scholarly journals Natural hybrid silica/protein superstructure at atomic resolution

2020 ◽  
Vol 117 (49) ◽  
pp. 31088-31093
Author(s):  
Stefan Görlich ◽  
Abisheik John Samuel ◽  
Richard Johannes Best ◽  
Ronald Seidel ◽  
Jean Vacelet ◽  
...  
Keyword(s):  
Tertiary Structure ◽  
Three Dimensional ◽  
Inorganic Materials ◽  
Atomic Resolution ◽  
Axial Filament ◽  
X Ray Crystallography ◽  
Organic Hybrid ◽  
Hybrid Silica ◽  
Transmission Electron

Formation of highly symmetric skeletal elements in demosponges, called spicules, follows a unique biomineralization mechanism in which polycondensation of an inherently disordered amorphous silica is guided by a highly ordered proteinaceous scaffold, the axial filament. The enzymatically active proteins, silicateins, are assembled into a slender hybrid silica/protein crystalline superstructure that directs the morphogenesis of the spicules. Furthermore, silicateins are known to catalyze the formation of a large variety of other technologically relevant organic and inorganic materials. However, despite the biological and biotechnological importance of this macromolecule, its tertiary structure was never determined. Here we report the atomic structure of silicatein and the entire mineral/organic hybrid assembly with a resolution of 2.4 Å. In this work, the serial X-ray crystallography method was successfully adopted to probe the 2-µm-thick filaments in situ, being embedded inside the skeletal elements. In combination with imaging and chemical analysis using high-resolution transmission electron microscopy, we provide detailed information on the enzymatic activity of silicatein, its crystallization, and the emergence of a functional three-dimensional silica/protein superstructure in vivo. Ultimately, we describe a naturally occurring mineral/protein crystalline assembly at atomic resolution.

scholarly journals Mice with cleavage-resistant N-cadherin exhibit synapse anomaly in the hippocampus and outperformance in spatial learning tasks

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
M. Asada-Utsugi ◽  
K. Uemura ◽  
M. Kubota ◽  
Y. Noda ◽  
Y. Tashiro ◽  
...  
Keyword(s):  
Memory Function ◽  
Three Dimensional ◽  
Excitatory Synapses ◽  
Missense Mutations ◽  
Glutamatergic Synapses ◽  
Learning Tasks ◽  
Transmission Electron ◽  
Nmdar Activation ◽  
And Function

AbstractN-cadherin is a homophilic cell adhesion molecule that stabilizes excitatory synapses, by connecting pre- and post-synaptic termini. Upon NMDA receptor (NMDAR) activation by glutamate, membrane-proximal domains of N-cadherin are cleaved serially by a-disintegrin-and-metalloprotease 10 (ADAM10) and then presenilin 1(PS1, catalytic subunit of the γ-secretase complex). To assess the physiological significance of the initial N-cadherin cleavage, we engineer the mouse genome to create a knock-in allele with tandem missense mutations in the mouse N-cadherin/Cadherin-2 gene (Cdh2R714G, I715D, or GD) that confers resistance on proteolysis by ADAM10 (GD mice). GD mice showed a better performance in the radial maze test, with significantly less revisiting errors after intervals of 30 and 300 s than WT, and a tendency for enhanced freezing in fear conditioning. Interestingly, GD mice reveal higher complexity in the tufts of thorny excrescence in the CA3 region of the hippocampus. Fine morphometry with serial section transmission electron microscopy (ssTEM) and three-dimensional (3D) reconstruction reveals significantly higher synaptic density, significantly smaller PSD area, and normal dendritic spine volume in GD mice. This knock-in mouse has provided in vivo evidence that ADAM10-mediated cleavage is a critical step in N-cadherin shedding and degradation and involved in the structure and function of glutamatergic synapses, which affect the memory function.

