and are equally displaced, in x y and z. The tubule membranes within layers merge giving rise to rings that tend to obey to quasi-rhombohedral symmetry. Merging may result either from close-association, hemifusion (one leaflet fusion) or from fusion (two leaflets fusion). Although the resolution of tomograms is not sufficient to distinguish these three cases, freeze-fracturing shows that hemifusion is a frequent process that leads to an reversible anastomosed membrane complex favoring communications, appearing as a major coupling factor of photosome light emission.
      Graphical abstract image

      PubDate: 2018-04-18T07:40:01Z
      DOI: 10.1016/j.jsb.2017.12.001
       
  • Structural changes of homodimers in the PDB
    • Authors: Ryotaro Koike; Takayuki Amemiya; Tatsuya Horii; Motonori Ota
      Pages: 42 - 50
      Abstract: Publication date: April 2018
      Source:Journal of Structural Biology, Volume 202, Issue 1
      Author(s): Ryotaro Koike, Takayuki Amemiya, Tatsuya Horii, Motonori Ota
      Protein complexes are involved in various biological phenomena. These complexes are intrinsically flexible, and structural changes are essential to their functions. To perform a large-scale automated analysis of the structural changes of complexes, we combined two original methods. An application, SCPC, compares two structures of protein complexes and decides the match of binding mode. Another application, Motion Tree, identifies rigid-body motions in various sizes and magnitude from the two structural complexes with the same binding mode. This approach was applied to all available homodimers in the Protein Data Bank (PDB). We defined two complex-specific motions: interface motion and subunit-spanning motion. In the former, each subunit of a complex constitutes a rigid body, and the relative movement between subunits occurs at the interface. In the latter, structural parts from distinct subunits constitute a rigid body, providing the relative movement spanning subunits. All structural changes were classified and examined. It was revealed that the complex-specific motions were common in the homodimers, detected in around 40% of families. The dimeric interfaces were likely to be small and flat for interface motion, while large and rugged for subunit-spanning motion. Interface motion was accompanied by a drastic change in contacts at the interface, while the change in the subunit-spanning motion was moderate. These results indicate that the interface properties of homodimers correlated with the type of complex-specific motion. The study demonstrates that the pipeline of SCPC and Motion Tree is useful for the massive analysis of structural change of protein complexes.

      PubDate: 2018-04-18T07:40:01Z
      DOI: 10.1016/j.jsb.2017.12.004
       
  • STEM tomography analysis of the trypanosome transition zone
    • Authors: Sylvain Trépout; Anne-Marie Tassin; Sergio Marco; Philippe Bastin
      Pages: 51 - 60
      Abstract: Publication date: April 2018
      Source:Journal of Structural Biology, Volume 202, Issue 1
      Author(s): Sylvain Trépout, Anne-Marie Tassin, Sergio Marco, Philippe Bastin
      The protist Trypanosoma brucei is an emerging model for the study of cilia and flagella. Here, we used scanning transmission electron microscopy (STEM) tomography to describe the structure of the trypanosome transition zone (TZ). At the base of the TZ, nine transition fibres irradiate from the B microtubule of each doublet towards the membrane. The TZ adopts a 9 + 0 structure throughout its length of ∼300 nm and its lumen contains an electron-dense structure. The proximal portion of the TZ has an invariant length of 150 nm and is characterised by a collarette surrounding the membrane and the presence of electron-dense material between the membrane and the doublets. The distal portion exhibits more length variation (from 55 to 235 nm) and contains typical Y-links. STEM analysis revealed a more complex organisation of the Y-links compared to what was reported by conventional transmission electron microscopy. Observation of the very early phase of flagellum assembly demonstrated that the proximal portion and the collarette are assembled early during construction. The presence of the flagella connector that maintains the tip of the new flagellum to the side of the old was confirmed and additional filamentous structures making contact with the membrane of the flagellar pocket were also detected. The structure and potential functions of the TZ in trypanosomes are discussed, as well as its mode of assembly.

      PubDate: 2018-04-18T07:40:01Z
      DOI: 10.1016/j.jsb.2017.12.005
       
  • Translocation of Epidermal Growth Factor (EGF) to the nucleus has distinct
           kinetics between adipose tissue-derived mesenchymal stem cells and a
           mesenchymal cancer cell lineage
    • Authors: Camila Cristina Fraga Faraco; Jerusa Araújo Quintão Arantes Faria; Marianna Kunrath-Lima; Marcelo Coutinho de Miranda; Mariane Izabella Abreu de Melo; Andrea da Fonseca Ferreira; Michele Angela Rodrigues; Dawidson Assis Gomes
      Pages: 61 - 69
      Abstract: Publication date: April 2018
      Source:Journal of Structural Biology, Volume 202, Issue 1
      Author(s): Camila Cristina Fraga Faraco, Jerusa Araújo Quintão Arantes Faria, Marianna Kunrath-Lima, Marcelo Coutinho de Miranda, Mariane Izabella Abreu de Melo, Andrea da Fonseca Ferreira, Michele Angela Rodrigues, Dawidson Assis Gomes
      Nuclear Epidermal Growth Factor Receptor (EGFR) has been associated with worse prognosis and treatment resistance for several cancer types. After Epidermal Growth Factor (EGF) binding, the ligand-receptor complex can translocate to the nucleus where it functions in oncological processes. By three-dimensional quantification analysis of super-resolution microscopy images, we verified the translocation kinetics of fluorescent conjugated EGF to the nucleus in two mesenchymal cell types: human adipose tissue-derived stem cells (hASC) and SK-HEP-1 tumor cells. The number of EGF clusters in the nucleus does not change after 10 min of stimulation with EGF in both cells. The total volume occupied by EGF clusters in the nucleus of hASC also does not change after 10 min of stimulation with EGF. However, the total volume of EGF clusters increases only after 20 min in SK-HEP-1 cells nuclei. In these cells the nuclear volume occupied by EGF is 3.2 times higher than in hASC after 20 min of stimulation, indicating that translocation kinetics of EGF differs between these two cell types. After stimulation, EGF clusters assemble in larger clusters in the cell nucleus in both cell types, which suggests specific sub-nuclear localizations of the receptor. Super-resolution microscopy images show that EGF clusters are widespread in the nucleoplasm, and can be localized in nuclear envelope invaginations, and in the nucleoli. The quantitative study of EGF-EGFR complex translocation to the nucleus may help to unravel its roles in health and pathological conditions, such as cancer.

      PubDate: 2018-04-18T07:40:01Z
      DOI: 10.1016/j.jsb.2017.12.007
       
  • Structural characterization of ribT from Bacillus subtilis reveals it as a
           GCN5-related N-acetyltransferase
    • Authors: Ritika Srivastava; Amanpreet Kaur; Charu Sharma; Subramanian Karthikeyan
      Pages: 70 - 81
      Abstract: Publication date: April 2018
      Source:Journal of Structural Biology, Volume 202, Issue 1
      Author(s): Ritika Srivastava, Amanpreet Kaur, Charu Sharma, Subramanian Karthikeyan
      In bacteria, biosynthesis of riboflavin occurs through a series of enzymatic steps starting with one molecule of GTP and two molecules of ribulose-5-phosphate. In Bacillus subtilis (B. subtilis) the genes (ribD/G, ribE, ribA, ribH and ribT) which are involved in riboflavin biosynthesis are organized in an operon referred as rib operon. All the genes of rib operon are characterized functionally except for ribT. The ribT gene with unknown function is found at the distal terminal of rib operon and annotated as a putative N-acetyltransferase. Here, we report the crystal structure of ribT from B. subtilis (bribT) complexed with coenzyme A (CoA) at 2.1 Å resolution determined by single wavelength anomalous dispersion method. Our structural study reveals that bribT is a member of GCN5-related N-acetyltransferase (GNAT) superfamily and contains all the four conserved structural motifs that have been in other members of GNAT superfamily. The members of GNAT family transfers the acetyl group from acetyl coenzyme A (AcCoA) to a variety of substrates. Moreover, the structural analysis reveals that the residues Glu-67 and Ser-107 are suitably positioned to act as a catalytic base and catalytic acid respectively suggesting that the catalysis by bribT may follow a direct transfer mechanism. Surprisingly, the mutation of a non-conserved amino acid residue Cys-112 to alanine or serine affected the binding of AcCoA to bribT, indicating a possible role of Cys-112 in the catalysis.

