Este trabajo fue apoyado por un Premio al Investigador Burroughs Wellcome para YM y por una beca postdoctoral de Brown-Coxe de la Universidad de Yale a MD.

Materiales: <ul><li> Muestra de la proteína - lisozima (50 mg / mL) </li><li> Colgando caída de 24 y la bandeja </li><li> Sentado caída de 24 y la bandeja </li><li> Batch de cristalización 96 bandeja y </li><li> Soluciones de cristalización (ya sea comercial, o de fabricación casera) </li><li> Grasa de silicona </li><li> 5 ml jeringa sin Luer-lock </li><li> Diapositivas con silicona cubrir </li><li> Cinta aislante óptico </li><li> Parafina </li><li> 0.1-2 l micropipeta con puntas de baja retención </li><li> Pinzas </li><li> Toallitas profesional </li></ul> 1. Colgando de estar / Procedimiento de caída: <ol><li> Para una pantalla inicial se puede utilizar pantallas disponibles en el mercado que aprovechan la matriz dispersa incompleta método factorial de las condiciones de ensayo. La matriz dispersa incompleta método factorial está sesgada por y seleccionados de las condiciones de cristalización de macromoléculas conocido (Carter y Carter, 1979; Cudney et al, 1994;. Jancarik y Kim, 1991;. Jancarik et al, 1991). Si la condición de cristalización que ya se sabe, una pantalla estrecha se puede montar, la variación de la concentración y el pH de precipitación de todo el éxito original. El precipitante más exitoso es el polietilenglicol (PEG), seguido de sulfato de amonio. En conjunto, estos dos factores precipitantes cuenta de aproximadamente el 60% de todos los factores precipitantes macromoleculares registrado utilizados para la cristalización (Gilliland et al, 1994;. Kimber et al, 2003;.. Page et al, 2003). </li><li> Prepare las soluciones de cristalización. Por lo general, estas condiciones se combinan de un agente de precipitación, la sal de algún tipo y un tampón para ajustar el pH. En algunos casos se pueden añadir aditivos específicamente para la proteína de interés. Todas solución madre debe ser filtrado con filtro de 0,22 micras. Algunas de la solución podría ser muy viscoso (50% PEG, glicerina, PEG-MME, etc.) <ul><li> Preparar 1.5 mL de 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 M NaCl en cada una de 0,1 M pH 4,0 NaOAc, 4.3, 4.6 y 4.9 (un total de 24 soluciones) </li></ul></li><li> Descongele la muestra proteínas y mantener en hielo. Reservas de proteínas debe ser almacenadas a -80 ° C a menos que la proteína de interés es sensible a la congelación / descongelación (si es así, se almacenan a 4 ° C). Las concentraciones típicas de proteínas están en el rango de 50-50 mg / mL, con concentraciones más altas suelen dar mejores resultados. </li><li> Girar la muestra 15min/18, 000xg / 4 ° C - algunas proteínas tienden a parcialmente agregan y precipitan tras un paso deshielo. Estos agregados pueden promover la precipitación amorfa del resto de las moléculas de proteína por lo tanto, giro completo hacia abajo se asegurará de que sólo las moléculas solubles en la muestra. Impiden que la proteína en hielo hasta la creación de la bandeja. </li><li> Determinar la concentración de proteínas a partir de su absorbancia a 280 nm utilizando un espectrofotómetro UV. La concentración de proteína puede ser calculada a partir de la lectura y el coeficiente de absorción de la proteína extinción. El coeficiente de extinción puede ser calculado con la herramienta ProtParam en el sitio web Expasy: <a href="http://ca.expasy.org/tools/protparam.html">http://ca.expasy.org/tools/protparam.html</a> </li><li> Llenar los pozos de los colgantes de 24 y / sala bandeja de caída con 500 l de la solución precipitante de acuerdo con el esquema de configuración de la bandeja (Figura 2B). Crear un anillo de grasa de silicona alrededor del borde del pozo. El anillo debe tener un pequeño espacio para evitar la acumulación de presión de aire en el pozo está sellado. En forma de gotas de estar, no hay necesidad de grasa ya que el pozo está sellado con cinta adhesiva (Figura 2A). </li><li> Tome una tapa deslizante y limpia con spray de aire condensado, o un profesional