scholarly journals Protein Folding: Search for Basic Physical Models

2003 ◽  
Vol 3 ◽  
pp. 623-635 ◽  
Author(s):  
Ivan Y. Torshin ◽  
Robert W. Harrison
Keyword(s):  
Protein Folding ◽  
Tertiary Structure ◽  
Three Dimensional ◽  
Physical Models ◽  
Dimensional Structure ◽  
Computational Techniques ◽  
Linear Sequence ◽  
Physical Forces

How a unique three-dimensional structure is rapidly formed from the linear sequence of a polypeptide is one of the important questions in contemporary science. Apart from biological context ofin vivoprotein folding (which has been studied only for a few proteins), the roles of the fundamental physical forces in thein vitrofolding remain largely unstudied. Despite a degree of success in using descriptions based on statistical and/or thermodynamic approaches, few of the current models explicitly include more basic physical forces (such as electrostatics and Van Der Waals forces). Moreover, the present-day models rarely take into account that the protein folding is, essentially, a rapid process that produces a highly specific architecture. This review considers several physical models that may provide more direct links between sequence and tertiary structure in terms of the physical forces. In particular, elaboration of such simple models is likely to produce extremely effective computational techniques with value for modern genomics.

scholarly journals Low-Zpolymer sample supports for fixed-target serial femtosecond X-ray crystallography

2015 ◽  
Vol 48 (4) ◽  
pp. 1072-1079 ◽  
Author(s):  
Geoffrey K. Feld ◽  
Michael Heymann ◽  
W. Henry Benner ◽  
Tommaso Pardini ◽  
Ching-Ju Tsai ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Three Dimensional ◽  
Two Dimensional ◽  
X Ray Diffraction ◽  
Fixed Target ◽  
X Ray ◽  
X Ray Crystallography ◽  
Transmission Electron ◽  
Linac Coherent Light Source ◽  
Efficient Alternative

X-ray free-electron lasers (XFELs) offer a new avenue to the structural probing of complex materials, including biomolecules. Delivery of precious sample to the XFEL beam is a key consideration, as the sample of interest must be serially replaced after each destructive pulse. The fixed-target approach to sample delivery involves depositing samples on a thin-film support and subsequent serial introductionviaa translating stage. Some classes of biological materials, including two-dimensional protein crystals, must be introduced on fixed-target supports, as they require a flat surface to prevent sample wrinkling. A series of wafer and transmission electron microscopy (TEM)-style grid supports constructed of low-Zplastic have been custom-designed and produced. Aluminium TEM grid holders were engineered, capable of delivering up to 20 different conventional or plastic TEM grids using fixed-target stages available at the Linac Coherent Light Source (LCLS). As proof-of-principle, X-ray diffraction has been demonstrated from two-dimensional crystals of bacteriorhodopsin and three-dimensional crystals of anthrax toxin protective antigen mounted on these supports at the LCLS. The benefits and limitations of these low-Zfixed-target supports are discussed; it is the authors' belief that they represent a viable and efficient alternative to previously reported fixed-target supports for conducting diffraction studies with XFELs.

scholarly journals Adding exogenous biglycan or decorin improves tendon formation for equine peritenon and tendon proper cells in vitro

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Monica Y. Pechanec ◽  
Tannah N. Boyd ◽  
Keith Baar ◽  
Michael J. Mienaltowski
Keyword(s):  
Material Properties ◽  
Quantitative Polymerase Chain Reaction ◽  
Tendon Repair ◽  
Three Dimensional ◽  
Collagen Content ◽  
Matrix Assembly ◽  
Post Injury ◽  
Transmission Electron

