The magic triangle goes MAD: experimental phasing with a bromine derivative

Beck, Tobias; Gruene, Tim; Sheldrick, George M.

doi:10.1107/s0907444909051609

Cited by 27 publications

(32 citation statements)

References 29 publications

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“…In order to overcome the problem of nonspecific binding of HAs to proteins, the Sheldrick group created compounds with a series of functional groups around a ring (Beck et al, 2008(Beck et al, , 2010. They used two carboxylates and an amine group, interspersed with either I or Br atoms, around a six-carbon ring: 5-amino-2,4,6-triiodoisophthalic acid (I3C; Beck et al, 2008) and 5-amino-2,4,6-tribromoisophthalic acid (B3C; Beck et al, 2010).…”

Section: Use Of Bromine and Iodine As Heavy Atomsmentioning

confidence: 99%

“…They used two carboxylates and an amine group, interspersed with either I or Br atoms, around a six-carbon ring: 5-amino-2,4,6-triiodoisophthalic acid (I3C; Beck et al, 2008) and 5-amino-2,4,6-tribromoisophthalic acid (B3C; Beck et al, 2010). Crystals are soaked in a high concentration (0.5 M) of one of these compounds for 10 s and then backsoaked in a cryosolution which does not contain the HA for 5 s before being cryocooled.…”

Section: Use Of Bromine and Iodine As Heavy Atomsmentioning

confidence: 99%

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An overview of heavy-atom derivatization of protein crystals

Pike

Garman

Krojer

et al. 2016

Acta Crystallogr D Struct Biol

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Heavy-atom derivatization is one of the oldest techniques for obtaining phase information for protein crystals and, although it is no longer the first choice, it remains a useful technique for obtaining phases for unknown structures and for low-resolution data sets. It is also valuable for confirming the chain trace in lowresolution electron-density maps. This overview provides a summary of the technique and is aimed at first-time users of the method. It includes guidelines on when to use it, which heavy atoms are most likely to work, how to prepare heavy-atom solutions, how to derivatize crystals and how to determine whether a crystal is in fact a derivative.

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Section: Use Of Bromine and Iodine As Heavy Atomsmentioning

confidence: 99%

Section: Use Of Bromine and Iodine As Heavy Atomsmentioning

confidence: 99%

An overview of heavy-atom derivatization of protein crystals

Pike

Garman

Krojer

et al. 2016

Acta Crystallogr D Struct Biol

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“…The resulting electron density map, however, was of poor quality and did not allow manual rebuilding of the model. After unsuccessful trials of the quick-soak approach using bromide or iodide anions ( 28 ), soaks with different heavy metals, as well as Magic Triangle compounds ( 29,30 ), were undertaken to obtain derivatized CspLOX1 crystals. Diffraction data from several potentially derivatized crystals were measured, but most of them appeared not to be suitable for single isomorphous replacement or single-wavelength anomalous diffraction approaches.…”

Section: Structure Determination and Refi Nementmentioning

confidence: 99%

Crystal structure of a lipoxygenase from Cyanothece sp. may reveal novel features for substrate acquisition

