Crystallization and preliminary crystallographic analysis of the chimeric protein LKB1–14-3-3ζ

SETMAR, a recently identified double strand break (DSB) repair enzyme in the human genome, contains an N-terminal SET domain and a C-terminal MAR domain. This chimeric protein arose through the fusion of a mariner-family DNA transposase gene, Hsmar1, downstream of a SET histone methyltransferase gene approximately 50 million years ago [1]. Although the SETMAR transposase domain retains the ability to bind terminal inverted repeat (TIR) DNA sequence, which is the hallmark of DNA transposons, it is no longer a functional transposase [2]. Nonetheless, the transposase domain with only 19 amino acid substitutions as compared to the ancestral Hsmar1 transposase has been under a strong selective evolutionary pressure suggesting that the transposase domain is functionally important. Determining how SETMAR interacts with DNA is central to understanding the molecular basis of its evolved DNA repair activity. Toward this goal, we have focused initially on the interaction of the DNA-binding domain (DBD) of the SETMAR transposase domain with TIR DNA. The DBD of SETMAR has been overexpressed and purified. A complex formed between SETMAR DBD and its transposon TIR DNA has been crystallized by using the hanging-drop vapor diffusion method. The crystals diffract to 3.15 Å resolution and exhibit orthorhombic symmetry (C2221), with unit-cell dimensions of a=72.233 Å, b=164.385 Å, and c=67.957 Å. As there is no suitable search model available, we are currently pursuing experimental phasing approaches in order to solve this structure. We anticipate the structural analysis of DBD of SETMAR bound to transposon DNA will provide insight into the mechanism by which SETMAR recognizes both TIR and non-TIR DNA.

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Quaternary structure of Limulus polyphemus hemocyanin

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100081747 ◽

1978 ◽

Vol 36 (2) ◽

pp. 176-177

Author(s):

T. Wichertjes ◽

E.J. Kwak ◽

E.F.J. Van Bruggen

Keyword(s):

Electron Microscopy ◽

Functional Properties ◽

Horseshoe Crab ◽

Temperature Jump ◽

Quaternary Structure ◽

Crystallographic Analysis ◽

Limulus Polyphemus ◽

Oxygen Binding ◽

X Ray ◽

Intact Molecule

Hemocyanin of the horseshoe crab (Limulus polyphemus) has been studied in nany ways. Recently the structure, dissociation and reassembly was studied using electron microscopy of negatively stained specimens as the method of investigation. Crystallization of the protein proved to be possible and X-ray crystallographic analysis was started. Also fluorescence properties of the hemocyanin after dialysis against Tris-glycine buffer + 0.01 M EDTA pH 8.9 (so called “stripped” hemocyanin) and its fractions II and V were studied, as well as functional properties of the fractions by NMR. Finally the temperature-jump method was used for assaying the oxygen binding of the dissociating molecule and of preparations of isolated subunits. Nevertheless very little is known about the structure of the intact molecule. Schutter et al. suggested that the molecule possibly consists of two halves, combined in a staggered way, the halves themselves consisting of four subunits arranged in a square.

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Some Observations on Intra-Mitochondrial Crystals

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100079486 ◽

1977 ◽

Vol 35 ◽

pp. 406-407

Author(s):

J. A. Clarke ◽

D. N. Landon ◽

P. R. Ward

Keyword(s):

Cytochrome B ◽

Mitochondrial Myopathy ◽

Crystallographic Analysis ◽

Mitochondrial Membranes ◽

Electron Microscopes ◽

Muscle Biopsies

Intra-mitochondrial crystals have been noted in muscle biopsies from patients in a wide variety of diseased states. As far as we are aware, none of these crystals have been subjected to detailed crystallographic analysis. Recently, similar crystals were observed in a biopsy from a patient with a mitochondrial myopathy, characterised by a deficiency in reducible cytochrome b (Morgan-Hughes, J. A., Darveniza, P., Kahn, S. N., Landon, D. N., Sherratt, R. M., Land, J. M. and Clark, J. B., 1977, Brain, In Press). Aldehyde-fixed, osmicated resin imbedded material was examined using Siemens, JEOL and Phillips electron microscopes with goniometer specimen stages. The crystals generally lay between the outer and inner mitochondrial membranes and measured 1 - 3 μm in length and 0.1 - 0.3 μm in width. Characteristically, these crystals revealed specific periodicities.