limpiar - el polvo para evitar facilitará la interpretación de la caída de la cristalización y eliminar la contaminación. Para sentarse gotas, deslice la cubierta no se utilizan. En cambio, el pozo tiene una estantería en la que se mezclan la proteína y precipitante. </li><li> Ponga volúmenes iguales de la muestra de la proteína y la solución de reserva de la tapa deslizante. Diferentes volúmenes de la proteína y precipitante puede ser tratado como parte del proceso de optimización. El uso de más proteína que los resultados de precipitación en una concentración neta de la proteína en la caída después del equilibrio, que puede ser deseable para diluir las muestras de proteínas, y para frenar la nucleación de cristales o de crecimiento. Cualquier otra sustancia volátil en la solución de colocar también se concentrará en el mismo factor. Cuando la proteína de pipeteo y soluciones de depósito en la tapa deslizante, extremar las precauciones para evitar la formación de burbujas. A menudo, esto ocurre cuando el aire es expulsado de la pipeta. <ul><li> Colgando caída: 2 l de carga de 50 mg / ml lisozima en 0,02 M de pH 4,9 a NaOAc el centro de la tapa deslizante y añadir a 2 l de la solución de reserva. </li><li> Sentado gota: 2 l de carga de 50 mg / ml de solución de lisozima en el centro de la plataforma y agregar a ella 2 l de solución de reserva. </li></ul></li><li> Voltear la tapa deslizante con cuidado y ponerla en el anillo de grasa en la parte superior del pozo. Presione con suavidad para que el aire puede escapar del pozo a través de la muesca de la grasa para prcaso de la acumulación de presión de aire en el pozo y mantener el bien cerrados. Acumulación de presión de aire en el pozo puede aumentar la tapa deslizante, lo que compromete el sello también. En el método de la gota sentada, cinta adhesiva transparente ópticamente transparente se utiliza para cubrir los pozos. Por lo tanto, en este método de sellado se llevará a cabo todas las filas / columnas. </li><li> Pasar a la siguiente, así hasta que la bandeja está completa. </li><li> Compruebe la bandeja de la configuración del - la eliminación de partículas de polvo y otros contaminantes puede reducir la identificación de falsos positivos de cristales de proteínas. </li><li> Use una hoja de puntuación para documentar su experiencia. </li><li> Coloque la bandeja en la temperatura de incubación deseado, por lo general entre 4 ° C y la temperatura ambiente. 20 ° C es el más exitoso. Tenga cuidado al manejar la bandeja con cuidado y evitar cualquier agitación. Tenga cuidado al abrir y cerrar la puerta de la incubadora. Vibraciones o cambios de temperatura durante el traslado o la incubación puede prevenir el crecimiento de cristales o afectar negativamente a la calidad del cristal. Un shock de material absorbente (espuma de empaque, por ejemplo) puede ser utilizado como relleno en las bandejas de cristal. </li><li> Compruebe la bandeja de cristales al día siguiente, y luego cada pocos días, siempre el manejo de las bandejas con cuidado. Documento las conclusiones y morfologías caída de una hoja de la bandeja de puntuación específica. Los cristales suelen tardar 2-5 días en aparecer, aunque en ocasiones aparecen cristales mucho antes (casi de inmediato) o posterior (hasta varios meses!). Una vez que las condiciones de cristalización se han identificado y la tasa de crecimiento de los cristales es conocida, lo mejor es dejar las bandejas tranquilo hasta que los cristales han aparecido y crecido por lo menos a la mitad de su tamaño final. Dejando las bandejas sin perturbaciones pueden reducir el número de eventos de nucleación de cristales, por lo tanto, producir un número menor de grandes cristales. </li></ol> 2. Batch de procedimiento: <ol><li> Muestra de la proteína y la preparación de precipitación son como se describió anteriormente. </li><li> Pistola de aire una bandeja batch nuevo para evitar el polvo y otras partículas grandes. </li><li> En la bandeja de batch, llenado de aceite de parafina hasta 3 mm de alto (lo suficiente para cubrir los