Los autores desean agradecer al personal PXRR al NSLS (Brookhaven National Laboratory) para la asistencia durante la recolección de datos y Pillon Mónica para ayuda en la purificación del ADN. Este trabajo fue financiado por los Institutos Canadienses de Investigación en Salud (RP 67189).

1. Purificación de proteínas El enlazador flexible que une el. N-(oligomerización) y C-terminal (de unión a ADN) dominios de SEQA SIDA el reconocimiento de hemimethylated repite GATC separados por uno a tres vueltas en el ADN Para este estudio, se utilizó una variante dimérica de SEQA (SeqAΔ (41-59)-A25R) con una mutación puntual en el dominio N-terminal que impide aún más la oligomerización y un enlazador acortado que restringe la unión al ADN en tándem para GATC repite separados exclusivamente por una vuelta en el ADN (Figura 1) 2,8. <ol><li> Transformar BL21 (DE3) células con el plásmido que codifica SEQA bajo el control del promotor T7, </li><li> Placa de la reacción de transformación en placas de agar LB-incluyendo 100 mg / ml de ampicilina, </li><li> Recoger colonias mixtas para inocular un cultivo durante la noche a pequeña escala (medio LB con 100 mg / ml de ampicilina), </li><li> A la mañana siguiente inocular un 1 L de medios de comunicación usando una dilución 1:100 de la overnight cultura, </li><li> Cultivar las células hasta una DO 600 de 0,7 ~ e inducir la producción de proteínas por adición de isopropil β-D-1-tiogalactopiranósido (IPTG) a una concentración final de 1 mM, </li><li> Continuar la incubación durante 3 horas a 37 ° C con agitación orbital y entonces recoger las células por centrifugación (10 min a 3.300 g), </li><li> Resuspender el sedimento celular en tampón de purificación A y lisar por sonicación, </li><li> Borrar el lisado por centrifugación (40 min a 39.000 g) y cargar el sobrenadante en una columna de heparina equilibrada con tampón de purificación, </li><li> SEQA Elute utilizando un gradiente lineal de NaCl 1 M (SEQA eluye a ~ NaCl 0,7 M), </li><li> Piscina las fracciones que contienen SEQA juntos, se diluye para disminuir la fuerza iónica de la muestra y de la carga en una columna de cromatografía de intercambio catiónico equilibrada con tampón de purificación, </li><li> Utilizando un gradiente lineal de sal, puro SEQA eluye a ~ NaCl 0,4 M, </li><li> Piscina las fracciones que contienen SEQA juntos, concentrane (3 mg / ml) y almacenar en tampón de almacenamiento. </li></ol> 2. Purificación de ADN <ol><li> Solicitar oligonucleótidos complementarios unmethylated metilado y de su compañía favorita, </li><li> Se disuelve 1 mol de cada cadena de ADN único liofilizado en 800 l de autoclave ddH 2 O, vórtice y dejar que repose durante 10-20 min, </li><li> Añadir 800 l de tampón de carga 2X precalentado a cada oligonucleótido, </li><li> Para oligonucleótidos 20-30 nucleótidos de largo, preparar una gran gel desnaturalizante 10% (160 x 250 x 1 mm): * Mezclar bien 80 ml de la mezcla PÁGINA 10%, 80 l TEMED y 800 l por persulfato de amonio gel y verter, * Una vez polimerizado, retire el peine y lave los pocillos con fondo ddH 2 O, * Reúne los geles de reparto gel incluyendo placa de enfriamiento y luego llenar los depósitos superior e inferior con tampón de corrida (1X TBE), * Pre-correr el gel a 700-750 V para calentar el gel hasta 55 ° C, * Detener la carrera y enjuague el thoro pozosughly con tampón de corrida. </li><li> Calentar los oligonucleótidos a 90 º C durante 2 min, </li><li> Vortex y centrifugar las muestras e inmediatamente antes de cargar el gel, </li><li> Correr el gel a 700 V ~ y detenerlo una vez que su oligonucleótido se ha desplazado hasta la mitad. (Tenga en cuenta que en un gel de poliacrilamida al 10% de azul de bromofenol co-migra con oligonucleótidos ~ 20 bases de largo y xileno cianol FF con ~ 60 bases de largo), </li><li> Detener el gel, se desmonte de la caja de gel y retirar los separadores, </li><li> En una superficie plana, retire una placa de vidrio y cubrir el gel con una envoltura de plástico, </li><li> Gire el gel alrededor, retire la placa de vidrio otra y se cubre con una envoltura de plástico, </li><li> Marque las bandas con luz UV y una placa fluorescente detrás del gel para ver la sombra de ADN, </li><li> Cortar la banda con una cuchilla de afeitar en trozos pequeños y los transfieren a un tubo estéril de 15 ml, </li><li> Añadir 9 ml de tampón de elución y eluir durante la noche a 37 ° C con agitación, </li><li> Transferir cuidadosamente la solucióna un tubo de centrífuga esterilizado en autoclave usando una pipeta de punta de carga de gel para evitar la transferencia de piezas de acrilamida y añadir 1 ml de acetato de sodio 3 M a pH 7 (dilución 1:10) y 25 ml de etanol enfriado 100% (2,5 volúmenes), </li><li> Incubar a -20 º C durante al menos 3 h, se </li><li> Decantar el sobrenadante y transferir a un tubo separado, </li><li> Secar el pellet de la velocidad-vac a fuego medio, </li><li> Resuspender el pellet en 400 l de autoclave ddH 2 O y transferir a un tubo nuevo, </li><li> Añadir 40 l de acetato de sodio 3 M a pH 7 y 1 ml de etanol al 100%, mezclar bien (vórtice) e incubar 30 min a temperatura ambiente, seguido por 30 min a -20 ° C, </li><li> Girar durante 15 minutos a 18.000 g y descartar el sobrenadante, </li><li> Enjuague el precipitado con 100 l de etanol al 70% frío para eliminar la sal residual del sedimento y centrifugado durante 6 min a 18.000 g. Deseche el etanol y secar el pellet de la velocidad-vac, </li><li> Resuspender el precipitado en un total de 100 l de autoclave ddH <sub&gt; 2 O. Medir la concentración del oligonucleótido, </li><li> Para reasociar los dúplex de ADN hemimethylated, mezclar concentraciones equimolares de las cadenas simples complementarias y calentar las mezclas a 95 ° C en un baño de agua durante 5 minutos y después se dejó enfriar lentamente a temperatura ambiente en el interior del baño de agua. </li></ol> 3. Proteína-ADN formación de complejos y Análisis <ol><li> Mezclar volúmenes iguales de SeqAΔ purificado (41-59)-A25 (81 mM) y ADN hemimetilado (81 mM), </li><li> Incubar a temperatura ambiente durante 15 min y se almacena a 4 º C hasta que esté listo para usarlo, </li><li> Pantalla de condiciones de cristalización utilizando escaso comerciales de matriz pantallas, </li><li> Una vez pistas iniciales de cristalización se han identificado, optimizar las condiciones para hacer crecer cristales de difracción de calidad, </li><li> Cryoprotect los resultantes de ADN SEQA-cristales, ya sea aumentando la cantidad de PEG 400 presente en la solución de cristalización a una concentración final de 25% (v / v) o la adición de glicerol al 20% (v / v) a la solución de cristalización, </li><li> Recoger los cristales individuales con un lazo de nylon, y los flash-congelar en nitrógeno líquido, </li><li> Pruebe el límite de difracción de cada cristal a 100 K. </li></ol> 4. Los resultados representativos Para obtener la estructura cristalina de SeqAΔ (41-59)-A25R unido al ADN hemimetilado, que consecutivamente tres parámetros optimizados en el ADN: (i) la separación entre hemimethylated secuencias GATC, (ii) la longitud del dúplex, y la (iii) ausencia / presencia de salientes 5 &#39;. Electro-ensayos de cambio de movilidad indican que SeqAΔ (41-59)-A25R se une preferentemente a repeticiones GATC separadas por 9-10 pares de bases (Figura 1). Por lo tanto, inicialmente proyectado dúplex 23-24 pares de bases (pb) de largo que contiene dos secuencias GATC hemimethylated separados por 9 o 10 puntos básicos. Tres dúplex produjo cristales bien formados (Figura 2). Alaunque ninguno de los cristales de difracción de alta resolución, el 23 bps largo dúplex con los dos sitios GATC separadas por 9 bps rayos X difractados mejor que el resto, lo que indica que una separación GATC de 9 bps se prefirió para la cristalización. Por lo tanto, se fijó la distancia entre GATC a 9 puntos básicos para todas las pantallas posteriores. En general, la cristalización se ve favorecida por ADN dúplex correspondientes a longitudes exactas vueltas helicoidales porque múltiples moléculas de ADN pueden apilar de cabeza a cola para formar un continuo B-DNA dentro del cristal 9. Por lo tanto, se acorta la longitud total de los dúplex a 21 bps (es decir, dos vueltas helicoidales). Mientras que un dúplex 21 bps con extremos romos no dió cristales de difracción de calidad (datos no mostrados), un dúplex 21 bps con un solo saliente 5 &#39;de nucleótidos en cada extremo que dió cristales difractan a 5 A en nuestros fuente de origen. La mejora en el límite de difracción sugerido que de extremo a extremo dúplex asociación fue en efecto favorecerING cristal de embalaje. Desde SEQA interactúa con secuencias GATC que están en la misma cara del ADN, la cara opuesta del ADN dúplex debe estar expuesto al disolvente. Para mejorar aún más los cristales del complejo, que modifica entonces la secuencia de los dúplex para incluir un dinucleótido CG entre los dos sitios GATC para promover la cadena principal de ranura interacciones con moléculas de ADN adyacentes en el cristal a través de la cara opuesta de la doble hélice, un método que se ha utilizado para mejorar la cristalización del ADN en el pasado 10. Sin embargo, el límite de difracción de los cristales crecidos con el dúplex que contiene CG era idéntica a las cultivadas con un dúplex de