      PubDate: 2018-04-18T07:40:01Z
      DOI: 10.1016/j.jsb.2017.12.006
       
  • Crystal structure of cystathionine β-synthase from honeybee Apis
           mellifera
    • Authors: Paula Giménez-Mascarell; Tomas Majtan; Iker Oyenarte; June Ereño-Orbea; Juraj Majtan; Jaroslav Klaudiny; Jan P. Kraus; Luis Alfonso Martínez-Cruz
      Pages: 82 - 93
      Abstract: Publication date: April 2018
      Source:Journal of Structural Biology, Volume 202, Issue 1
      Author(s): Paula Giménez-Mascarell, Tomas Majtan, Iker Oyenarte, June Ereño-Orbea, Juraj Majtan, Jaroslav Klaudiny, Jan P. Kraus, Luis Alfonso Martínez-Cruz
      Cystathionine β-synthase (CBS), the key enzyme in the transsulfuration pathway, links methionine metabolism to the biosynthesis of cellular redox controlling molecules. CBS catalyzes the pyridoxal-5′-phosphate-dependent condensation of serine and homocysteine to form cystathionine, which is subsequently converted into cysteine. Besides maintaining cellular sulfur amino acid homeostasis, CBS also catalyzes multiple hydrogen sulfide-generating reactions using cysteine and homocysteine as substrates. In mammals, CBS is activated by S-adenosylmethionine (AdoMet), where it can adopt two different conformations (basal and activated), but exists as a unique highly active species in fruit fly Drosophila melanogaster. Here we present the crystal structure of CBS from honeybey Apis mellifera, which shows a constitutively active dimeric species and let explain why the enzyme is not allosterically regulated by AdoMet. In addition, comparison of available CBS structures unveils a substrate-induced closure of the catalytic cavity, which in humans is affected by the AdoMet-dependent regulation and likely impaired by the homocystinuria causing mutation T191M.

      PubDate: 2018-04-18T07:40:01Z
      DOI: 10.1016/j.jsb.2017.12.008
       
  • Building the atomic model of a boreal lake virus of unknown fold in a
           3.9 Å cryo-EM map
    • Authors: Luigi De Colibus; David I. Stuart
      Pages: 94 - 99
      Abstract: Publication date: April 2018
      Source:Journal of Structural Biology, Volume 202, Issue 1
      Author(s): Luigi De Colibus, David I. Stuart
      We report here the protocol adopted to build the atomic model of the newly discovered virus FLiP ( Flavobacterium infecting, lipid-containing phage) into 3.9 Å cryo-electron microscopy (cryo-EM) maps. In particular, this report discusses the combination of density modification procedures, automatic model building and bioinformatics tools applied to guide the tracing of the major capsid protein (MCP) of this virus. The protocol outlined here may serve as a reference for future structural determination by cryo-EM of viruses lacking detectable structural homologues.

      PubDate: 2018-04-18T07:40:01Z
      DOI: 10.1016/j.jsb.2017.10.010
       
  • New crystal forms of the integral membrane Escherichia coli
           quinol:fumarate reductase suggest that ligands control domain movement
    • Authors: C.A. Starbird; Thomas M. Tomasiak; Prashant K. Singh; Victoria Yankovskaya; Elena Maklashina; Michael Eisenbach; Gary Cecchini; T.M. Iverson
      Pages: 100 - 104
      Abstract: Publication date: April 2018
      Source:Journal of Structural Biology, Volume 202, Issue 1
      Author(s): C.A. Starbird, Thomas M. Tomasiak, Prashant K. Singh, Victoria Yankovskaya, Elena Maklashina, Michael Eisenbach, Gary Cecchini, T.M. Iverson
      Quinol:fumarate reductase (QFR) is an integral membrane protein and a member of the respiratory Complex II superfamily. Although the structure of Escherichia coli QFR was first reported almost twenty years ago, many open questions of catalysis remain. Here we report two new crystal forms of QFR, one grown from the lipidic cubic phase and one grown from dodecyl maltoside micelles. QFR crystals grown from the lipid cubic phase processed as P1, merged to 7.5 Å resolution, and exhibited crystal packing similar to previous crystal forms. Crystals grown from dodecyl maltoside micelles processed as P21, merged to 3.35 Å resolution, and displayed a unique crystal packing. This latter crystal form provides the first view of the E. coli QFR active site without a dicarboxylate ligand. Instead, an unidentified anion binds at a shifted position. In one of the molecules in the asymmetric unit, this is accompanied by rotation of the capping domain of the catalytic subunit. In the other molecule, this is associated with loss of interpretable electron density for this same capping domain. Analysis of the structure suggests that the ligand adjusts the position of the capping domain.
      Graphical abstract image

      PubDate: 2018-04-18T07:40:01Z
      DOI: 10.1016/j.jsb.2017.11.004
       
  • Journal of Structural Biology – Paper of the year 2017
    • Authors: Andrei N. Lupas; Alasdair C. Steven
      Pages: 185 - 186
      Abstract: Publication date: March 2018
      Source:Journal of Structural Biology, Volume 201, Issue 3
      Author(s): Andrei N. Lupas, Alasdair C. Steven


      PubDate: 2018-02-26T00:37:46Z
      DOI: 10.1016/j.jsb.2018.01.008
       
  • Open questions on the 3D structures of collagen containing vertebrate
           mineralized tissues: A perspective
    • Authors: Ron Shahar; Steve Weiner
      Pages: 187 - 198
      Abstract: Publication date: March 2018
      Source:Journal of Structural Biology, Volume 201, Issue 3
      Author(s): Ron Shahar, Steve Weiner
      Our current understanding of the structures of vertebrate mineralized tissues is largely based on light microscopy/histology and projections of 3D structures onto 2D planes using electron microscopy. We know little about the fine details of these structures in 3D at the length scales of their basic building blocks, the inherent variations of structure within a tissue and the cell-extracellular tissue interfaces. This limits progress in understanding tissue formation, relating structure to mechanical and metabolic functions, and obtaining deeper insights into pathologies and the evolution of these tissues. In this perspective we identify and discuss a series of open questions pertaining to collagen containing vertebrate mineralized tissues that can be addressed using appropriate 3D structural determination methods. By so doing we hope to encourage more research into the 3D structures of mineralized vertebrate tissues.

      PubDate: 2018-02-26T00:37:46Z
      DOI: 10.1016/j.jsb.2017.11.008
       
  • Identification of interfaces involved in weak interactions with
           application to F-actin-aldolase rafts
    • Authors: Guiqing Hu; Dianne W. Taylor; Jun Liu; Kenneth A. Taylor
      Pages: 199 - 209
      Abstract: Publication date: March 2018
      Source:Journal of Structural Biology, Volume 201, Issue 3
      Author(s): Guiqing Hu, Dianne W. Taylor, Jun Liu, Kenneth A. Taylor
      Macromolecular interactions occur with widely varying affinities. Strong interactions form well defined interfaces but weak interactions are more dynamic and variable. Weak interactions can collectively lead to large structures such as microvilli via cooperativity and are often the precursors of much stronger interactions, e.g. the initial actin-myosin interaction during muscle contraction. Electron tomography combined with subvolume alignment and classification is an ideal method for the study of weak interactions because a 3-D image is obtained for the individual interactions, which subsequently are characterized collectively. Here we describe a method to characterize heterogeneous F-actin-aldolase interactions in 2-D rafts using electron tomography. By forming separate averages of the two constituents and fitting an atomic structure to each average, together with the alignment information which relates the raw motif to the average, an atomic model of each crosslink is determined and a frequency map of contact residues is computed. The approach should be applicable to any large structure composed of constituents that interact weakly and heterogeneously.

      PubDate: 2018-02-26T00:37:46Z
      DOI: 10.1016/j.jsb.2017.11.005
       
  • Mapping of recent brachiopod microstructure: A tool for environmental
           studies
    • Authors: Facheng Ye; Gaia Crippa; Lucia Angiolini; Uwe Brand; GianCarlo Capitani; Maggie Cusack; Claudio Garbelli; Erika Griesshaber; Elizabeth Harper; Wolfgang Schmahl
      Pages: 221 - 236
      Abstract: Publication date: March 2018
      Source:Journal of Structural Biology, Volume 201, Issue 3
      Author(s): Facheng Ye, Gaia Crippa, Lucia Angiolini, Uwe Brand, GianCarlo Capitani, Maggie Cusack, Claudio Garbelli, Erika Griesshaber, Elizabeth Harper, Wolfgang Schmahl
      Shells of brachiopods are excellent archives for environmental reconstructions in the recent and distant past as their microstructure and geochemistry respond to climate and environmental forcings. We studied the morphology and size of the basic structural unit, the secondary layer fibre, of the shells of several extant brachiopod taxa to derive a model correlating microstructural patterns to environmental conditions. Twenty-one adult specimens of six recent brachiopod species adapted to different environmental conditions, from Antarctica, to New Zealand, to the Mediterranean Sea, were chosen for microstructural analysis using SEM, TEM and EBSD. We conclude that: 1) there is no significant difference in the shape and size of the fibres between ventral and dorsal valves, 2) there is an ontogenetic trend in the shape and size of the fibres, as they become larger, wider, and flatter with increasing age. This indicates that the fibrous layer produced in the later stages of growth, which is recommended by the literature to be the best material for geochemical analyses, has a different morphostructure and probably a lower organic content than that produced earlier in life. In two species of the same genus living in seawater with different temperature and carbonate saturation state, a relationship emerged between the microstructure and environmental conditions. Fibres of the polar Liothyrella uva tend to be smaller, rounder and less convex than those of the temperate Liothyrella neozelanica, suggesting a relationship between microstructural size, shell organic matter content, ambient seawater temperature and calcite saturation state.

