Agradecemos a nuestros colaboradores para proporcionar valiosas muestras de proteínas, lo que contribuyó a algunas de nuestras experiencias relacionadas con los métodos y observaciones. Günther Schmalzing amablemente ofreció la oportunidad de FR a unirse a este proyecto. Barbara Armbruster, Brink y Jacob Mills Deryck se les agradece por su gran ayuda y de entrada en el equipo. El financiamiento fue proporcionado por el NIH subvención HL090630.

1. Preparación de la red de ensayos de cristalización 2D <ol><li> Carbono recubiertos de malla de cobre de 400 redes de EM son preparados por tinción negativa. Acetato de uranilo es de uso frecuente y proporciona una mancha de larga duración en términos de almacenamiento de la solución durante varios meses antes de su uso, así como la idoneidad para el almacenamiento a largo plazo de las redes. En contraste, otros negativos, tales como manchas de formato de uranilo, mientras que proporciona una excelente tinción, deben estar recién hecho 1. Para la preparación rápida de un gran número de redes que se utilizan para la detección de los ensayos de cristalización 2D, una versión modificada de tinción negativa se utiliza. Un volumen de 2 l de la muestra es una pipeta en carbono cubierta rejilla EM y se incubó durante 60 s. Esto es seguido por transferencia desde el borde con un trozo de papel Whatman # 4 filtros (Figura 1, video), y luego 2 l de acetato de uranilo al 1% se aplican inmediatamente, que son borrados de nuevo al borde de la parrilla después de 30 s. Tocar la red en el borde con el borde roto del papel de filtro garantiza la eliminación óptima de líquido sin necesidad de retirar proteoliposomas. Además, el secado en el borde de la red asegura la mejor conservación de la película de carbono. Se debe tener cuidado en la preparación y manipulación de las redes, la rotura de la película de carbono delicada previene la adherencia de la muestra y puede resultar en una representación inexacta de la muestra. Si bien tradicionalmente grandes volúmenes de muestra de 5 l se y se utilizan habitualmente para la preparación de la red, preciosas muestras pueden ser salvados por la reducción del volumen de 2 l o menos 2. </li><li> Para producir la mayor membranas es posible, algunas de nuestras muestras requieren altas concentraciones de glicerol o sacarosa (10-20%) para estar presente en el tampón de diálisis. Esto puede tener un efecto negativo en la preparación de la red, como el glicerol o sacarosa es muy viscosa y el acetato de uranilo previene de penetrar adecuadamente el buffer, y por lo tanto incompleta tinción de las membranas. En consecuencia, una posible red se oscurecerán. El búfer se pueden intercambiar por centrifugación de las muestras y su sustitución por glicerol / sacarosa sin buffer (no se muestra), o las rejillas se pueden lavar con tampón o glicerol / sacarosa sin búfer en uno o varios ciclos antes de la tinción negativa similar a la una técnica utilizada para la crio-ME 3,4. </li></ol> 2. La evaluación de los ensayos de cristalización 2D por EM <ol><li> Dependiendo del tipo de soporte de la muestra, ya sea una o varias redes se cargan. Un aumento de 2-10K, que se hace referencia aquí como ampliación intermedia, permite una primera impresión de la distribución de media y el grado de dispersión de las membranas, la morfología y tamaño, que es tomado en consideración en el cuaderno de laboratorio 5. Zonas adecuadas se registran como una visión general representativa con la ayuda de una cámara CCD, o si una cámara CCD no está disponible, en la película. </li><li> Bajo aumento en el rango de alrededor de 400-800x se utiliza en momentos en que una visión general de toda la muestra / red que se desea. Aunque no todos los empleados con la red, bajo aumento da información valiosa de ayudar en la evaluación de la preparación de la red en cuanto a varios aspectos: tanto en tinción negativa y la rotura parcial del potencial de carbón, concentración de la muestra en la parrilla, y la distribución proteoliposoma posiblemente irregular. Cuadrados individuales de la red puede ser visto con los prismáticos o una cámara CCD. Con algunos mercados emergentes, es posible guardar las posiciones de interés particular, que pueden ser llamadas para una inspección posterior a mayores aumentos. </li><li> Una vez que el área de la cuadrícula de interés ha sido identificado, ya sea en aumento bajo o intermedio, la ampliación se cambia a aproximadamente 50K-60K. En función de la membrana, el cristal y el tamaño de la celda unitaria, si se conoce, aumentos tan bajo como 30K y 80K tan alto como se utilizan. El rango de aumento de 30-80K se conoce como una gran ampliación a los efectos de la detección de cristales 2D. Centrándose se produce ya sea en el ajuste del enfoque de la dosis baja de puesta en marcha, con el interruptor posterior a la creación de imagen / foto, o en las proximidades de la zona de interés. <ol class="lalpha"><li> En los casos de ambigüedad en si el área de interés es de hecho una membrana, se efectúa una prueba de carbono-film en el tamaño de proteoliposomas, mica, o de otros artefactos. Para ello, los bordes revelan plegables y morfología típicos. </li><li> Ahora el área de interés es inspeccionado con una cámara CCD. Una imagen CCD se recoge en el 30K-80K ampliación, dependiendo del tamaño de