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Mohamed Fathi, Adel R Moawad, Magdy R Badr
Cryopreservation of oocytes would serve as an alternative to overcome the limited availability of dromedary camel oocytes and facilitate improvements in IVP techniques in this species. Our goal was to develop a protocol for the vitrification of camel oocytes at the germinal vesicle (GV) stage using different cryoprotectant combinations: 20% EG and 20% DMSO (VS1), 25% EG plus 25% DMSO (VS2) or 25% EG and 25% glycerol (VS3) and various cryo-carriers; straws or open pulled-straw (OPS) or solid surface vitrification (SSV); and Cryotop...
2018: PloS One
Michael E Paulaitis, Kitty Agarwal, Serge Patrick Nana-Sinkam
A model is proposed for characterizing exosome size distributions based on dynamic scaling of domain growth on the limiting membrane of multivesicular bodies in the established exosome biogenesis pathway. The scaling exponent in this model captures the asymmetry of exosome size distributions, which are notably right-skewed to larger vesicles independent of the minimum detectable vesicle size. Analyses of exosome size distributions obtained by cryo-TEM imaging and nanoparticle tracking (NTA) show, respectively, that the scaling exponent is sensitive to the state of the cell source for exosomes in cell culture supernatants, and can distinguish exosome size distributions in serum samples taken from cancer patients relative to those from healthy donors...
March 15, 2018: Langmuir: the ACS Journal of Surfaces and Colloids
Zhe Du, Shujun Wang, Bing Yue, Ying Wang, You Wang
T-cells, second only to macrophages, are often considered as the potential cells involved in debris-related failure of arthroplasty. Here, we assessed the effects of particulate wear debris on T-cells and inflammatory reactions. Blood samples from 25 donors were incubated with polyether-ether-ketone (PEEK) and cobalt-chromium-molybdenum (CoCrMo) particles generated by custom cryo-milling and pulverization. The T-cell phenotypes were assessed using immunostaining and flow cytometry. For the in vivo study, 0...
February 16, 2018: Oncotarget
Verity A Jackson, Dimphna H Meijer, Maria Carrasquero, Laura S van Bezouwen, Edward D Lowe, Colin Kleanthous, Bert J C Janssen, Elena Seiradake
Teneurins are ancient cell-cell adhesion receptors that are vital for brain development and synapse organisation. They originated in early metazoan evolution through a horizontal gene transfer event when a bacterial YD-repeat toxin fused to a eukaryotic receptor. We present X-ray crystallography and cryo-EM structures of two Teneurins, revealing a ~200 kDa extracellular super-fold in which eight sub-domains form an intricate structure centred on a spiralling YD-repeat shell. An alternatively spliced loop, which is implicated in homophilic Teneurin interaction and specificity, is exposed and thus poised for interaction...
March 14, 2018: Nature Communications
Tomohiro Kubo, Yuqing Hou, Deborah A Cochran, George B Witman, Toshiyuki Oda
Motility of cilia/flagella is generated by a coordinated activity of thousands of dyneins. Inner dynein arms (IDAs) are particularly important for the formation of ciliary/flagellar waveforms, but the molecular mechanism of IDA regulation is poorly understood. Here, we show using cryo-electron tomography and biochemical analyses of Chlamydomonas flagella that a conserved protein FAP44 forms a complex that tethers IDA f (I1 dynein) head domains to the A-tubule of the axonemal outer doublet microtubule. In wild-type flagella, IDA f showed little nucleotide-dependent movement except for a tilt in the f β head perpendicular to the microtubule-sliding direction...
March 14, 2018: Molecular Biology of the Cell
Nazario Martin, Alicia López-Andarias, Javier López-Andarias, Carmen Atienza, Francisco J Chichón, José L Carrascosa
Supramolecular chemistry has evolved from the traditional focus on thermodynamic on-pathways to the complex study of kinetic off-pathways, which are strongly dependent of the environmental conditions. Moreover, the control over pathway complexity allows obtaining nanostructures that are inaccessible through spontaneous thermodynamic processes. Here, we present a family of peptide-based π-extended tetrathiafulvalene (exTTF) molecules which show two self-assembly pathways leading to two distinct J-aggregates, namely metastable (M) and thermodynamic (T), with different spectroscopic, chiroptical and electrochemical behavior...
March 14, 2018: Chemistry: a European Journal
Jonathan M Grimes, David R Hall, Alun W Ashton, Gwyndaf Evans, Robin L Owen, Armin Wagner, Katherine E McAuley, Frank von Delft, Allen M Orville, Thomas Sorensen, Martin A Walsh, Helen M Ginn, David I Stuart
Macromolecular crystallography (MX) has been a motor for biology for over half a century and this continues apace. A series of revolutions, including the production of recombinant proteins and cryo-crystallography, have meant that MX has repeatedly reinvented itself to dramatically increase its reach. Over the last 30 years synchrotron radiation has nucleated a succession of advances, ranging from detectors to optics and automation. These advances, in turn, open up opportunities. For instance, a further order of magnitude could perhaps be gained in signal to noise for general synchrotron experiments...
