Exploiting tertiary structure through local folds for crystallographic phasing

Sammito, Massimo; Millán, Claudia; Rodríguez, Daniel Salazar; Ilarduya, Iñaki M. de; Meindl, Kathrin; Marino, Ivan De; Petrillo, Giovanna; Buey, Rubén M.; Pereda, José M. de; Zeth, Kornelius; Sheldrick, George M.; Usón, Isabel

doi:10.1038/nmeth.2644

Cited by 69 publications

(68 citation statements)

References 18 publications

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“…However, we were unable to phase the structure using the anomalous signals, most likely because all three methionines in the protein lie in the very N- and C-termini of the A46(1–83) construct and, consequently, are located in flexible regions. Finally, the phases were obtained by ARCIMBOLDO_BORGES [25] crystallographic software. The program exploits tertiary structure libraries extracted from the Protein Data Bank for ab initio phasing.…”

Section: Resultsmentioning

confidence: 99%

“…However, the regular crystal packing allowed high-resolution data sets to be obtained that were initially processed at 1.55 Å resolution. This high resolution data, together with the short length of the protein, allowed the phases to be solved using ab initio methods [25]. In this method, which has been used successfully for numerous α-helical structures [34], fragments of known structures are employed as small search models.…”

Section: Discussionmentioning

confidence: 99%

“…Six secondary structure elements were aligned involving 57 residues at an rmsd of 3.39 Å for 45 C α ; however, the mutual orientation of both sheets is markedly different and the CS domain seen in NudC does not show oligomerisation. Therefore, we searched for similar local folds of the same connectivity using the same core of 45 residues with the program BORGES [25]. The closest match for the strands of ligand bound subunit A was extracted from 2XN2, with 3.09 Å rmsd, whereas for subunit B, a fold extracted from 2OQE gave 2.42 Å.…”

Section: Discussionmentioning

confidence: 99%

“…The structure was solved by ARCIMBOLDO_BORGES ab initio phasing software [25] combining fragment search with Phaser [26] and density modification with SHELXE [46] on the supercomputer Gordon at the SDSC.…”

Section: Methodsmentioning

confidence: 99%

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Vaccinia Virus Immunomodulator A46: A Lipid and Protein-Binding Scaffold for Sequestering Host TIR-Domain Proteins

et al. 2016

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Vaccinia virus interferes with early events of the activation pathway of the transcriptional factor NF-kB by binding to numerous host TIR-domain containing adaptor proteins. We have previously determined the X-ray structure of the A46 C-terminal domain; however, the structure and function of the A46 N-terminal domain and its relationship to the C-terminal domain have remained unclear. Here, we biophysically characterize residues 1–83 of the N-terminal domain of A46 and present the X-ray structure at 1.55 Å. Crystallographic phases were obtained by a recently developed ab initio method entitled ARCIMBOLDO_BORGES that employs tertiary structure libraries extracted from the Protein Data Bank; data analysis revealed an all β-sheet structure. This is the first such structure solved by this method which should be applicable to any protein composed entirely of β-sheets. The A46(1–83) structure itself is a β-sandwich containing a co-purified molecule of myristic acid inside a hydrophobic pocket and represents a previously unknown lipid-binding fold. Mass spectrometry analysis confirmed the presence of long-chain fatty acids in both N-terminal and full-length A46; mutation of the hydrophobic pocket reduced the lipid content. Using a combination of high resolution X-ray structures of the N- and C-terminal domains and SAXS analysis of full-length protein A46(1–240), we present here a structural model of A46 in a tetrameric assembly. Integrating affinity measurements and structural data, we propose how A46 simultaneously interferes with several TIR-domain containing proteins to inhibit NF-κB activation and postulate that A46 employs a bipartite binding arrangement to sequester the host immune adaptors TRAM and MyD88.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Vaccinia Virus Immunomodulator A46: A Lipid and Protein-Binding Scaffold for Sequestering Host TIR-Domain Proteins

et al. 2016

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“…In the case of the program, most collections of fragments remain a "still-life", but some are correct enough for density modification and main chain tracing to reveal the protein's true portrait. Beyond alpha-helices, other fragments can be exploited in analogous way: libraries of helices with modeled side-chains, beta strands, predictable fragments such as DNA-binding folds or fragments selected from distant homologs up to libraries of small local folds that are used to enforce non-specific tertiary structure, thus restoring the ab initio nature of the method (Sammito et al, 2013). Using these methods, a number of unknown macromolecules with a few thousand atoms and resolutions around 2 Å have been solved.…”

Section: Macromolecular Ab Initio Phasingmentioning

confidence: 99%

Crystallographic structure solution

Millán¹,

Usón²

2015

Arbor

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The three-dimensional view of molecules at the atomic level provided by X-ray crystallography is not only extremely informative but is also easily and intuitively understood by humans, who very much rely on their vision. However, unlike microscopy, this technique does not directly yield an image. The structural model cannot be directly calculated from the diffraction data, as only the intensities of scattered beams and not their phases are experimentally accessible. In order to obtain the 3-dimensional structure phases have to be obtained by either additional experimental or computational methods. This is known as the phase problem in crystallography. In this manuscript we provide an overview of major milestones along the quest for the lost phases.KEYWORDS: phase problem; constraints; structure factors; Fourier maps; search; minimization; maximum-likelihood; optimization; restraints; X-rays. RESUMEN: La cristalografía proporciona una visión tridimensional de las moléculas a un nivel de detalle atómico, que no sólo resulta muy informativa sino que además puede ser fácil e intuitivamente comprendida por seres tan predominantemente visuales como solemos ser los humanos. Sin embargo, al contrario que la microscopía, esta técnica no ofrece directamente una imagen y el modelo estructural no puede calcularse directamente a partir de los datos de difracción, ya que solamente las intensidades de los rayos difractados y no sus fases son accesibles a la medida experimental. Para determinar la estructura tridimensional las fases deben ser obtenidas por medio de métodos adicionales, bien experimentales o computacionales. Esto constituye el problema de la fase en cristalografía. En este artículo ofreceremos una visión general de los principales hitos en la búsqueda de las fases perdidas.PALABRAS CLAVE: problema de la fase; constricciones; factores de estructura; mapas de Fourier; métodos de búsqueda; minimización; máxima verosimilitud; optimización; restricciones; rayos-X.

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