Abstract Background Tendon injuries amount to one of the leading causes of career-ending injuries in horses due to the inability for tendon to completely repair and the high reinjury potential. As a result, novel therapeutics are necessary to improve repair with the goal of decreasing leg lameness and potential reinjury. Small leucine-rich repeat proteoglycans (SLRPs), a class of regulatory molecules responsible for collagen organization and maturation, may be one such therapeutic to improve tendon repair. Before SLRP supplementation can occur in vivo, proper evaluation of the effect of these molecules in vitro needs to be assessed. The objective of this study was to evaluate the effectiveness of purified bovine biglycan or decorin on tendon proper and peritenon cell populations in three-dimensional tendon constructs. Methods Equine tendon proper or peritenon cell seeded fibrin three-dimensional constructs were supplemented with biglycan or decorin at two concentrations (5 nM or 25 nM). The functionality and ultrastructural morphology of the constructs were assessed using biomechanics, collagen content analysis, transmission electron microscopy (TEM), and gene expression by real time – quantitative polymerase chain reaction (RT-qPCR). Results SLRP supplementation affected both tendon proper and peritenon cells-seeded constructs. With additional SLRPs, material and tensile properties of constructs strengthened, though ultrastructural analyses indicated production of similar-sized or smaller fibrils. Overall expression of tendon markers was bolstered more in peritenon cells supplemented with either SLRP, while supplementation of SLRPs to TP cell-derived constructs demonstrated fewer changes in tendon and extracellular matrix markers. Moreover, relative to non-supplemented tendon proper cell-seeded constructs, SLRP supplementation of the peritenon cells showed increases in mechanical strength, material properties, and collagen content. Conclusions The SLRP-supplemented peritenon cells produced constructs with greater mechanical and material properties than tendon proper seeded constructs, as well as increased expression of matrix assembly molecules. These findings provide evidence that SLRPs should be further investigated for their potential to improve tendon formation in engineered grafts or post-injury.

Synopses from the poster exhibition organized by I. M. Kerr, 1. Interferon: a tertiary structure predicted from amino acid sequences

1982 ◽  
Vol 299 (1094) ◽  
pp. 125-127 ◽  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Tertiary Structure ◽  
Three Dimensional ◽  
Amino Acid Sequences ◽  
Dimensional Structure ◽  
X Ray ◽  
Three Dimensional Structure ◽  
X Ray Crystallography ◽  
Functional Studies

Functional studies on interferon would be helped by a three-dimensional structure for the molecule. However, it may be several years before the structure of the protein is determined by X-ray crystallography. We have therefore used available methods for predicting the secondary - and the tertiary - structure of a protein from its amino acid sequence to propose a tertiary model involving the packing of four a-helices. Details of this work have been published elsewhere (Sternberg & Cohen 1982).

Chiral Helices in a 3D Network of Zn2(en)V2O7 (en = Ethylenediamine)

2008 ◽  
Vol 55-57 ◽  
pp. 669-672 ◽  
Author(s):  
S. Krachodnok ◽  
K.J. Haller ◽  
I.D. Williams
Keyword(s):  
Thermal Stability ◽  
Three Dimensional ◽  
Monoclinic Space Group ◽  
Tg Analysis ◽  
X Ray ◽  
X Ray Crystallography ◽  
Organic Hybrid ◽  
Dimensional Network ◽  
3D Network ◽  
Thermal Stability Of

An inorganic-organic hybrid vanadate complex, Zn2(en)V2O7 (en = ethylenediamine) has been hydrothermally synthesized and characterized by single-crystal X-ray crystallography, TG analysis, IR spectroscopy, and elemental analysis. The title compound crystallizes in the monoclinic space group P21 with a = 7.0885(7), b = 8.6040(9), c = 8.1869(8) Å, b = 112.224(2)° and V = 462.22(8) Å3, T = 298±2 K and R1[I>2s(I)] = 0.0291 for 2170 observed reflections. The structure consists of undulating sheets constructed from helical chains that share Zn coordination centers with repeating units propagated into a helix by the two-fold screw axes at 0, y, ½ and pillared with alternating VO4 tetrahedra to generate a 3D chiral network. Neighboring helices are interconnected by µ2-O bridges to form the three-dimensional network. The strength of the network is reinforced by hydrogen bonding. TG analysis shows thermal stability of the network up to 283 °C.