      PubDate: 2018-02-26T00:37:46Z
      DOI: 10.1016/j.jsb.2017.11.011
       
  • Bone morphogenetic protein signaling through ACVR1 and BMPR1A negatively
           regulates bone mass along with alterations in bone composition
    • Authors: Ce Shi; Gurjit S. Mandair; Honghao Zhang; Gloria G. Vanrenterghem; Ryan Ridella; Akira Takahashi; Yanshuai Zhang; David H. Kohn; Michael D. Morris; Yuji Mishina; Hongchen Sun
      Pages: 237 - 246
      Abstract: Publication date: March 2018
      Source:Journal of Structural Biology, Volume 201, Issue 3
      Author(s): Ce Shi, Gurjit S. Mandair, Honghao Zhang, Gloria G. Vanrenterghem, Ryan Ridella, Akira Takahashi, Yanshuai Zhang, David H. Kohn, Michael D. Morris, Yuji Mishina, Hongchen Sun
      Bone quantity and bone quality are important factors in determining the properties and the mechanical functions of bone. This study examined the effects of disrupting bone morphogenetic protein (BMP) signaling through BMP receptors on bone quantity and bone quality. More specifically, we disrupted two BMP receptors, Acvr1 and Bmpr1a, respectively, in Osterix-expressing osteogenic progenitor cells in mice. We examined the structural changes to the femora from 3-month old male and female conditional knockout (cKO) mice using micro-computed tomography (micro-CT) and histology, as well as compositional changes to both cortical and trabecular compartments of bone using Raman spectroscopy. We found that the deletion of Acvr1 and Bmpr1a, respectively, in an osteoblast-specific manner resulted in higher bone mass in the trabecular compartment. Disruption of Bmpr1a resulted in a more significantly increased bone mass in the trabecular compartment. We also found that these cKO mice showed lower mineral-to-matrix ratio, while tissue mineral density was lower in the cortical compartment. Collagen crosslink ratio was higher in both cortical and trabecular compartments of male cKO mice. Our study suggested that BMP signaling in osteoblast mediated by BMP receptors, namely ACVR1 and BMPR1A, is critical in regulating bone quantity and bone quality.

      PubDate: 2018-02-26T00:37:46Z
      DOI: 10.1016/j.jsb.2017.11.010
       
  • Development of a new protein labeling system to map subunits and domains
           of macromolecular complexes for electron microscopy
    • Authors: Zuben P. Brown; Takao Arimori; Kenji Iwasaki; Junichi Takagi
      Pages: 247 - 251
      Abstract: Publication date: March 2018
      Source:Journal of Structural Biology, Volume 201, Issue 3
      Author(s): Zuben P. Brown, Takao Arimori, Kenji Iwasaki, Junichi Takagi
      Several gene fusion technologies have been successfully applied to label particular subunits or domains within macromolecular complexes to enable positional mapping of electron microscopy (EM) density maps, but exogenous fusion of a protein domain into the target polypeptide can cause unwanted structural and functional outcomes. Fab fragments from antibodies can be used as labeling reagents during EM visualization without gene manipulation of the target protein, but this method requires a panel of high-affinity antibodies that recognize a wide variety of epitopes. Linear peptide tags and their anti-tag antibodies can be used but they have a limited mapping ability as their placement is usually limited to the terminal regions of a protein. The PA dodecapeptide epitope tag (GVAMPGAEDDVV), forms a tight β-turn in the antigen binding pocket of its antibody (NZ-1). This capability allows for insertion of the PA tag into various surface-exposed loops within a multi-domain cell adhesion receptor, αIIbβ3 integrin. We confirmed that the purified PA-tagged integrin ectodomain fragments can form a stable complex with NZ-1 Fab. Negative stain EM of the various integrin-NZ-1 complexes revealed that a majority of the particles exhibited a clear density corresponding to the NZ-1 Fab; and the positions of the bound Fab were in good agreement with the predicted location of the inserted PA tag. The high-affinity and insertion-compatibility of the PA tag system allowed us to develop a new EM labeling methodology applicable to proteins for which good antibodies are not available.

      PubDate: 2018-02-26T00:37:46Z
      DOI: 10.1016/j.jsb.2017.11.006
       
  • MRCZ – A file format for cryo-TEM data with fast compression
    • Authors: Robert A. McLeod; Ricardo Diogo Righetto; Andy Stewart; Henning Stahlberg
      Pages: 252 - 257
      Abstract: Publication date: March 2018
      Source:Journal of Structural Biology, Volume 201, Issue 3
      Author(s): Robert A. McLeod, Ricardo Diogo Righetto, Andy Stewart, Henning Stahlberg
      The introduction of fast CMOS detectors is moving the field of transmission electron microscopy into the computer science field of big data. Automated data pipelines control the instrument and initial processing steps which imposes more onerous data transfer and archiving requirements. Here we conduct a technical demonstration whereby storage and read/write times are improved 10× at a dose rate of 1 e−/pix/frame for data from a Gatan K2 direct-detection device by combination of integer decimation and lossless compression. The example project is hosted at github.com/em-MRCZ and released under the BSD license.

      PubDate: 2018-02-26T00:37:46Z
      DOI: 10.1016/j.jsb.2017.11.012
       
  • New zonal structure and transition of the membrane to mammillae in the
           eggshell of chicken Gallus domesticus
    • Authors: Yunong Li; Yang Li; Shirong Liu; Yang Tang; Bing Mo; Hui Zhou
      Abstract: Publication date: Available online 30 April 2018
      Source:Journal of Structural Biology
      Author(s): Yunong Li, Yang Li, Shirong Liu, Yang Tang, Bing Mo, Hui Zhou
      Avian eggshell is a typical bio-engineered ceramics characterized by layer structures. These layers were divided mainly by the form of crystalline calcite. Whether there exist other layer structures, how the membrane layer was transformed to the carbonate one, what form the carbonate takes after the transition. These questions remain to be clarified. Here we examined the eggshell of chicken Gallus domesticus by optical microscope, scanning electron microscope and transmission electron microscope. We found that there exists another layer structure defined by variation of organic matrices. The transition between the membrane to the mammillary cones were realized through the calcium reserve assemblies or the mammillary cores. The strength of the transitional structure was weakened by removal of the calcium reserve, which was completed in about 10 days of incubation. As the first deposited carbonate layer after the transition, the mammillary cones were composed of amorphous calcium carbonate and clusters/assemblies of calcite crystallites the size about a nanometer, plus bubble pores extending preferentially in the lateral direction. Our results provided new insights into the structure and component of the avian eggshell, and may help decipher the constitution of the bio-ceramics in the perspective of material science.