pozos). Eliminar el acceso de petróleo. </li><li> Cargar una L de solución de proteína (50 mg / ml lisozima) en el aceite precargada así - asegúrese de que la solución de pipeta directamente al fondo del pozo (Figura 2). </li><li> Cargar una L de solución precipitante en el acuerdo, así que el esquema de configuración de la bandeja (Figura 2B) - asegúrese de que la caída se hunde hasta el fondo del pozo y se funde con la gota de proteínas. </li><li> Pasar a la siguiente, así hasta que la bandeja está completa. </li><li> Siga los pasos 11-14 de la cortina de estar / drop procedure. </li></ol> 3. Los resultados representativos: La cristalización es generalmente conocido como el cuello de botella de la cristalografía de rayos X. Una matriz dispersa incompleta pantalla factorial de las condiciones de precipitación suele producir muchos tipos diferentes de agregación de la proteína y las precipitaciones, entre ellos grandes monocristales. Si la proteína o las concentraciones de precipitación son muy altos se puede ver la materia de color marrón con forma distinta y no el tamaño (precipitación amorfa). Cuando la solución es insaturada, la caída suele ser del todo claro y carente de cualquier tipo de precipitación. La Figura 3 muestra varios ejemplos de los fenómenos de precipitación y cristales (Dessau et al., 2006). Más fenómenos de precipitación con una interpretación más detallada se puede encontrar en <a href="http://xray.bmc.uu.se/terese/tutorials.html">http://xray.bmc.uu.se/terese/tutorials.html</a> . <img src="/files/ftp_upload/2285/2285fig1.jpg" alt="Figura 1" /> Figura 1. El principio de la cristalización de proteínas. El principio de la cristalización de proteínas. En un experimento de difusión de vapor (A) volúmenes iguales de precipitación y las proteínas están presentes en la caída. El agua se difundirá a cabo y tanto la precipitación y la concentración de proteínas se duplicará hasta que se alcanza el equilibrio entre la gota y la solución de reserva. En la cristalización por lotes (B) de la precipitación y la concentración de proteínas no cambian durante el experimento. Punto A - proteína permanece undersaturated cristales no se pueden formar, punto B - nucleación de proteínas se producen, los cristales comenzaron a formarse y la concentración de proteínas en solución se reduce a la saturación. El punto C - proteína se precipita, pero los cristales pueden crecer todavía. (C) la cristalización de diálisis con un gel capilar conectado. Empiezan a aparecer cristales de la sal se difunde fuera de la muestra de proteína (y / o precipitante difunde en la muestra de proteína) a través del tapón de gel. <img src="/files/ftp_upload/2285/2285fig2.jpg" alt="Figura 2" /> Figura 2. Esquema típico de experimentos de cristalización de proteínas. (A) Procedimientos para colgar caída de la difusión del vapor, sentado caída de la difusión del vapor, y la cristalización de proteínas batch. En cada caso, un pequeño volumen de muestra de proteína concentrada se mezcla con un volumen igual o menor de precipitant solución y se dejó equilibrar. (B) Para la cristalización de la lisozima de pollo, una bandeja de 24 pocillos se configura con diferentes concentraciones de cloruro de sodio (la precipitación) y con 0,1 M de acetato de sodio como el tampón a pH diferentes (4,0 a 4,9). <img src="/files/ftp_upload/2285/2285fig3.jpg" alt="Figura 3" /> Figura 3. Resultados típicos de un experimento de cristalización de proteínas. (A) la precipitación amorfo. Cuando la proteína o factor precipitante (o ambos) se encuentran en alta concentración. (B) La separación de fases. Proteínas o detergentes pueden separar a una fase diferente cuando se mezcla con ciertos factores precipitantes en alta concentración. (C) los cristales en forma de barra de proteínas de AtCSN7 obtenidos en polietilenglicol 8000 y el acetato de magnesio (Dessau et al., 2006). (D) lisozima cristales obtenidos en 1,0 M NaCl y el pH de acetato de sodio 4.9. (E) En virtud de las gotas saturadas por lo general siguen siendo claras. Fotografía por Moshe Dessau.