February 1, 2018: Acta Crystallographica. Section D, Structural Biology
Kevin Cowtan, Jon Agirre
Refinement is a critical step in the determination of a model which explains the crystallographic observations and thus best accounts for the missing phase components. The scattering density is usually described in terms of atomic parameters; however, in macromolecular crystallography the resolution of the data is generally insufficient to determine the values of these parameters for individual atoms. Stereochemical and geometric restraints are used to provide additional information, but produce interrelationships between parameters which slow convergence, resulting in longer refinement times...
February 1, 2018: Acta Crystallographica. Section D, Structural Biology
Oliver S Smart, Vladimír Horský, Swanand Gore, Radka Svobodová Vařeková, Veronika Bendová, Gerard J Kleywegt, Sameer Velankar
Realising the importance of assessing the quality of the biomolecular structures deposited in the Protein Data Bank (PDB), the Worldwide Protein Data Bank (wwPDB) partners established Validation Task Forces to obtain advice on the methods and standards to be used to validate structures determined by X-ray crystallography, nuclear magnetic resonance spectroscopy and three-dimensional electron cryo-microscopy. The resulting wwPDB validation pipeline is an integral part of the wwPDB OneDep deposition, biocuration and validation system...
March 1, 2018: Acta Crystallographica. Section D, Structural Biology
Elizabeth L Guenther, Peng Ge, Hamilton Trinh, Michael R Sawaya, Duilio Cascio, David R Boyer, Tamir Gonen, Z Hong Zhou, David S Eisenberg
Proteins in the fibrous amyloid state are a major hallmark of neurodegenerative disease. Understanding the multiple conformations, or polymorphs, of amyloid proteins at the molecular level is a challenge of amyloid research. Here, we detail the wide range of polymorphs formed by a segment of human TAR DNA-binding protein 43 (TDP-43) as a model for the polymorphic capabilities of pathological amyloid aggregation. Using X-ray diffraction, microelectron diffraction (MicroED) and single-particle cryo-EM, we show that the247 DLIIKGISVHI257 segment from the second RNA-recognition motif (RRM2) forms an array of amyloid polymorphs...
March 12, 2018: Nature Structural & Molecular Biology
Andrea Arsiccio, Roberto Pisano
Biopharmaceuticals are frequently stored in the frozen state to avoid rapid degradation. Moreover, therapeutic proteins are frequently made into a dried form to provide long-term storage. However, both freezing and drying stresses can result in protein unfolding and aggregation. Thus, a proper formulation, containing suitable excipients, must be used to avoid loss of activity. Here, the conformational stability of a model protein, human growth hormone, is studied during freezing, and in the dried state as well, using molecular dynamics...
March 12, 2018: Physical Chemistry Chemical Physics: PCCP
Roman I Koning, Abraham J Koster, Thomas H Sharp
Cryo-electron tomography (CET) utilizes a combination of specimen cryo-fixation and multi-angle electron microscopy imaging to produce three-dimensional (3D) volume reconstructions of native-state macromolecular and subcellular biological structures with nanometer-scale resolution. In recent years, cryo-electron microscopy (cryoEM) has experienced a dramatic increase in the attainable resolution of 3D reconstructions, resulting from technical improvements of electron microscopes, improved detector sensitivity, the implementation of phase plates, automated data acquisition schemes, and improved image reconstruction software and hardware...
March 8, 2018: Annals of Anatomy, Anatomischer Anzeiger: Official Organ of the Anatomische Gesellschaft
M G Herrera, D S Vazquez, R Sreij, M Drechsler, Y Hertle, T Hellweg, V I Dodero
Alpha-gliadin is a highly immunogenic protein from wheat, which is associated with many human diseases, like celiac disease and non-celiac gluten sensitivity. Because of that, gliadin solution is subject to intense biomedical research. However, the physicochemical nature of the employed gliadin solution at physiological pH is not understood. Herein, we present a supramolecular evaluation of the alpha-gliadin protein in water at pH 3.0 by dynamic light scattering (DLS), cryo-transmission electron microscopy (cryo-TEM) and small-angle-...