      PubDate: 2018-05-02T08:18:34Z
      DOI: 10.1016/j.jsb.2018.04.006
       
  • Analytical symmetry detection in protein assemblies. I. Cyclic symmetries
    • Authors: Guillaume Pagès; Elvira Kinzina; Sergei Grudinin
      Abstract: Publication date: Available online 27 April 2018
      Source:Journal of Structural Biology
      Author(s): Guillaume Pagès, Elvira Kinzina, Sergei Grudinin
      Symmetry in protein, and, more generally, in macromolecular assemblies is a key point to understand their structure, stability and function. Many symmetrical assemblies are currently present in the Protein Data Bank (PDB) and some of them are among the largest solved structures, thus an efficient computational method is needed for the exhaustive analysis of these. The cyclic symmetry groups represent the most common assemblies in the PDB. These are also the building blocks for higher-order symmetries. This paper presents a mathematical formulation to find the position and the orientation of the symmetry axis in a cyclic symmetrical protein assembly, and also to assess the quality of this symmetry. Our method can also detect symmetries in partial assemblies. We provide an efficient C++ implementation of the method and demonstrate its efficiency on several examples including partial assemblies and pseudo symmetries. We also compare the method with two other published techniques and show that it is significantly faster on all the tested examples. Our method produces results with a machine precision, its cost function is solely based on 3D Euclidean geometry, and most of the operations are performed analytically. The method is available at http://team.inria.fr/nano-d/software/ananas. The graphical user interface of the method built for the SAMSON platform is available at http://samson-connect.net.
      Graphical abstract image

      PubDate: 2018-05-02T08:18:34Z
      DOI: 10.1016/j.jsb.2018.04.004
       
  • Human Skin Barrier Structure and Function Analyzed by Cryo-EM and
           Molecular Dynamics Simulation
    • Authors: Magnus Lundborg; Ali Narangifard; Christian L. Wennberg; Erik Lindahl; Bertil Daneholt; Lars Norlén
      Abstract: Publication date: Available online 24 April 2018
      Source:Journal of Structural Biology
      Author(s): Magnus Lundborg, Ali Narangifard, Christian L. Wennberg, Erik Lindahl, Bertil Daneholt, Lars Norlén
      In the present study we have analyzed the molecular structure and function of the human skin’s permeability barrier using molecular dynamics simulation validated against cryo-electron microscopy data from near native skin. The skin’s barrier capacity is located to an intercellular lipid structure embedding the cells of the superficial most layer of skin – the stratum corneum. According to the splayed bilayer model (Iwai et al., 2012) the lipid structure is organized as stacked bilayers of ceramides in a splayed chain conformation with cholesterol associated with the ceramide sphingoid moiety and free fatty acids associated with the ceramide fatty acid moiety. However, knowledge about the lipid structure’s detailed molecular organization, and the roles of its different lipid constituents, remains circumstantial. Starting from a molecular dynamics model based on the splayed bilayer model, we have, by stepwise structural and compositional modifications, arrived at a thermodynamically stable molecular dynamics model expressing simulated electron microscopy patterns matching original cryo-electron microscopy patterns from skin extremely closely. Strikingly, the closer the individual molecular dynamics models’ lipid composition was to that reported in human stratum corneum, the better was the match between the models’ simulated electron microscopy patterns and the original cryo-electron microscopy patterns. Moreover, the closest-matching model’s calculated water permeability and thermotropic behaviour were found compatible with that of human skin. The new model may facilitate more advanced physics-based skin permeability predictions of drugs and toxicants. The proposed procedure for molecular dynamics based analysis of cellular cryo-electron microscopy data might be applied to other biomolecular systems.
      Graphical abstract image

      PubDate: 2018-04-25T08:00:21Z
      DOI: 10.1016/j.jsb.2018.04.005
       
  • Image processing techniques for high-resolution structure determination
           from badly ordered 2D crystals
    • Authors: Nikhil Biyani; Sebastian Scherer; Ricardo D. Righetto; Julia Kowal; Mohamed Chami; Henning Stahlberg
      Abstract: Publication date: Available online 22 April 2018
      Source:Journal of Structural Biology
      Author(s): Nikhil Biyani, Sebastian Scherer, Ricardo D. Righetto, Julia Kowal, Mohamed Chami, Henning Stahlberg
      2D electron crystallography can be used to study small membrane proteins in their native environment. Obtaining highly ordered 2D crystals is difficult and time-consuming. However, 2D crystals diffracting to only 10-12 Å can be prepared relatively conveniently in most cases. We have developed image-processing algorithms allowing to generate a high resolution 3D structure from cryo-electron crystallography images of badly ordered crystals. These include movie-mode unbending, refinement over sub-tiles of the images in order to locally refine the sample tilt geometry, implementation of different CTF correction schemes, and an iterative method to apply known constraints in the real and reciprocal space to approximate amplitudes and phases in the so-called missing cone regions. These algorithms applied to a dataset of the potassium channel MloK1 show significant resolution improvements to better than 5Å.

      PubDate: 2018-04-25T08:00:21Z
      DOI: 10.1016/j.jsb.2018.03.013
       
  • Protein-Solvent Interfaces in Human Y145Stop Prion Protein Amyloid Fibrils
           Probed by Paramagnetic Solid-State NMR Spectroscopy
    • Authors: Darryl Aucoin; Yongjie Xia; Theint Theint; Philippe S. Nadaud; Krystyna Surewicz; Witold K. Surewicz; Christopher P. Jaroniec
      Abstract: Publication date: Available online 18 April 2018
      Source:Journal of Structural Biology
      Author(s): Darryl Aucoin, Yongjie Xia, Theint Theint, Philippe S. Nadaud, Krystyna Surewicz, Witold K. Surewicz, Christopher P. Jaroniec
      The C-terminally truncated Y145Stop variant of prion protein (PrP23-144), which is associated with heritable PrP cerebral amyloid angiopathy in humans and also capable of triggering a transmissible prion disease in mice, serves as a useful in vitro model for investigating the molecular and structural basis of amyloid strains and cross-seeding specificities. Here, we determine the protein-solvent interfaces in human PrP23-144 amyloid fibrils generated from recombinant 13C,15N-enriched protein and incubated in aqueous solution containing paramagnetic Cu(II)-EDTA, by measuring residue-specific 15N longitudinal paramagnetic relaxation enhancements using two-dimensional magic-angle spinning solid-state NMR spectroscopy. To further probe the interactions of the amyloid core residues with solvent molecules we perform complementary measurements of amide hydrogen/deuterium exchange detected by solid-state NMR and solution NMR methods. The solvent accessibility data are evaluated in the context of the structural model for human PrP23-144 amyloid.

      PubDate: 2018-04-18T07:40:01Z
      DOI: 10.1016/j.jsb.2018.04.002
       
  • Comparison of the 3D Structures of Mouse and Human α-Synuclein Fibrils by
           Solid-State NMR and STEM
    • Authors: Songhwan Hwang; Pascal Fricke; Maximilian Zinke; Karin Giller; Joseph S. Wall; Dietmar Riedel; Stefan Becker; Adam Lange
      Abstract: Publication date: Available online 17 April 2018
      Source:Journal of Structural Biology
      Author(s): Songhwan Hwang, Pascal Fricke, Maximilian Zinke, Karin Giller, Joseph S. Wall, Dietmar Riedel, Stefan Becker, Adam Lange
      Intra-neuronal aggregation of α-synuclein into fibrils is the molecular basis for α -synucleinopathies, such as Parkinson’s disease. The atomic structure of human α -synuclein (hAS) fibrils was recently determined by Tuttle et al. using solid-state NMR (ssNMR). The previous study found that hAS fibrils are composed of a single protofilament. Here, we have investigated the structure of mouse α -synuclein (mAS) fibrils by STEM and isotope-dilution ssNMR experiments. We found that in contrast to hAS, mAS fibrils consist of two or even three protofilaments which are connected by rather weak interactions in between them. Although the number of protofilaments appears to be different between hAS and mAS, we found that they have a remarkably similar secondary structure and protofilament 3D structure as judged by secondary chemical shifts and intra-molecular distance restraints. We conclude that the two mutant sites between hAS and mAS (positions 53 and 87) in the fibril core region are crucial for determining the quaternary structure of α -synuclein fibrils.

      PubDate: 2018-04-18T07:40:01Z
      DOI: 10.1016/j.jsb.2018.04.003
       
  • Delivery of femtolitre droplets using surface acoustic wave based
           atomisation for cryo-EM grid preparation
    • Authors: Dariush Ashtiani; Hari Venugopal; Matthew Belousoff; Bradley Spicer; Johnson Mak; Adrian Neild; Alex de Marco
      Abstract: Publication date: Available online 6 April 2018
      Source:Journal of Structural Biology
      Author(s): Dariush Ashtiani, Hari Venugopal, Matthew Belousoff, Bradley Spicer, Johnson Mak, Adrian Neild, Alex de Marco
      Cryo-Electron Microscopy (cryo-EM) has become an invaluable tool for structural biology. Over the past decade, the advent of direct electron detectors and automated data acquisition has established cryo-EM as a central method in structural biology. However, challenges remain in the reliable and efficient preparation of samples in a manner which is compatible with high time resolution. The delivery of sample onto the grid is recognized as a critical step in the workflow as it is a source of variability and loss of material due to the blotting which is usually required. Here, we present a method for sample delivery and plunge freezing based on the use of Surface Acoustic Waves to deploy 6-8 µm droplets to the EM grid. This method minimises the sample dead volume and ensures vitrification within 52.6 ms from the moment the sample leaves the microfluidics chip. We demonstrate a working protocol to minimize the atomised volume and apply it to plunge freeze three different samples and provide proof that no damage occurs due to the interaction between the sample and the acoustic waves.