<ol>
	<li>Bergfors, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Protein crystallization : techniques, strategies, and tips : a laboratory manual. , (1999).</li><li>Carter, C. W., Carter, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+crystallization+using+incomplete+factorial+experiments.">Protein crystallization using incomplete factorial experiments.</a> J Biol Chem. 254, 12219-12223 (1979).</li><li>Cudney, R., Patel, S., Weisgraber, K., Newhouse, Y., McPherson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+and+optimization+strategies+for+macromolecular+crystal+growth.">Screening and optimization strategies for macromolecular crystal growth.</a> Acta Crystallogr D Biol Crystallogr. 50, 414-423 (1994).</li><li>Dessau, M., Chamovitz, D. A., Hirsch, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression,+purification+and+crystallization+of+a+PCI+domain+from+the+COP9+signalosome+subunit+7+(CSN7).">Expression, purification and crystallization of a PCI domain from the COP9 signalosome subunit 7 (CSN7).</a> Acta Crystallogr Sect F Struct Biol Cryst Commun. 62, 1138-1140 (2006).</li><li>Gilliland, G. L., Tung, M., Blakeslee, D. M., Ladner, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+Macromolecule+Crystallization+Database,+Version+3.0:+new+features,+data+and+the+NASA+archive+for+protein+crystal+growth+data.">Biological Macromolecule Crystallization Database, Version 3.0: new features, data and the NASA archive for protein crystal growth data.</a> Acta Crystallogr D Biol Crystallogr. 50, 408-413 (1994).</li><li>Jancarik, J., Kim, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sparse+matrix+sampling:+a+screening+method+for+crystallization+of+proteins.">Sparse matrix sampling: a screening method for crystallization of proteins.</a> J Appl Cryst. 23, 409-411 (1991).</li><li>Jancarik, J., Scott, W. G., Milligan, D. L., Koshland, D. E., Kim, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+and+preliminary+X-ray+diffraction+study+of+the+ligand-binding+domain+of+the+bacterial+chemotaxis-mediating+aspartate+receptor+of+Salmonella+typhimurium.">Crystallization and preliminary X-ray diffraction study of the ligand-binding domain of the bacterial chemotaxis-mediating aspartate receptor of Salmonella typhimurium.</a> J Mol Biol. 221, 31-34 (1991).</li><li>Kimber, M. S., Vallee, F., Houston, S., Necakov, A., Skarina, T., Evdokimova, E., Beasley, S., Christendat, D., Savchenko, A., Arrowsmith, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Data+mining+crystallization+databases:+knowledge-based+approaches+to+optimize+protein+crystal+screens.">Data mining crystallization databases: knowledge-based approaches to optimize protein crystal screens.</a> Proteins. 51, 562-568 (2003).</li><li>Lorber, B., Sauter, C., Theobald-Dietrich, A., Moreno, A., Schellenberger, P., Robert, M. C., Capelle, B., Sanglier, S., Potier, N., Giege, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+growth+of+proteins,+nucleic+acids,+and+viruses+in+gels.">Crystal growth of proteins, nucleic acids, and viruses in gels.</a> Prog Biophys Mol Biol. 101, 13-25 (2009).</li><li>McPherson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Crystallization of biological macromolecules. , (1999).</li><li>McPherson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+protein+crystallization.">Introduction to protein crystallization.</a> Methods. 34, 254-265 (2004).</li><li>Page, R., Grzechnik, S. K., Canaves, J. M., Spraggon, G., Kreusch, A., Kuhn, P., Stevens, R. C., Lesley, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shotgun+crystallization+strategy+for+structural+genomics:+an+optimized+two-tiered+crystallization+screen+against+the+Thermotoga+maritima+proteome.">Shotgun crystallization strategy for structural genomics: an optimized two-tiered crystallization screen against the Thermotoga maritima proteome.</a> Acta Crystallogr D Biol Crystallogr. 59, 1028-1037 (2003).</li></ol>