February 27, 2018: Colloids and Surfaces. B, Biointerfaces
Jan Michael Schuller, Sebastian Falk, Lisa Fromm, Ed Hurt, Elena Conti
The RNA exosome complex processes and degrades a wide range of transcripts, including ribosomal RNAs. We used cryo-EM to visualize the yeast nuclear exosome holo-complex captured on a precursor large ribosomal subunit (pre-60S) during 7S-to-5.8S rRNA processing. The cofactors of the nuclear exosome are sandwiched between the ribonuclease core complex (Exo-10) and the remodeled "foot" structure of the pre-60S particle, which harbors the 5.8S rRNA precursor. The exosome-associated helicase Mtr4 recognizes the preribosomal substrate by docking to specific sites on the 25S rRNA, captures the 3' extension of the 5...
March 8, 2018: Science
Katharina Braunger, Stefan Pfeffer, Shiteshu Shrimal, Reid Gilmore, Otto Berninghausen, Elisabet C Mandon, Thomas Becker, Friedrich Förster, Roland Beckmann
Protein synthesis, transport and N-glycosylation are coupled at the mammalian endoplasmic reticulum (ER) by complex formation between the ribosome, the Sec61 protein-conducting channel and the oligosaccharyltransferase (OST). Here, we used different cryo-electron microscopy approaches to determine structures of native and solubilized ribosome-Sec61-OST complexes. A molecular model for the catalytic OST subunit revealed how STT3A is integrated into the OST and how STT3 paralog specificity for translocon-associated OST is achieved...
March 8, 2018: Science
Karen M Davies, Thorsten B Blum, Werner Kühlbrandt
We used electron cryo-tomography and subtomogram averaging to investigate the structure of complex I and its supramolecular assemblies in the inner mitochondrial membrane of mammals, fungi, and plants. Tomographic volumes containing complex I were averaged at ∼4 nm resolution. Principal component analysis indicated that ∼60% of complex I formed a supercomplex with dimeric complex III, while ∼40% were not associated with other respiratory chain complexes. The mutual arrangement of complex I and III2 was essentially conserved in all supercomplexes investigated...
March 8, 2018: Proceedings of the National Academy of Sciences of the United States of America
David E Timm, Valorie Bowman, Russell Madsen, Charles Rauch
Protein arginine methyl transferase 5 (PRMT5) is a signaling protein and histone modifying enzyme that is important in many cellular processes, including regulation of eukaryotic gene transcription. Reported here is a 3.7 Å structure of PRMT5, solved in complex with regulatory binding subunit MEP50 (methylosome associated protein 50, WDR77, p44), by single particle (SP) cryo-Electron Microscopy (cryo-EM) using micrographs of particles that are visibly crowded and aggregated. Despite suboptimal micrograph appearance, this cryo-EM structure is in good agreement with previously reported crystal structures of the complex, which revealed a 450 kDa hetero-octameric assembly having internal D2 symmetry...
2018: PloS One
Lin Chen, Jing He, Salim Sazzed, Rayshawn Walker
Cryo-electron microscopy (cryo-EM) is a structure determination method for large molecular complexes. As more and more atomic structures are determined using this technique, it is becoming possible to perform statistical characterization of side-chain conformations. Two data sets were involved to characterize block lengths for each of the 18 types of amino acids. One set contains 9131 structures resolved using X-ray crystallography from density maps with better than or equal to 1.5 Å resolutions, and the other contains 237 protein structures derived from cryo-EM density maps with 2-4 Å resolutions...
March 8, 2018: Molecules: a Journal of Synthetic Chemistry and Natural Product Chemistry
Oksana O Gerlits, James C Campbell, Matthew P Blakeley, Choel Kim, Andrey Y Kovalevsky
As one of main receptors of a second messenger, cGMP, cGMP-dependent protein kinase (PKG) isoforms I and II regu-late distinct physiological processes. Design of isoform specific activators is thus of great biomedical importance and requires detailed structural information on PKG isoforms bound with activators, including accurate positions of hydrogen atoms and description of hydrogen bonding and water architecture. Here, we determined a 2.2 Å room-temperature joint X-ray/neutron (XN) structure of the human PKG II carboxyl cyclic nucleotide binding (CNB-B) domain bound with a potent PKG II activator, 8-pCPT-cGMP...
March 8, 2018: Biochemistry
Gang Fu, Qian Wang, Nhan Phan, Paulina Urbanska, Ewa Joachimiak, Jianfeng Lin, Dorota Wloga, Daniela Nicastro
Motile cilia are essential for propelling cells and moving fluids across tissues. The activity of axonemal dynein motors must be precisely coordinated to generate ciliary motility, but their regulatory mechanisms are not well understood. The tether and tether head (T/TH) complex was hypothesized to provide mechanical feedback during ciliary beating because it links the motor domains of the regulatory I1 dynein to the ciliary doublet microtubule. Combining genetic and biochemical approaches with cryo-electron tomography, we identified FAP44 and FAP43 (plus the algae-specific, FAP43-redundant FAP244) as T/TH components...
March 7, 2018: Molecular Biology of the Cell
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