      PubDate: 2018-04-18T07:40:01Z
      DOI: 10.1016/j.jsb.2018.03.012
       
  • Substrate-bound structures of a ketoreductase from amphotericin modular
           polyketide synthase
    • Authors: Chenguang Liu; Meijuan Yuan; Xu Xu; Lei Wang; Adrian T. Keatinge-Clay; Zixin Deng; Shuangjun Lin; Jianting Zheng
      Abstract: Publication date: Available online 4 April 2018
      Source:Journal of Structural Biology
      Author(s): Chenguang Liu, Meijuan Yuan, Xu Xu, Lei Wang, Adrian T. Keatinge-Clay, Zixin Deng, Shuangjun Lin, Jianting Zheng
      Ketoreductase (KR) domains of modular polyketide synthases (PKSs) control the stereochemistry of C2 methyl and C3 hydroxyl substituents of polyketide intermediates. To understand the molecular basis of stereocontrol exerted by KRs, the crystal structure of a KR from the second module of the amphotericin PKS (AmpKR2) complexed with NADP+ and 2-methyl-3-oxopentanoyl-pantetheine was solved. This first ternary structure provides direct evidence to the hypothesis that a substrate enters into the active site of an A-type KR from the side opposite the coenzyme to generate an L-hydroxyl substituent. A comparison with the ternary complex of a G355T/Q364H mutant sheds light on the structural basis for stereospecificity toward the substrate C2 methyl substituent. Functional assays suggest the pantetheine handle shows obvious influence on the catalytic efficiency and the stereochemical outcome. Together, these findings extend our current understanding of the stereochemical control of PKS KR domains.
      Graphical abstract image

      PubDate: 2018-04-18T07:40:01Z
      DOI: 10.1016/j.jsb.2018.04.001
       
  • Structural and functional analysis of Erwinia amylovora SrlD. The first
           crystal structure of a sorbitol-6-phosphate 2-dehydrogenase
    • Authors: Marco Salomone-Stagni; Joseph D. Bartho; Eeshan Kalita; Martin Rejzek; Robert A. Field; Dom Bellini; Martin A. Walsh; Stefano Benini
      Abstract: Publication date: Available online 29 March 2018
      Source:Journal of Structural Biology
      Author(s): Marco Salomone-Stagni, Joseph D. Bartho, Eeshan Kalita, Martin Rejzek, Robert A. Field, Dom Bellini, Martin A. Walsh, Stefano Benini
      Sorbitol-6-phosphate 2-dehydrogenases (S6PDH) catalyze the interconversion of d-sorbitol 6-phosphate to d-fructose 6-phosphate. In the plant pathogen Erwinia amylovora the S6PDH SrlD is used by the bacterium to utilize sorbitol, which is used for carbohydrate transport in the host plants belonging to the Amygdaloideae subfamily (e.g., apple, pear, and quince). We have determined the crystal structure of S6PDH SrlD at 1.84 Å resolution, which is the first structure of an EC 1.1.1.140 enzyme. Kinetic data show that SrlD is much faster at oxidizing d-sorbitol 6-phosphate than in reducing d-fructose 6-phosphate, however, equilibrium analysis revealed that only part of the d-sorbitol 6-phosphate present in the in vitro environment is converted into d-fructose 6-phosphate. The comparison of the structures of SrlD and Rhodobacter sphaeroides sorbitol dehydrogenase showed that the tetrameric quaternary structure, the catalytic residues and a conserved aspartate residue that confers specificity for NAD+ over NADP+ are preserved. Analysis of the SrlD cofactor and substrate binding sites identified residues important for the formation of the complex with cofactor and substrate and in particular the role of Lys42 in selectivity towards the phospho-substrate. The comparison of SrlD backbone with the backbone of 302 short-chain dehydrogenases/reductases showed the conservation of the protein core and identified the variable parts. The SrlD sequence was compared with 500 S6PDH sequences selected by homology revealing that the C-terminal part is more conserved than the N-terminal, the consensus of the catalytic tetrad (Y[SN]AGXA) and a not previously described consensus for the NAD(H) binding.

      PubDate: 2018-04-18T07:40:01Z
      DOI: 10.1016/j.jsb.2018.03.010
       
  • A new HIV-1 Rev structure optimizes interaction with target RNA (RRE) for
           nuclear export
    • Authors: Norman R. Watts; Elif Eren; Xiaolei Zhuang; Yun-Xing Wang; Alasdair C. Steven; Paul T. Wingfield
      Abstract: Publication date: Available online 29 March 2018
      Source:Journal of Structural Biology
      Author(s): Norman R. Watts, Elif Eren, Xiaolei Zhuang, Yun-Xing Wang, Alasdair C. Steven, Paul T. Wingfield
      HIV-1 Rev mediates the nuclear export of unspliced and partially-spliced viral transcripts for the production of progeny genomes and structural proteins. In this process, four (or more) copies of Rev assemble onto a highly-structured 351-nt region in such viral transcripts, the Rev response element (RRE). How this occurs is not known. The Rev assembly domain has a helical-hairpin structure which associates through three (A-A, B-B and C-C) interfaces. The RRE has the topology of an upper-case letter A, with the two known Rev binding sites mapping onto the legs of the A. We have determined a crystal structure for the Rev assembly domain at 2.25 Å resolution, without resort to either mutations or chaperones. It shows that B-B dimers adopt an arrangement reversed relative to that previously reported, and join through a C-C interface to form tetramers. The new subunit arrangement shows how four Rev molecules can assemble on the two sites on the RRE to form the specificity checkpoint, and how further copies add through A-A interactions. Residues at the C-C interface, specifically the Pro31-Trp45 axis, are a potential target for intervention.

      PubDate: 2018-04-18T07:40:01Z
      DOI: 10.1016/j.jsb.2018.03.011
       
  • Amyloid structure of high-order assembly of Leucine-rich amelogenin
           revealed by solid-state NMR
    • Authors: Cheng-Wei Ma; Jing Zhang; Xing-Qi Dong; Jun-Xia Lu
      Abstract: Publication date: Available online 28 March 2018
      Source:Journal of Structural Biology
      Author(s): Cheng-Wei Ma, Jing Zhang, Xing-Qi Dong, Jun-Xia Lu
      High-order assemblies of amelogenin, the major protein in enamel protein matrix, are believed to act as the template for enamel mineral formation. The Leucine-rich amelogenin (LRAP) is a natural splice-variant of amelogenin, a functional protein in vivo, containing conserved domains of amelogenin. In this work, we showed LRAP aggregates hierarchically into assemblies with various sizes including scattered beads, beads-on-a-string and gel-like precipitations in the presence of both calcium and phosphate ions. Solid-state NMR combined with X-ray diffraction and microscopic techniques, was applied to give a picture of LRAP self-assemblies at the atomic level. Our results, for the first time, confirmed LRAP assemblies with different sizes all contained a consistent rigid segment with β-sheet secondary structure (residues 12–27) and the β-sheet segment would further assemble into amyloid-like structures.

      PubDate: 2018-04-18T07:40:01Z
      DOI: 10.1016/j.jsb.2018.03.009
       
  • Structure and electrochemistry of proteins harboring iron-sulfur clusters
           of different nuclearities. Part III. [4Fe-4S], [3Fe-4S] and [2Fe-2S]
           iron-sulfur proteins
    • Authors: Piero Zanello
      Abstract: Publication date: Available online 27 March 2018
      Source:Journal of Structural Biology
      Author(s): Piero Zanello
      A systematic rationalization of the hundreds of proteins harboring iron-sulfur clusters and able to exhibit the most diverse biological functions is missing. In this picture we have already reviewed structure/electrochemistry of metalloproteins expressing single types of iron-sulfur centres [namely, {Fe(Cys)4}, {[Fe2S2](Cys)4}, {[Fe2S2](Cys)3(X)} (X = Asp, Arg, His), {[Fe2S2](Cys)2(His)2}, {[Fe3S4](Cys)3}, {[Fe4S4](Cys)4} and {[Fe4S4](Sγ Cys)3(nonthiolate ligand)}] and their synthetic analogs. Recently we are focussing on structure/electrochemistry of metalloproteins containing iron-sulfur centres of different nuclearities. Having started such a subject with proteins harboring [4Fe-4S] and [2Fe-2S] (Zanello, 2017c) as well as [4Fe-4S] and [3Fe-4S] (Zanello, in press) clusters, we now provide the state of art of proteins harboring [4Fe-4S], [3Fe-4S] and [2Fe-2S] clusters, a subject that resulted strictly limited to enzymes active in the respiratory Complex II.
      Graphical abstract image