ADN similares que no contienen el dinucleótido CG (Figura 3). Este resultado indica que la cadena principal de ranura interacciones no fueron importantes en este caso. Estamos posteriormente optimizado la longitud de los salientes mediante la comparación de los cristales crecidos con un dúplex de ADN que tenía dos nucleótidos adicionales en cadaExtremo 5 &#39;. Este cambio tuvo un efecto drástico en la morfología del cristal, así como, límite de difracción adicional que indica que el nucleótido cambiado drásticamente los contactos moleculares y organización cristalina mejorada (Figura 3). La estructura cristalina de SeqAΔ (41-59)-A25R unido a esta última dúplex de ADN confirmó que la cara libre de la doble hélice de ADN no participa en contactos de cristal, como el esperado del efecto limitado de la introducción de un dinucleótido CG-. A pesar del tamaño relativo de las proteínas y el ADN, la mayoría de las interacciones entre los compañeros de simetría están mediadas por interacciones proteína-proteína y proteína-DNA-(Figura 4). Curiosamente, en este caso particular, el efecto beneficioso de un voladizo dinucleótido 5 &#39;no es debido a la formación de un ADN de pseudo-continuo. En cambio, el extremo 5 &#39;de los proyectos de cadena metilada de distancia de los ejes del ADN e interacciona con la molécula SEQA proximal del complejo, que explica por qué dos nucleótidos w<html> ere estrictamente necesario para cambiar el cristal embalaje 8,11. <img alt="Figura 1" src="/files/ftp_upload/4266/4266fig1.jpg" /> Figura 1. La unión de SeqAΔ (41-59)-A25R a hemimethylated ADN. (A) Representación esquemática de los dominios de oligomerización SEQA que median la proteína y unión de ADN. (B) la movilidad electroforética cambio de ensayo de SeqAΔ (41-59)-A25R con los ADN que contienen dos secuencias GATC hemimethylated separados por un número cada vez mayor de pares de bases ( X). El carril de más a la izquierda (con la etiqueta en blanco) contiene una mezcla equimolar de los ADN con 5, 7, 12, 21, 25 y 34 pares de bases entre las dos secuencias GATC en ausencia de SeqAΔ (41-59)-A25R. (C) Diagrama que representa las tres variables optimizados en los dúplex de ADN para obtener cristales de difracción de calidad. upload/4266/4266fig2.jpg &quot;fo: content-width =&quot; 6in &quot;fo: src =&quot; / files/ftp_upload/4266/4266fig2highres.jpg &quot;/&gt; Figura 2. Efecto de la variación de la distancia inter-GATC. Resumen de los oligonucleótidos utilizados y los cristales obtenidos con 23-24 pb de largo dúplex que contienen dos sitios GATC hemimethylated separadas por 9 o 10 pares de bases. Todas las imágenes de los cristales fueron tomadas con el mismo aumento y la barra de escala indica 100 micras. El límite de resolución de cada SeqAΔ (41-59)-A25R-DNA cristal se basa en imágenes de difracción recogidos en un Rigaku RU-300 sistema de generador de rayos-X. <a href="http://www.jove.com/files/ftp_upload/4266/4266fig2large.jpg" target="_blank">Haga clic aquí para ampliar la cifra</a> . <img alt="Figura 3" src="/files/ftp_upload/4266/4266fig3.jpg" fo:content-width="6in" fo:src="/files/ftp_upload/4266/4266fig3highres.jpg" /> Figura 3. Efecto de la variación de los extremos de ADN. Resumen de tél oligonucleótidos utilizados y obtenidos cristales con 21 dúplex bps largos que contiene dos sitios GATC hemimethylated separadas por 9 pares de bases e incluyendo 0, 1 o 2 nucleótidos salientes 5 &#39;finales. Todas las imágenes de los cristales fueron tomadas con el mismo aumento y la barra de escala indica 100 micras. El límite de resolución de cada SeqAΔ (41-59)-A25R-DNA cristal se basa en imágenes de difracción recogidos en líneas de luz y X12C X29 (NSLS, BNL). <a href="http://www.jove.com/files/ftp_upload/4266/4266fig3large.jpg" target="_blank">Haga clic aquí para ampliar la cifra</a> . <img alt="Figura 4" src="/files/ftp_upload/4266/4266fig4.jpg" /> Figura 4. Embalaje de cristal de SeqAΔ (41-59)-A25R unido al ADN con saliente dinucleótido: Visto desde la parte superior (A) y lateral (B). La unidad asimétrica contiene dos SeqAΔ (41-59) de ADN A25R complejos donde se muestra la proteína en gris, mientras que el ADN se muestra en color naranja. Symmetria relacionada SeqAΔ (41-59)-A25R moléculas de ADN se muestran en blanco para la proteína y amarillo para el ADN. Esta cifra se preparó utilizando PyMOL 12. Esta cifra está relacionada con película 1. <a href="http://www.jove.com/files/ftp_upload/4266/4266fig4large.jpg" target="_blank">Haga clic aquí para ampliar la cifra</a> . Película 1. <a href="http://www.jove.com/files/ftp_upload/4266/4266movie1.avi" target="_blank">Haga clic aquí para ver la película</a> .