En este artículo se describen y demuestran en general los protocolos actuales para la cristalización de proteínas. Ya que un procedimiento de múltiples pasos que hay algunas consideraciones hay que tener en cuenta. Cuando se trabaja con volúmenes muy pequeños (0.5-2 l), el secado de la caída debido a la evaporación es una preocupación importante. Por lo tanto, se recomienda trabajar en un entorno bien controlado (con flujo de aire de baja, alta humedad y control de la temperatura ajustada) y la adopción de una...

<table border="1">
		<tbody>
<tr>
<td>New Item</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Lysozyme</td>
<td>&nbsp;</td>
<td>Sigma-aldrich</td>
<td>L6876-1G</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>24 well VDX Plate</td>
<td>&nbsp;</td>
<td>Hampton research</td>
<td>HR3-142</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>24 well Cryschem Plate</td>
<td>&nbsp;</td>
<td>Hampton research</td>
<td>HR3-158</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Dow Corning Vacuum Grease</td>
<td>&nbsp;</td>
<td>Hampton research</td>
<td>HR3-510</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Siliconized glass circle coverslides</td>
<td>&nbsp;</td>
<td>Hampton research</td>
<td>HR3-231</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>100% paraffin oil</td>
<td>&nbsp;</td>
<td>Hampton research</td>
<td>HR3-411</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>1.88 inch wide Crystal Clear Sealing Tape</td>
<td>&nbsp;</td>
<td>Hampton research</td>
<td>HR3-511</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>96 Well Imp@ct Plate (Microbatch plate)</td>
<td>&nbsp;</td>
<td>Hampton research</td>
<td>HR3-098</td>
<td>&nbsp;</td>	</tr>	</tbody>
		</table>

protein crystallization for x-ray crystallography

Usando la estructura tridimensional de macromoléculas biológicas para deducir su funcionamiento es uno de los campos más importantes de la biología moderna. La disponibilidad de las estructuras de resolución atómica proporciona una comprensión profunda y única de la función de proteínas, y ayuda a desentrañar el funcionamiento interno de la célula viva. Hasta la fecha, el 86% de la Protein Data Bank (PDB-RCSB) las entradas son estructuras macromoleculares que se determinaron utilizando cristalografía de rayos X. Para obtener cristales adecuados para los estudios de cristalografía, la macromolécula (por ejemplo, proteínas, ácidos nucleicos y complejos proteína-proteína o proteína-ácido nucleico complejo) debe ser purificado a homogeneidad, o lo más cerca posible a la homogeneidad. La homogeneidad de la preparación es un factor clave en la obtención de cristales que se difractan a alta resolución (Bergfors, 1999; McPherson, 1999). La cristalización requiere llevar la macromolécula a la sobresaturación. La muestra, por tanto, concentra a la mayor concentración posible, sin causar la agregación o precipitación de la macromolécula (generalmente 20-50 mg / ml). La introducción de la muestra a un agente precipitante no puede promover la nucleación de cristales de proteínas en la solución, lo cual puede resultar en grandes cristales tridimensionales de crecimiento de la solución. Existen dos técnicas principales para obtener cristales: la difusión del vapor y la cristalización por lotes. En la difusión del vapor, una gota que contiene una mezcla de soluciones de precipitación y de la proteína se encuentra sellado en una cámara con precipitación puro. El vapor de agua se difunde fuera de la gota hasta que la osmolaridad de la gota y la precipitación son iguales (fig. 1A). La deshidratación de la caída provoca una lenta concentración de proteínas y precipitación hasta que se alcanza el equilibrio, a ser posible en la zona de la nucleación de cristales del diagrama de fase. El método se basa en lotes con lo que el de proteínas directamente en la zona de la nucleación de la proteína se mezcla con la cantidad apropiada de precipitación (fig. 1B). Este método se realiza generalmente bajo una parafina / mezcla de aceite mineral para evitar la difusión de agua de la gota. Aquí se muestran dos tipos de montaje experimental para la difusión del vapor, colgando caer y caer sentado, además de la cristalización por lotes en el aceite.

Crystallization is usually referred to as the bottleneck of X-ray crystallography. A sparse matrix incomplete factorial screen of precipitating conditions typically produces many different types of protein aggregation and precipitation, among them large single crystals. If the protein or precipitant concentrations are too high one can see brown matter with no distinct shape and size (amorphous precipitation). When the solution is undersaturated, the drop will often be completely clear and devoid of any kind of precipitat...