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Morphology of Σ3{/70.53°} grain boundaries in a C15 intermetallic compound

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100171158 ◽

1994 ◽

Vol 52 ◽

pp. 684-685

Author(s):

Fuming Chu ◽

D. P. Pope ◽

D. S. Zhou ◽

T. E. Mitchell

Keyword(s):

Grain Boundary ◽

Grain Boundaries ◽

Crystallographic Analysis ◽

Laves Phase ◽

Second Phase ◽

Bright Field Image ◽

Boundary Edge ◽

Arc Melting ◽

Fcc Lattice ◽

Diffraction Patterns

A C15 Laves phase, HfV2+Nb, shows promising mechanical properties and here we describe the structure of its grain boundaries. The C15 Laves phase has a fcc lattice with a=7.4Å. An alloy of composition Hf14V64Nb22 (including a C15 matrix and a second phase of V-rich bcc solution) was made by arc-melting. The alloy was homogenized at 1200°C for 120h. Preliminary study concentrated on Σ3{<110>/70.53°} grain boundaries in the C15 phase using Philips 400T and CM 30 microscopes.The most-commonly observed morphology of Σ3{<110>/70.53°} grain boundaries in the C15 phase is a faceted boundary. A bright field image (BFI) of the faceted boundary and the corresponding diffraction patterns with the grain boundary edge-on are shown in Fig. 1(a). From the diffraction patterns using a <110> zone axis for both grains, it is obvious that this is a Σ3{<110>/70.53°} grain boundary. Crystallographic analysis shows that the Σ3{<110>/70.53°} grain boundaries selectively facet with the following relationships between the two grains: {111}1//{111}2, {112}1//{112}2, {111}1//{115}2, and {001}1//{221}2.

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A crystallographic analysis of the CoSi/Si(111) interface using Transmission Electron Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100176009 ◽

1990 ◽

Vol 48 (4) ◽

pp. 574-575

Author(s):

A.C. Daykin ◽

C.J. Kiely ◽

R.C. Pond ◽

J.L. Batstone

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Defect Structure ◽

Room Temperature ◽

Theoretical Method ◽

Crystallographic Analysis ◽

Si Substrate ◽

Transmission Electron ◽

Theoretical Predictions

When CoSi2 is grown onto a Si(111) surface it can form in two distinct orientations. A-type CoSi2 has the same orientation as the Si substrate and B-type is rotated by 180° degrees about the [111] surface normal.One method of producing epitaxial CoSi2 is to deposit Co at room temperature and anneal to 650°C.If greater than 10Å of Co is deposited then both A and B-type CoSi2 form via a number of intermediate silicides .The literature suggests that the co-existence of A and B-type CoSi2 is in some way linked to these intermediate silicides analogous to the NiSi2/Si(111) system. The phase which forms prior to complete CoSi2 formation is CoSi. This paper is a crystallographic analysis of the CoSi2/Si(l11) bicrystal using a theoretical method developed by Pond. Transmission electron microscopy (TEM) has been used to verify the theoretical predictions and to characterise the defect structure at the interface.

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Lamin B receptor: role on chromatin structure, cellular senescence and possibly aging

Biochemical Journal ◽

10.1042/bcj20200165 ◽

2020 ◽

Vol 477 (14) ◽

pp. 2715-2720

Author(s):

Susana Castro-Obregón

Keyword(s):

Cellular Senescence ◽

Nuclear Membrane ◽

Chromatin Structure ◽

Cholesterol Synthesis ◽

Reductase Activity ◽

Chimeric Protein ◽

Nuclear Lamina ◽

Lamin B ◽

Inner Nuclear Membrane ◽

Lamin B Receptor

The nuclear envelope is composed by an outer nuclear membrane and an inner nuclear membrane, which is underlain by the nuclear lamina that provides the nucleus with mechanical strength for maintaining structure and regulates chromatin organization for modulating gene expression and silencing. A layer of heterochromatin is beneath the nuclear lamina, attached by inner nuclear membrane integral proteins such as Lamin B receptor (LBR). LBR is a chimeric protein, having also a sterol reductase activity with which it contributes to cholesterol synthesis. Lukasova et al. showed that when DNA is damaged by ɣ-radiation in cancer cells, LBR is lost causing chromatin structure changes and promoting cellular senescence. Cellular senescence is characterized by terminal cell cycle arrest and the expression and secretion of various growth factors, cytokines, metalloproteinases, etc., collectively known as senescence-associated secretory phenotype (SASP) that cause chronic inflammation and tumor progression when they persist in the tissue. Therefore, it is fundamental to understand the molecular basis for senescence establishment, maintenance and the regulation of SASP. The work of Lukasova et al. contributed to our understanding of cellular senescence establishment and provided the basis that lead to the further discovery that chromatin changes caused by LBR reduction induce an up-regulated expression of SASP factors. LBR dysfunction has relevance in several diseases and possibly in physiological aging. The potential bifunctional role of LBR on cellular senescence establishment, namely its role in chromatin structure together with its enzymatic activity contributing to cholesterol synthesis, provide a new target to develop potential anti-aging therapies.

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