      PubDate: 2018-04-18T07:40:01Z
      DOI: 10.1016/j.jsb.2018.03.008
       
  • Blind estimation of DED camera gain in Electron Microscopy
    • Authors: C.O.S. Sorzano; E. Fernández-Giménez; V. Peredo-Robinson; J. Vargas; T. Majtner; G. Caffarena; J. Otón; J.L. Vilas; J.M. de la Rosa-Trevín; R. Melero; J. Gómez-Blanco; J. Cuenca; L. del Cano; P. Conesa; R. Marabini; J.M. Carazo
      Abstract: Publication date: Available online 15 March 2018
      Source:Journal of Structural Biology
      Author(s): C.O.S. Sorzano, E. Fernández-Giménez, V. Peredo-Robinson, J. Vargas, T. Majtner, G. Caffarena, J. Otón, J.L. Vilas, J.M. de la Rosa-Trevín, R. Melero, J. Gómez-Blanco, J. Cuenca, L. del Cano, P. Conesa, R. Marabini, J.M. Carazo
      The introduction of Direct Electron Detector (DED) videos in the Electron Microscope field has boosted Single Particle Analysis to a point in which it is currently considered to be a key technique in Structural Biology. In this article we introduce an approach to estimate the DED camera gain at each pixel from the movies themselves. This gain is needed to have the set of recorded frames into a coherent gray level range, homogeneous over the whole image. The algorithm does not need any other input than the DED movie itself, being capable of providing an estimate of the camera gain image, helping to identify dead pixels and cases of incorrectly calibrated cameras. We propose the algorithm to be used either to validate the experimentally acquired gain image (for instance, to follow its possible change over time) or to verify that there is no residual gain image after experimentally correcting for the camera gain. We show results for a number of DED camera models currently in use (DE, Falcon II, Falcon 3, and K2).

      PubDate: 2018-04-18T07:40:01Z
      DOI: 10.1016/j.jsb.2018.03.007
       
  • Structure of a prokaryotic SEFIR domain reveals two novel SEFIR-SEFIR
           interaction modes
    • Authors: Hui Yang; Yun Zhu; Xing Chen; Xiaoxia Li; Sheng Ye; Rongguang Zhang
      Abstract: Publication date: Available online 14 March 2018
      Source:Journal of Structural Biology
      Author(s): Hui Yang, Yun Zhu, Xing Chen, Xiaoxia Li, Sheng Ye, Rongguang Zhang
      SEFIR domain-containing proteins are crucial for mammalian adaptive immunity. As a unique intracellular signaling domain, the SEFIR-SEFIR interactions mediate physical protein-protein interactions in the immune signaling network, especially the IL-17- and IL-25-mediated pathways. However, due to the lack of structural information, the detailed molecular mechanism for SEFIR-SEFIR assembly remains unclear. In the present study, we solved the crystal structures of a prokaryotic SEFIR domain from Bacillus cereus F65185 (BcSEFIR), where the SEFIR domain is located at the N terminus. The structure of BcSEFIR revealed two radically distinct SEFIR-SEFIR interaction modes. In the asymmetric form, the C-terminal tail of one SEFIR binds to the helix αA and βB–αB′ segment of the other one, while in the symmetric form, the helices ηC and αE and the DE-segment compose the inter-protomer interface. The C-terminal tail of BcSEFIR, critical for asymmetric interaction, is highly conserved among the SEFIR domains of Act1 orthologs from different species, in particular three absolutely conserved residues that constitute an EXXXXPP motif. In the symmetric interaction mode, the most significant contacts made by residues on helix αE are highly conserved in Act1 SEFIR domains, constituted an RLI/LXE motif. The two novel SEFIR-SEFIR interaction modes might explain the structural basis for SEFIR domain-mediated complex assembly in signaling pathways.

      PubDate: 2018-04-18T07:40:01Z
      DOI: 10.1016/j.jsb.2018.03.005
       
  • Baculovirus-driven protein expression in insect cells: A benchmarking
           study
    • Authors: Peggy Stolt-Bergner; Christian Benda; Tim Bergbrede; Hüseyin Besir; Patrick H.N. Celie; Cindy Chang; David Drechsel; Ariane Fischer; Arie Geerlof; Barbara Giabbai; Joop van den Heuvel; Georg Huber; Wolfgang Knecht; Anita Lehner; Regis Lemaitre; Kristina Nordén; Gwynn Pardee; Ines Racke; Kim Remans; Astrid Sander; Judith Scholz; Magda Stadnik; Paola Storici; Daniel Weinbruch; Isabel Zaror; Linda H.L. Lua; Sabine Suppmann
      Abstract: Publication date: Available online 12 March 2018
      Source:Journal of Structural Biology
      Author(s): Peggy Stolt-Bergner, Christian Benda, Tim Bergbrede, Hüseyin Besir, Patrick H.N. Celie, Cindy Chang, David Drechsel, Ariane Fischer, Arie Geerlof, Barbara Giabbai, Joop van den Heuvel, Georg Huber, Wolfgang Knecht, Anita Lehner, Regis Lemaitre, Kristina Nordén, Gwynn Pardee, Ines Racke, Kim Remans, Astrid Sander, Judith Scholz, Magda Stadnik, Paola Storici, Daniel Weinbruch, Isabel Zaror, Linda H.L. Lua, Sabine Suppmann
      Baculovirus-insect cell expression system has become one of the most widely used eukaryotic expression systems for heterologous protein production in many laboratories. The availability of robust insect cell lines, serum-free media, a range of vectors and commercially-packaged kits have supported the demand for maximizing the exploitation of the baculovirus-insect cell expression system. Naturally, this resulted in varied strategies adopted by different laboratories to optimize protein production. Most laboratories have preference in using either the E. coli transposition-based recombination bacmid technology (e.g. Bac-to-Bac®) or homologous recombination transfection within insect cells (e.g. flashBAC™). Limited data is presented in the literature to benchmark the protocols used for these baculovirus vectors to facilitate the selection of a system for optimal production of target proteins. Taking advantage of the Protein Production and Purification Partnership in Europe (P4EU) scientific network, a benchmarking initiative was designed to compare the diverse protocols established in thirteen individual laboratories. This benchmarking initiative compared the expression of four selected intracellular proteins (mouse Dicer-2, 204 kDa; human ABL1 wildtype, 126 kDa; human FMRP, 68 kDa; viral vNS1-H1, 76 kDa). Here, we present the expression and purification results on these proteins and highlight the significant differences in expression yields obtained using different commercially-packaged baculovirus vectors. The highest expression level for difficult-to-express intracellular protein candidates were observed with the EmBacY baculovirus vector system.

      PubDate: 2018-04-18T07:40:01Z
      DOI: 10.1016/j.jsb.2018.03.004
       
  • Solution structure of an archaeal DUF61 family protein SSO0941 encoded by
           a gene in the operon of box C/D RNA protein complexes
    • Authors: Tao Zhou; Xingzhe Yao; Jinfeng Wang; Yingang Feng
      Abstract: Publication date: Available online 8 March 2018
      Source:Journal of Structural Biology
      Author(s): Tao Zhou, Xingzhe Yao, Jinfeng Wang, Yingang Feng
      Domain of unknown function 61 (DUF61) family proteins widely exist in archaea and the genes of DUF61 proteins in crenarchaea are in an operon containing two genes of box C/D RNA protein complexes. Here we report the solution NMR structure of DUF61 family member protein SSO0941, from the hyperthermophilic archaeon Sulfolobus solfataricus. SSO0941 has a rigid core structure and flexible N- and C-terminal regions as well as a negatively-charged independent C-terminal helix. The core structure consists of N- and C-terminal subdomains, in which the C-terminal subdomain shows significant structural similarity with several nucleic acid binding proteins. The structure of SSO0941 is the first representative structure of DUF61 family proteins.
      Graphical abstract image

      PubDate: 2018-04-18T07:40:01Z
      DOI: 10.1016/j.jsb.2018.03.003
       
  • Gaussian-input Gaussian mixture model for representing density maps and
           atomic models
    • Authors: Takeshi Kawabata
      Abstract: Publication date: Available online 6 March 2018
      Source:Journal of Structural Biology
      Author(s): Takeshi Kawabata
      A new Gaussian mixture model (GMM) has been developed for better representations of both atomic models and electron microscopy 3D density maps. The standard GMM algorithm employs an EM algorithm to determine the parameters. It accepted a set of 3D points with weights, corresponding to voxel or atomic centers. Although the standard algorithm worked reasonably well; however, it had three problems. First, it ignored the size (voxel width or atomic radius) of the input, and thus it could lead to a GMM with a smaller spread than the input. Second, the algorithm had a singularity problem, as it sometimes stopped the iterative procedure due to a Gaussian function with almost zero variance. Third, a map with a large number of voxels required a long computation time for conversion to a GMM. To solve these problems, we have introduced a Gaussian-input GMM algorithm, which considers the input atoms or voxels as a set of Gaussian functions. The standard EM algorithm of GMM was extended to optimize the new GMM. The new GMM has identical radius of gyration to the input, and does not suddenly stop due to the singularity problem. For fast computation, we have introduced a down-sampled Gaussian functions (DSG) by merging neighboring voxels into an anisotropic Gaussian function. It provides a GMM with thousands of Gaussian functions in a short computation time. We also have introduced a DSG-input GMM: the Gaussian-input GMM with the DSG as the input. This new algorithm is much faster than the standard algorithm.