Watch this Scientific Journal Video about Protein Crystallization for X-ray Crystallography at JoVE.com

Protein Crystallization for X-ray Crystallography

La estructura 3-D de una molécula proporciona un conocimiento único de cómo funciona la molécula. El principal método para la determinación de la estructura en casi atómica resolución cristalografía de rayos X. En este sentido, demostrar los métodos actuales de obtención de cristales tridimensionales de cualquier macromolécula, dado que son adecuados para la determinación de la estructura por cristalografía de rayos X.

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 ...

protein-crystallization-for-x-ray-crystallography

Research

JoVE Journal

Biology

63.1K vistas.  Yale University. La estructura 3-D de una molécula proporciona un conocimiento único de cómo funciona la molécula. El principal método para la determinación de la estructura en casi atómica resolución cristalografía de rayos X. En este sentido, demostrar los métodos actuales de obtención de cristales tridimensionales de cualquier macromolécula, dado que son adecuados para la determinación de la estructura por cristalografía de rayos X.

Video: Protein Crystallization for X-ray Crystallography

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.
We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.

No invasivo en 3D de visualización con la Sub-micron resolución mediante sincrotrón de rayos X de la tomografía

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

Investigación

Biología

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Tinción de las proteínas en geles con Coomassie G-250, sin ácido disolvente orgánico y el acético

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Utilizando el sistema de fraccionamiento GELFREE 8100 de peso molecular basado en Fraccionamiento con la recuperación de la fase líquida

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

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 ...

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 ...

3D Printing of Preclinical X-ray Computed Tomographic Data Sets

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing. Traditional, mold-injection methods to create models or parts have several limitations, the most important of which is a difficulty in making highly complex products in a timely, cost-effective manner.1 However, gradual improvements in three-dimensional printing technology have resulted in both high-end and economy instruments that are now available for the facile production of customized models.2 These printers have the ability to extrude high-resolution objects with enough detail to accurately represent in vivo images generated from a preclinical X-ray CT scanner. With proper data collection, surface rendering, and stereolithographic editing, it is now possible and inexpensive to rapidly produce detailed skeletal and soft tissue structures from X-ray CT data. Even in the early stages of development, the anatomical models produced by three-dimensional printing appeal to both educators and researchers who can utilize the technology to improve visualization proficiency. 3, 4 The real benefits of this method result from the tangible experience a researcher can have with data that cannot be adequately conveyed through a computer screen. The translation of pre-clinical 3D data to a physical object that is an exact copy of the test subject is a powerful tool for visualization and communication, especially for relating imaging research to students, or those in other fields. Here, we provide a detailed method for printing plastic models of bone and organ structures derived from X-ray CT scans utilizing an Albira X-ray CT system in conjunction with PMOD, ImageJ, Meshlab, Netfabb, and ReplicatorG software packages.

Using modern plastic extrusion and printing technologies, it is now possible to quickly and inexpensively produce physical models of X-ray CT data taken in a laboratory. The three -dimensional printing of tomographic data is a powerful visualization, research, and educational tool that may now be accessed by the preclinical imaging community.

Impresión 3D de Preclínica de rayos X de datos tomografía computarizada Establece

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing.  Traditional, ...

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

Random microseed matrix screening (rMMS) is a protein crystallization technique in which seed crystals are added to random screens. By increasing the likelihood that crystals will grow in the metastable zone of a protein's phase diagram, extra crystallization leads are often obtained, the quality of crystals produced may be increased, and a good supply of crystals for data collection and soaking experiments is provided. Here we describe a general method for rMMS that may be applied to either sitting drop or hanging drop vapor diffusion experiments, established either by hand or using liquid handling robotics, in 96-well or 24-well tray format.

Here we describe a general method for random microseed matrix screening. This technique is shown to significantly increase the success rate of protein crystallization screening experiments, reduce the need for optimization, and provide a reliable supply of crystals for data collection and ligand-soaking experiments.

La mejora de la tasa de éxito de cristalización de proteínas por Random Matrix Microseed Screening

Random microseed matrix screening  (rMMS) is a protein crystallization technique in which seed crystals are added  to random screens. By increasing the ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Fluorescente verde a base de proteína Expresión Proyección de proteínas de membrana en<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

Efficient Expresión células de mamíferos y Single-step La purificación de glicoproteínas extracelular para la cristalización

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...