<ol>
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No hay conflictos de interés declarado.

Uno de los mayores retos en macromolecular cristalografía de rayos X es la obtención de cristales de difracción de calidad. En el caso de complejos de proteínas o ADN-proteína, este reto se agrava debido a las variables adicionales que deben ser optimizados. Se cree ampliamente que la longitud del ADN y la presencia de voladizos pegajosas para mejorar la asociación de moléculas de ADN vecinos en un tiempo pseudo-duplex son los principales parámetros a optimizar. Sin embargo, hemos demostrado que la naturaleza y ...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Catálogo # </td><td> Comentarios (opcional) </td></tr><tr><td> TRIS </td><td> Bioshop </td><td> TRS003.5 </td><td></td></tr><tr><td> Ácido etilendiaminotetraacético (EDTA) </td><td> Fisher Scientific </td><td> E478-500 </td><td></td></tr><tr><td> Dithiotheitrol (TDT) </td><td> Bio Basic Inc. </td><td> DB0058 </td><td></td></tr><tr><td> NaCl </td><td> Bioshop </td><td> SOD002.10 </td><td></td></tr><tr><td> Glicerol </td><td> Caledon </td><td> 5350-1 </td><td></td></tr><tr><td> Sacarosa </td><td> Sigma-Aldrich </td><td> S5016-500G </td><td></td></tr><tr><td> Dodecil sulfato de sodio (SDS) </td><td> Bioshop </td><td> SDS001.500 </td><td></td></tr><tr><td> Urea </td><td> Bioshop </td><td> URE001.5 </td><td></td></tr><tr><td> 40% Bis 29:1 / acrilamida </td><td> Bio Basic Inc. </td><td> A0007-500ml </td><td> Almacenar a 4 ° C </td></tr><tr><td> El ácido bórico </td><td> EMD </td><td> BX0865-1 </td><td></td></tr><tr><td> Xileno FF cianol </td><td> Bio-Rad </td><td> 161-0423 </td><td></td></tr><tr><td> Azul de bromofenol </td><td> Bioshop </td><td> BR0222 </td><td></td></tr><tr><td> Sistema Dual Adjustable Gel Vertical </td><td> CBC Scientific Company Inc. </td><td> DASG-250 </td><td></td></tr><tr><td> Índice de pantalla cristalización </td><td> Hampton Research </td><td> HR2-144 </td><td> Almacenar a 4 ° C </td></tr><tr><td> Asistente Yo pantalla cristalización </td><td> BioSystems Esmeralda </td><td> EBS-WIZ-1 </td><td> Almacenar a 4 ° C </td></tr><tr><td> Wizard II pantalla de cristalización </td><td&gt; Emerald BioSystems </td><td> EBS-WIZ-2 </td><td> Almacenar a 4 ° C </td></tr><tr><td> Clásicos pantalla cristalización </td><td> Qiagen </td><td> 130701 </td><td> Almacenar a 4 ° C </td></tr><tr><td> Intelliplate bandejas </td><td> Art Robbins Instrumentos </td><td> 102-0001-00 </td><td></td></tr><tr><td></td><td></td><td></td><td> Soluciones Proteína tampón de purificación: 100 mM Tris pH 8, 2 mM EDTA, 2 mM DTT y 5% de glicerol. Proteína tampón de almacenamiento: 20 mM Tris pH 8, 150 mM NaCl, 5 mM DTT, 0,5 mM EDTA y 5% de glicerol. Gel mezcla de carga: Añadir 20 g de sacarosa, 25 mg de azul de bromofenol, 25 mg de xileno cianol FF, 1 ml de 10% w / v de SDS y 10 ml de TBE 10X a 70 ml de ddH autoclave 2 O. Se agita con calentamiento suave hasta que la sacarosa se disuelve y ajustar la finalvolumen a 100 ml con ddH autoclave 2 O. Almacenar a 4 ° C. Tampón de carga 2X: Añadir 11 g de urea a 10 ml de mezcla de carga de gel. Se agita sobre una placa caliente hasta que se disuelve la urea. Dividir en partes alícuotas en tubos de 2 ml y se almacena a 4 º C. Mezcla PÁGINA 10X: Mix 420,4 g de urea, 100 ml de TBE 10X (autoclave), 250 ml de 40% 29:1 Bis / acrilamida en ddH 2 O. Agitar hasta que esté totalmente disuelto y ajustar el volumen a 1 litro. Almacene en botellas oscuras a 4 ° C. 10X TBE: Disolver 108 g de TRIS, 55 g de ácido bórico y 9,3 g de EDTA en 1 litro de ddH 2 O. Autoclave y almacenar a temperatura ambiente. Tampón de elución: Diluir 8 ml de 5 M NaCl, 2 ml de 1 M Tris pH 7,5, 0,4 ml de 0,5 M EDTA pH 8 en 200 ml de ddH 2 O. Autoclave y almacenar a temperatura ambiente. </td></tr></tbody></table&gt;