      PubDate: 2018-04-18T07:40:01Z
      DOI: 10.1016/j.jsb.2018.03.002
       
  • Structural basis for partition of the cyclodipeptide synthases into two
           subfamilies
    • Authors: Gabrielle Bourgeois; Jérôme Seguin; Morgan Babin; Pascal Belin; Mireille Moutiez; Yves Mechulam; Muriel Gondry; Emmanuelle Schmitt
      Abstract: Publication date: Available online 2 March 2018
      Source:Journal of Structural Biology
      Author(s): Gabrielle Bourgeois, Jérôme Seguin, Morgan Babin, Pascal Belin, Mireille Moutiez, Yves Mechulam, Muriel Gondry, Emmanuelle Schmitt
      Cyclodipeptide synthases (CDPSs) use two aminoacyl-tRNAs to catalyze the formation of two peptide bonds leading to cyclodipeptides that can be further used for the synthesis of diketopiperazines. It was shown that CDPSs fall into two subfamilies, NYH and XYP, characterized by the presence of specific sequence signatures. However, current understanding of CDPSs only comes from studies of enzymes from the NYH subfamily. The present study reveals the crystal structures of three CDPSs from the XYP subfamily. Comparison of the XYP and NYH enzymes shows that the two subfamilies mainly differ in the first half of their Rossmann fold. This gives a structural basis for the partition of CDPSs into two subfamilies. Despite these differences, the catalytic residues adopt similar positioning regardless of the subfamily suggesting that the XYP and NYH motifs correspond to two structural solutions to facilitate the reactivity of the catalytic serine residue.
      Graphical abstract image

      PubDate: 2018-04-18T07:40:01Z
      DOI: 10.1016/j.jsb.2018.03.001
       
  • αCGRP, another amyloidogenic member of the CGRP family
    • Authors: Paraskevi L. Tsiolaki; Georgia I. Nasi; Fotis A. Baltoumas; Nikolaos N. Louros; Vassiliki Magafa; Stavros J. Hamodrakas; Vassiliki A. Iconomidou
      Abstract: Publication date: Available online 1 March 2018
      Source:Journal of Structural Biology
      Author(s): Paraskevi L. Tsiolaki, Georgia I. Nasi, Fotis A. Baltoumas, Nikolaos N. Louros, Vassiliki Magafa, Stavros J. Hamodrakas, Vassiliki A. Iconomidou
      The Calcitonin-gene related peptide (CGRP) family is a group of peptide hormones, which consists of IAPP, calcitonin, adrenomedullin, intermedin, αCGRP and βCGRP. IAPP and calcitonin have been extensively associated with the formation of amyloid fibrils, causing Type 2 Diabetes and Medullary Thyroid Carcinoma, respectively. In contrast, the potential amyloidogenic properties of αCGRP still remain unexplored, although experimental trials have indicated its presence in deposits, associated with the aforementioned disorders. Therefore, in this work, we investigated the amyloidogenic profile of αCGRP, a 37-residue-long peptide hormone, utilizing both biophysical experimental techniques and Molecular Dynamics simulations. These efforts unravel a novel amyloidogenic member of the CGRP family and provide insights into the mechanism underlying the αCGRP polymerization.

      PubDate: 2018-04-18T07:40:01Z
      DOI: 10.1016/j.jsb.2018.02.008
       
  • Allosteric effects in bacteriophage HK97 procapsids revealed directly from
           covariance analysis of cryo EM data
    • Authors: Nan Xu; David Veesler; Peter C. Doerschuk; John E. Johnson
      Abstract: Publication date: Available online 10 January 2018
      Source:Journal of Structural Biology
      Author(s): Nan Xu, David Veesler, Peter C. Doerschuk, John E. Johnson
      The information content of cryo EM data sets exceeds that of the electron scattering potential (cryo EM) density initially derived for structure determination. Previously we demonstrated the power of data variance analysis for characterizing regions of cryo EM density that displayed functionally important variance anomalies associated with maturation cleavage events in Nudaurelia Omega Capensis Virus and the presence or absence of a maturation protease in bacteriophage HK97 procapsids. Here we extend the analysis in two ways. First, instead of imposing icosahedral symmetry on every particle in the data set during the variance analysis, we only assume that the data set as a whole has icosahedral symmetry. This change removes artifacts of high variance along icosahedral symmetry axes, but retains all of the features previously reported in the HK97 data set. Second we present a covariance analysis that reveals correlations in structural dynamics (variance) between the interior of the HK97 procapsid with the protease and regions of the exterior (not seen in the absence of the protease). The latter analysis corresponds well with hydrogen deuterium exchange studies previously published that reveal the same correlation.

      PubDate: 2018-01-14T02:41:59Z
      DOI: 10.1016/j.jsb.2017.12.013
       
  • Structure of the Bacillus anthracis dTDP-l-rhamnose biosynthetic pathway
           enzyme: dTDP-α-d-glucose 4,6-dehydratase, RfbB
    • Authors: Trevor Gokey; Andrei S. Halavaty; George Minasov; Wayne F. Anderson; Misty L. Kuhn
      Abstract: Publication date: Available online 10 January 2018
      Source:Journal of Structural Biology
      Author(s): Trevor Gokey, Andrei S. Halavaty, George Minasov, Wayne F. Anderson, Misty L. Kuhn
      Many bacteria require l-rhamnose as a key cell wall component. This sugar is transferred to the cell wall using an activated donor dTDP-l-rhamnose, which is produced by the dTDP-l-rhamnose biosynthetic pathway. We determined the crystal structure of the second enzyme of this pathway dTDP-α-d-glucose 4,6-dehydratase (RfbB) from Bacillus anthracis. Interestingly, RfbB only crystallized in the presence of the third enzyme of the pathway RfbC; however, RfbC was not present in the crystal. Our work represents the first complete structural characterization of the four proteins of this pathway in a single Gram-positive bacterium.

      PubDate: 2018-01-14T02:41:59Z
      DOI: 10.1016/j.jsb.2018.01.006
       
  • Optimizing “Self-Wicking” Nanowire Grids
    • Authors: Hui Wei; Venkata P. Dandey; Zhening Zhang; Ashleigh Raczkowski; Willam J. Rice; Bridget Carragher; Clinton S. Potter
      Abstract: Publication date: Available online 6 January 2018
      Source:Journal of Structural Biology
      Author(s): Hui Wei, Venkata P. Dandey, Zhening Zhang, Ashleigh Raczkowski, Willam J. Rice, Bridget Carragher, Clinton S. Potter
      We have developed a self-blotting TEM grid for use with a novel instrument for vitrifying samples for cryo-electron microscopy (cryoEM). Nanowires are grown on the copper surface of the grid using a simple chemical reaction and the opposite smooth side is used to adhere to a holey sample substrate support, for example carbon or gold. When small volumes of sample are applied to the nanowire grids the wires effectively act as blotting paper to rapidly wick away the liquid, leaving behind a thin film. In this technical note, we present a detailed description of how we make these grids using a variety of substrates fenestrated with either lacey or regularly spaced holes. We explain how we characterize the quality of the grids and we describe their behavior under a variety of conditions.