scholarly journals Introduction to high-resolution cryo-electron microscopy

2016 ◽  
Vol 62 (3) ◽  
pp. 383-394
Author(s):  
Mariusz Czarnocki-Cieciura ◽  
Marcin Nowotny
Keyword(s):  
Electron Microscopy ◽  
Nmr Spectroscopy ◽  
Structural Biology ◽  
Atomic Resolution ◽  
Biological Macromolecules ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
Electron Microscopes

For many years two techniques have dominated structural biology – X-ray crystallography and NMR spectroscopy. Traditional cryo-electron microscopy of biological macromolecules produced macromolecular reconstructions at resolution limited to 6–10 Å. Recent development of transmission electron microscopes, in particular the development of direct electron detectors, and continuous improvements in the available software, have led to the “resolution revolution” in cryo-EM. It is now possible to routinely obtain near-atomic-resolution 3D maps of intact biological macromolecules as small as ~100 kDa. Thus, cryo-EM is now becoming the method of choice for structural analysis of many complex assemblies that are unsuitable for structure determination by other methods.

scholarly journals Determining protein structures using genetics

2018 ◽  
Author(s):  
Jörn M. Schmiedel ◽  
Ben Lehner
Keyword(s):  
Structure Determination ◽  
Low Cost ◽  
Protein Structures ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Biological Research ◽  
X Ray Crystallography ◽  
Close Relationship ◽  
And Function

SummaryDetermining the three dimensional structures of macromolecules is a major goal of biological research because of the close relationship between structure and function. Structure determination usually relies on physical techniques including x-ray crystallography, NMR spectroscopy and cryo-electron microscopy. Here we present a method that allows the high-resolution three-dimensional structure of a biological macromolecule to be determined only from measurements of the activity of mutant variants of the molecule. This genetic approach to structure determination relies on the quantification of genetic interactions (epistasis) between mutations and the discrimination of direct from indirect interactions. This provides a new experimental strategy for structure determination, with the potential to reveal functional and in vivo structural conformations at low cost and high throughput.

scholarly journals Mice with a cleavage-resistant N-cadherin exhibit synapse anomaly in the hippocampus and outperformance in spatial learning tasks

2021 ◽  
Author(s):  
Megumi Asada-Utsugi ◽  
Kengo Uemura ◽  
Masakazu Kubota ◽  
Yasuha Noda ◽  
Yasutaka Tashiro ◽  
...  
Keyword(s):  
Memory Function ◽  
Three Dimensional ◽  
Excitatory Synapses ◽  
Missense Mutations ◽  
Glutamatergic Synapses ◽  
Learning Tasks ◽  
Transmission Electron ◽  
Nmdar Activation ◽  
And Function

Abstract N-cadherin is a homophilic cell adhesion molecule that stabilizes excitatory synapses, by connecting pre- and post-synaptic termini. Upon NMDA receptor (NMDAR) activation by glutamate, membrane-proximal domains of N-cadherin are cleaved serially by a-disintegrin-and-metalloprotease 10 (ADAM10) and then presenilin 1(PS1, catalytic subunit of the γ-secretase complex). To assess the physiological significance of the initial N-cadherin cleavage, we engineer the mouse genome to create a knock-in allele with tandem missense mutations in the mouse N-cadherin/Cadherin-2 gene (Cdh2 R714G, I715D, or GD) that confers resistance on proteolysis by ADAM10 (GD mice). GD mice showed a better performance in the radial maze test, with significantly less revisiting errors after intervals of 30 and 300 sec than WT, and a tendency for enhanced freezing in fear conditioning. Interestingly, GD mice reveal higher complexity in the tufts of thorny excrescence in the CA3 region of the hippocampus. Fine morphometry with serial section transmission electron microscopy (ssTEM) and three-dimensional (3D) reconstruction reveals significantly higher synaptic density, significantly smaller PSD area, and normal dendritic spine volume in GD mice. This knock-in mouse has provided in vivo evidence that ADAM10-mediated cleavage is a critical step in N-cadherin shedding and degradation and involved in the structure and function of glutamatergic synapses, which affect the memory function.

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