iterative optimization of dna duplexes for crystallization of seqa-dna complexes

Escherichia coli SEQA es un regulador negativo de la replicación del ADN que impide prematuros eventos reiniciación por secuestrantes GATC grupos hemimethylated dentro del origen de replicación 1. Más allá del origen, SEQA se encuentra en las horquillas de replicación, donde se organiza ADN recién replicado en mayores estructuras ordenadas 2. Asociados SEQA sólo débilmente con las secuencias GATC individuales, sino que forma complejos de alta afinidad con dúplex de ADN que contienen múltiples sitios GATC. La unidad mínima funcional y estructural de SEQA es un dímero, lo que explica el requisito de al menos dos secuencias GATC para formar un complejo de alta afinidad con el ADN hemimethylated 3. Además, la arquitectura SEQA, con la oligomerización y dominios de unión al ADN separados por un enlazador flexible, permite la unión a repeticiones GATC separados por hasta tres vueltas helicoidales. Por lo tanto, la comprensión de la función de SEQA a nivel molecular estructural requiere la analisis de SEQA unido a múltiples secuencias GATC. En la cristalización de proteínas-ADN, el ADN puede tener ninguno a un efecto excepcional en las interacciones de embalaje en función de los tamaños relativos y la arquitectura de la proteína y el ADN. Si la proteína es más grande que el ADN o huellas de la mayoría del ADN, el cristal de embalaje es principalmente mediada por interacciones proteína-proteína. A la inversa, cuando la proteína es del mismo tamaño o más pequeño que el ADN o sólo cubre una fracción de las interacciones ADN, ADN-ADN y ADN-proteína dominar cristal de embalaje. Por lo tanto, la cristalización de proteínas de ADN complejos requiere el cribado sistemático de ADN de longitud y 4 extremos de ADN (romo o saliente) 5-7. En este reporte se describe cómo diseñar, optimizar, purificar y cristalizar hemimethylated dúplex de ADN que contienen repeticiones en tándem GATC en el complejo con una variante dimérica de SEQA (SeqAΔ (41-59)-A25R) para obtener cristales adecuados para la determinación de la estructura.