      PubDate: 2018-01-14T02:41:59Z
      DOI: 10.1016/j.jsb.2018.01.001
       
  • Comparative structural and enzymatic studies on Salmonella typhimurium
           diaminopropionate ammonia lyase reveal its unique features
    • Authors: G. Deka; S. Bisht; H.S. Savithri; M.R.N. Murthy
      Abstract: Publication date: Available online 30 December 2017
      Source:Journal of Structural Biology
      Author(s): G. Deka, S. Bisht, H.S. Savithri, M.R.N. Murthy
      Cellular metabolism of amino acids is controlled by a large number of pyridoxal 5′-phosphate (PLP) dependent enzymes. Diaminopropionate ammonia lyase (DAPAL), a fold type II PLP-dependent enzyme, degrades both the D and L forms of diaminopropionic acid (DAP) to pyruvate and ammonia. Earlier studies on the Escherichia coli DAPAL (EcDAPAL) had suggested that a disulfide bond located close to the active site may be crucial for maintaining the geometry of the substrate entry channel and the active site. In order to obtain further insights into the catalytic properties of DAPAL, structural and functional studies on Salmonella typhimurium DAPAL (StDAPAL) were initiated. The three-dimensional X-ray crystal structure of StDAPAL was determined at 2.5 Å resolution. As expected, the polypeptide fold and dimeric organization of StDAPAL is similar to those of EcDAPAL. A phosphate group was located in the active site of StDAPAL and expulsion of this phosphate is probably essential to bring Asp125 to a conformation suitable for proton abstraction from the substrate (D-DAP). The unique disulfide bond of EcDAPAL was absent in StDAPAL, although the enzyme displayed comparable catalytic activity. Site directed mutagenesis of the cysteine residues involved in disulfide bond formation in EcDAPAL followed by functional and biophysical studies further confirmed that the disulfide bond is not necessary either for substrate binding or for catalysis. The activity of StDAPAL but not EcDAPAL was enhanced by monovalent cations suggesting subtle differences in the active site geometries of these two closely related enzymes.

      PubDate: 2018-01-14T02:41:59Z
      DOI: 10.1016/j.jsb.2017.12.012
       
  • A convolutional autoencoder approach for mining features in cellular
           electron cryo-tomograms and weakly supervised coarse segmentation
    • Authors: Xiangrui Zeng; Miguel Ricardo Leung; Tzviya Zeev-Ben-Mordehai; Min Xu
      Abstract: Publication date: Available online 28 December 2017
      Source:Journal of Structural Biology
      Author(s): Xiangrui Zeng, Miguel Ricardo Leung, Tzviya Zeev-Ben-Mordehai, Min Xu
      Cellular electron cryo-tomography enables the 3D visualization of cellular organization in the near-native state and at submolecular resolution. However, the contents of cellular tomograms are often complex, making it difficult to automatically isolate different in situ cellular components. In this paper, we propose a convolutional autoencoder-based unsupervised approach to provide a coarse grouping of 3D small subvolumes extracted from tomograms. We demonstrate that the autoencoder can be used for efficient and coarse characterization of features of macromolecular complexes and surfaces, such as membranes. In addition, the autoencoder can be used to detect non-cellular features related to sample preparation and data collection, such as carbon edges from the grid and tomogram boundaries. The autoencoder is also able to detect patterns that may indicate spatial interactions between cellular components. Furthermore, we demonstrate that our autoencoder can be used for weakly supervised semantic segmentation of cellular components, requiring a very small amount of manual annotation.

      PubDate: 2018-01-14T02:41:59Z
      DOI: 10.1016/j.jsb.2017.12.015
       
  • A novel small molecule displays two different binding modes during
           inhibiting H1N1 influenza A virus neuraminidases
    • Authors: Shanshan Guan; Yan Xu; Yongbo Qiao; Ziyu Kuai; Mengdan Qian; Xiaoyu Jiang; Song Wang; Hao Zhang; Wei Kong; Yaming Shan
      Abstract: Publication date: Available online 28 December 2017
      Source:Journal of Structural Biology
      Author(s): Shanshan Guan, Yan Xu, Yongbo Qiao, Ziyu Kuai, Mengdan Qian, Xiaoyu Jiang, Song Wang, Hao Zhang, Wei Kong, Yaming Shan
      Neuraminidase (NA) inhibitors can suppress NA activity to block the release of progeny virions and are effective against influenza viruses. As potential anti-flu drugs with unique functions, NA inhibitors are greatly concerned by the worldwide scientists. It has been reported recently that one of the novel quindoline derivatives named 7a, could inhibit both A/Puerto Rico/8/34 (H1N1) NA (NAPR) and A/California/04/09 (H1N1) NA (NACA). However, potential structure differences in the active site could be easily detected between the NAPR and NACA according to the flexibilities of their 150-loops located catalytic site. And no obvious 150-cavity could be observed in NACA crystal structure. In order to explore whether 7a could trigger the inhibition against these two NAs in the same way, a serial molecular dynamics simulation approach were applied in this study. The results indicated that 7a could be adopted under a relatively extended pose in the active center of NAPR. While in NACA-7a complex, the derivate preferred to be recognized and located on the side of active center. Interestingly, the potential of 7a was also found to be able to change the flexibility of the 150-loop in NACA that is absent of 150-cavity. Furthermore, a 150-cavity-like architecture could be induced in the active site of NACA. The results of this study revealed two kinds of binding modes of this novel small molecule inhibitor against NAs that might provide a theoretical basis for proposing novel inhibition mechanism and developing future influenza A virus inhibitors.
      Graphical abstract image

      PubDate: 2018-01-14T02:41:59Z
      DOI: 10.1016/j.jsb.2017.12.014
       
  • High resolution crystal structures of Clostridium botulinum neurotoxin A3
           and A4 binding domains
    • Authors: Jonathan R. Davies; Jay Rees; Sai Man Liu; K. Ravi Acharya
      Abstract: Publication date: Available online 26 December 2017
      Source:Journal of Structural Biology
      Author(s): Jonathan Davies, Jay Rees, Sai Man Liu, K. Ravi Acharya
      Clostridium botulinum neurotoxins (BoNTs) cause the life-threatening condition, botulism. However, while they have the potential to cause serious harm, they are increasingly being utilised for therapeutic applications. BoNTs comprise of seven distinct serotypes termed BoNT/A through BoNT/G, with the most widely characterised being sub-serotype BoNT/A1. Each BoNT consists of three structurally distinct domains, a binding domain (HC), a translocation domain (HN), and a proteolytic domain (LC). The HC domain is responsible for the highly specific targeting of the neurotoxin to neuronal cell membranes. Here, we present two high-resolution structures of the binding domain of subtype BoNT/A3 (HC/A3) and BoNT/A4 (HC/A4) at 1.6 Å and 1.34 Å resolution, respectively. The structures of both proteins share a high degree of similarity to other known BoNT HC domains whilst containing some subtle differences, and are of benefit to research into therapeutic neurotoxins with novel characteristics.

      PubDate: 2017-12-27T04:45:50Z
      DOI: 10.1016/j.jsb.2017.12.010
       
  • Structural Characterization of SpoIIIAB Sporulation-essential Protein in
           Bacillus subtilis
    • Authors: N. Zeytuni; K.A. Flanagan; L.J. Worrall; S.C. Massoni; A.H. Camp; N.C.J. Strynadka
      Abstract: Publication date: Available online 26 December 2017
      Source:Journal of Structural Biology
      Author(s): N. Zeytuni, K.A. Flanagan, L.J. Worrall, S.C. Massoni, A.H. Camp, N.C.J. Strynadka
      Endospore formation in the Gram-positive bacterium Bacillus subtilis initiates in response to nutrient depletion and involves a series of morphological changes that result in the creation of a dormant spore. Early in this developmental process, the cell undergoes an asymmetric cell division that produces the larger mother cell and smaller forespore, the latter destined to become the mature spore. The mother cell septal membrane then engulfs the forespore, at which time an essential channel, the so-called feeding-tube apparatus, is thought to cross both membranes to create a direct conduit between the cells. At least nine proteins are required to form this channel including SpoIIQ under forespore control and SpoIIIAA-AH under the mother cell control. Several of these proteins share similarity to components of Type-II, -III and -IV secretion systems as well as the flagellum from Gram-negative bacteria. Here we report the X-ray crystallographic structure of the cytosolic domain of SpoIIIAB to 2.3 Å resolution. This domain adopts a conserved, secretion-system related fold of a six membered anti-parallel helical bundle with a positively charged membrane-interaction face at one end and a small groove at the other end that may serve as a binding site for partner proteins in the assembled apparatus. We analyzed and identified potential interaction interfaces by structure-guided mutagenesis in vivo. Furthermore, we were able to identify a remarkable structural homology to the C-subunit of a bacterial V-ATPase. Collectively, our data provides new insight into the possible roles of SpoIIIAB protein within the secretion-like apparatus essential to bacterial sporulation.

      PubDate: 2017-12-27T04:45:50Z
      DOI: 10.1016/j.jsb.2017.12.009
       
 
 
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