Newie¹,

Andreou²,

Neumann

et al. 2016

Journal of Lipid Research

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This article is available online at http://www.jlr.org PUFA oxygenation is a process that leads to the formation of bioactive lipid compounds with a diversity of biological functions in plants and animals ( 1, 2 ). Oxidation of PUFAs may be catalyzed by two major classes of enzymes, cyclooxygenases or ␣ -dioxygenases and lipoxygenases (LOXs) ( 3 ). Of the two, LOXs are nonheme iron-containing dioxygenases that are widely found in higher plants and animals, but have also been detected in some corals, mosses, fungi, and a number of bacteria ( 4, 5 ). Members of the LOX family catalyze the regio-and stereospecifi c oxygenation of PUFAs with one or more (1 Z ,4 Z )-pentadiene moieties leading to the formation of hydroperoxy PUFAs ( 6 ). LOX hydroperoxide products are precursors of important signaling compounds such as aldehydes and jasmonates in plants and leukotrienes, resolvins, and lipoxins in mammals ( 3 ). These signaling molecules play an important role in wound and defense responses as well as in aspects of plant development ( 7 ), while in mammals they function in infl ammation, asthma, and the development of atherosclerosis and cancer ( 1 ). Other than in higher organisms, very little is still known regarding the overall function of LOX products in prokaryotes and fungi ( 4, 5 ).As the regio-and stereospecifi city of the LOX reaction has an infl uence on the biological function of the product, many studies have focused on the molecular basis of this specifi city. In the case of arachidonic acid [20:4(n-6)], which is a typical mammalian LOX substrate, several Abstract In eukaryotes, oxidized PUFAs, so-called oxylipins, are vital signaling molecules. The fi rst step in their biosynthesis may be catalyzed by a lipoxygenase (LOX), which forms hydroperoxides by introducing dioxygen into PUFAs. Here we characterized CspLOX1, a phylogenetically distant LOX family member from Cyanothece sp. PCC 8801 and determined its crystal structure. In addition to the classical two domains found in plant, animal, and coral LOXs, we identifi ed an N-terminal helical extension, reminiscent of the long ␣ -helical insertion in Pseudomonas aeruginosa LOX. In liposome fl otation studies, this helical extension, rather than the ␤ -barrel domain, was crucial for a membrane binding function. Additionally, CspLOX1 could oxygenate 1,2-diarachidonyl-sn -glycero-3-phosphocholine, suggesting that the enzyme may act directly on membranes and that fatty acids bind to the active site in a tail-fi rst orientation. This binding mode is further supported by the fact that CspLOX1 catalyzed oxygenation at the n-10 position of both linoleic and arachidonic acid, resulting in 9 R -and 11 R -hydroperoxides, respectively. Together these results reveal unifying structural features of LOXs and their function. While the core of the active site is important for lipoxygenation and thus highly conserved, peripheral domains functioning in membrane and substrate binding are more variable.

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“…For SAD phasing, a 1 M stock solution of B3C was obtained by dissolving the solid material in 2 M lithium hydroxide solution (Beck et al, 2010). The B3C stock solution was added to the protein solution to produce a final concentration of 0.2 M B3C along with 30 mM NaF, 5 mM BeCl 2 , 3%(w/v) 1,5-diaminopentane dihydrochloride.…”

Section: Crystallizationmentioning

confidence: 99%

“…The experimental phasing was obtained by single-wavelength anomalous dispersion (SAD) using 5-amino-2,4,6-tribromoisophthalic acid (B3C; Beck et al, 2009). B3C contains three functional groups (two carboxylate groups and one amino group) that interact with proteins via hydrogen bonds, and three Br atoms suitable for anomalous dispersion phasing (Beck et al, 2010). The B3C molecules bound to the proteins provide a strong anomalous signal which can be used for MAD (multiwavelength anomalous diffraction) phasing (Beck et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

Crystallization and preliminary X-ray diffraction analysis of full-length spr1814, a response regulator ofStreptococcus pneumoniae, in complex with a phosphoryl analogue

Park

Min

et al. 2014

Acta Cryst Sect F

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Spr1814 of Streptococcus pneumoniae is a signal transduction response regulator belonging to the NarL/FixJ subfamily, which has a helix-turn-helix DNAbinding effector domain. To understand how the phosphorylation of the conserved aspartic acid residue induces conformational changes in spr1814 allowing binding to the target promoter, recombinant spr1814 expressed in Escherichia coli was crystallized with the phosphoryl analogue beryllium fluoride BeF 3 À by the hanging-drop vapour-diffusion method. The crystals diffracted to 1.9 Å resolution and belonged to space group P2 1 , with unit-cell parameters a = 40.2, b = 114.5, c = 50.1 Å , = 92.1. Structure determination by the SAD method using the bromine derivative 5-amino-2,4,6-tribromoisophthalic acid (B3C) is under way.

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The magic triangle goes MAD: experimental phasing with a bromine derivative

Cited by 27 publications

References 29 publications

An overview of heavy-atom derivatization of protein crystals

An overview of heavy-atom derivatization of protein crystals

Crystal structure of a lipoxygenase from Cyanothece sp. may reveal novel features for substrate acquisition

Crystallization and preliminary X-ray diffraction analysis of full-length spr1814, a response regulator ofStreptococcus pneumoniae, in complex with a phosphoryl analogue

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