Watch this Scientific Journal Video about Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes at JoVE.com

Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

Estructura cristalina de los complejos de proteínas de ADN puede proporcionar información sobre la función de proteínas, el mecanismo, así como, la naturaleza de la interacción específica. Aquí reportamos cómo optimizar la longitud, la secuencia y los extremos del ADN dúplex en co-cristalización con<em&gt; Escherichia coli</em&gt; SEQA, un regulador negativo de la iniciación de la replicación.

Iterativo de optimización de dúplex de ADN para la cristalización de SEQA-ADN complejos

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters ...

iterative-optimization-dna-duplexes-for-crystallization-seqa-dna

Research

JoVE Journal

Biology

9.9K vistas.  McMaster University. Estructura cristalina de los complejos de proteínas de ADN puede proporcionar información sobre la función de proteínas, el mecanismo, así como, la naturaleza de la interacción específica. Aquí reportamos cómo optimizar la longitud, la secuencia y los extremos del ADN dúplex en co-cristalización con Escherichia coli SEQA, un regulador negativo de la iniciación de la replicación.

Video: Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.
By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.
While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 &#x2013; 90kDa.

La evaluación de dos dimensiones ensayos de cristalización de proteínas de membrana pequeña de Estudios de Biología Estructural de Cristalografía de electrones

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

Investigación

Biología

Protein Crystallization for X-ray Crystallography

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The availability of atomic resolution structures provides a deep and unique understanding of protein function, and helps to unravel the inner workings of the living cell. To date, 86% of the Protein Data Bank (rcsb-PDB) entries are macromolecular structures that were determined using X-ray crystallography.
To obtain crystals suitable for crystallographic studies, the macromolecule (e.g. protein, nucleic acid, protein-protein complex or protein-nucleic acid complex) must be purified to homogeneity, or as close as possible to homogeneity. The homogeneity of the preparation is a key factor in obtaining crystals that diffract to high resolution (Bergfors, 1999; McPherson, 1999).
Crystallization requires bringing the macromolecule to supersaturation. The sample should therefore be concentrated to the highest possible concentration without causing aggregation or precipitation of the macromolecule (usually 2-50 mg/ mL). Introducing the sample to precipitating agent can promote the nucleation of protein crystals in the solution, which can result in large three-dimensional crystals growing from the solution. There are two main techniques to obtain crystals: vapor diffusion and batch crystallization. In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuses out of the drop until the osmolarity of the drop and the precipitant are equal (Figure 1A). The dehydration of the drop causes a slow concentration of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram. The batch method relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant (Figure 1B). This method is usually performed under a paraffin/mineral oil mixture to prevent the diffusion of water out of the drop.
Here we will demonstrate two kinds of experimental setup for vapor diffusion, hanging drop and sitting drop, in addition to batch crystallization under oil.

The 3-D structure of a molecule provides a unique understanding of how the molecule functions. The principal method for structure determination at near-atomic resolution is X-ray crystallography. Here, we demonstrate the current methods for obtaining three-dimensional crystals of any given macromolecule that are suitable for structure determination by X-ray crystallography.

De cristalización de proteínas para la cristalografía de rayos X

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

La cristalización de proteínas de membrana en mesofases lipídico

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

De alto rendimiento de cristalización de proteínas de membrana Utilizando el Método lipídico Bicelle

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

El uso de un robot para la cristalización de alto rendimiento de proteínas de membrana en mesofases lipídicas

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Identificación de modificaciones post-traduccionales de la proteína vegetal Complejos

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

La optimización y utilización de<em&gt; Agrobacterium</em&gt; Mediada por la producción de proteína transitoria en<em&gt; Nicotiana</em

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

Cartográficas basadas en AFM de las propiedades elásticas de las paredes celulares: en tejido, celular, y las Resoluciones Subcelulares

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

Imágenes en tiempo real de Soltero Engineered ARN transcritos en células vivas utilizando Ratiometric bimoleculares Balizas

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

Generación y purificación de Humanos INO80 remodelación de la cromatina complejos y Subcomplejos

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...