la membrana, proteínas o el tamaño de la celda unitaria, o zona conocida como cristalino. El entramado de una proteína de membrana más pequeño y / o hidrófobos en su mayoría no es necesariamente visible mediante la evaluación visual de la imagen CCD de sí mismo. Ya sea toda la imagen se utiliza para transformar una línea de Fourier (FT, o rápido FT-FFT). Esta FT contienen una cantidad significativa de ruido, sin embargo, si el arreglo ordenado es pequeño. Por lo tanto, una reducción del tamaño de imagen en caja va a permitir una mejor relación señal-ruido de un Smaller de cristal y una identificación más fácil. Para ello, la caja se mueve sobre la imagen y una M en directo se evalúa. La intensidad de brillo / de la viga se ajusta teniendo en dosis bajas condiciones de la muestra, así como los ajustes de cámara CCD en mente. Dependiendo de la cámara CCD utilizada, la gamma de la FT en vivo se ajusta para la identificación óptima de arreglos ordenados. Un valor demasiado alto puede ocultar manchas debido a las contribuciones de ruido, y un valor de gamma excesivamente baja evitará más débiles manchas de ser identificados. Estos puntos débiles puede ser debido a pequeños arreglos cristalinos con manchas en el Financial Times apenas por encima del nivel de ruido. Si bien los datos de mayor resolución que se ha recogido de un pequeño número de muestras de 6, normalmente la resolución de los cristales 2D con tinción negativa se limita o no se debe esperar a ser mejor que la resolución de aproximadamente 15 bis. Con un desenfoque de aproximadamente -400 nm, no más de 1-3 órdenes de los puntos se espera que sean fáciles de identificar. Las muestras generalmente no están evaluados para su resolución, como la crio-ME de recopilación de datos le dará una indicación correcta de la más alta resolución posible. Nitidez de las manchas y mosaicidad posibles señalar, no obstante. </li></ol></li><li> Diferentes membranas, así como la morfología de la membrana, son evaluados por el orden a gran aumento. Esto es especialmente importante al principio o etapas intermedias de los ensayos de cristalización en 2D, como proteoliposomas más pequeños en vez de más grande o más parches de membrana puede contener las áreas más prometedoras. Porcentajes muy bajos de cristales 2D requerirá la adquisición de imágenes y el FT, o la difracción óptica, de un gran número de imágenes desde la identificación inicial de arreglos ordenados con frecuencia conducen a una rápida mejora de su tamaño y calidad de 2,4,7. </li></ol> 3. Resultados representante Proteoliposomas Lo ideal sería que ordenó la pantalla fácilmente reconocibles, los puntos fuerte. Los cristales grandes y bien ordenada son identificados fácilmente por Internet-FT de imágenes CCD o la difracción óptica de microfotografías. El ejemplo muestra cristales 2D de una proteína de membrana pequeña de 18kDa que son de hasta varias micras de tamaño. Las manchas en el FT son fáciles de identificar y nítidas. El movimiento de la caja de vivir-M demuestra que la red es continuo, sin mosaicidad. El enrejado de una proteína más grande, con un dominio más amplio soluble se pueden identificar en la pequeña pantalla de la EM. Colección de imágenes CCD y FT es necesario para proporcionar un medio para mejorar la evaluación y para obtener información sobre, por ejemplo, mosaicidad, es posible (mostrar FT). En el cálculo de una FT de un proteoliposoma que no se impone, el ruido puede ser inicialmente confundido con manchas. Mientras que la caja para el live-M se mueve, sin embargo, las manchas desaparecerán. Por otro lado, arreglos pequeños, con cristalinidad cuestionable, tendrá sus puntos restantes inmóvil cuando el live-M se mueve ligeramente sobre el área de la imagen. Además, estos pequeños cristales se pueden reconocer por lo general tienen el tamaño de la misma unidad celular, y las distancias entre los puntos en diferentes FTS se puede medir de varias maneras, como con un círculo de un tamaño específico. Cristales de lípidos mostrar una morfología de red distintas y FT. No es raro encontrar la precipitación en los ensayos iniciales. Aquí la precipitación de proteínas sin reconstitución debe distinguirse de lípidos agregados pequeña sin embargo. Las muestras que parecen ser precipitados a bajo aumento con frecuencia resultan ser agregados de lípidos cuando se ve a mayor aumento. Tras la inspección a 30-50K, los bordes de estas estructuras oscuras revelan que se compondrá de las membranas sin precipitación de la proteína. Estas observaciones son importantes como los agregados de lípidos podría ser mayor en tamaño a las membranas grandes en los siguientes experimentos. Los pobres resultados obtenidos en las muestras de la evaluación a veces se conecta a una concentración baja de membrana que impide la adecuada selección y rápido. Con frecuencia, esto se puede superar con el uso de una concentración de proteína más alto para la cristalización 2D mediante diálisis. Por otra parte, las membranas se deja reposar durante unos días en la parte inferior del tubo de Eppendorf durante el almacenamiento. En algunos casos la solución rápida, o casi instantánea de las membranas se produce y pipeteo de la parte inferior del tubo se traducirá en una mayor densidad de la membrana de la parrilla. Otra opción más rápida es la centrifugación (en 3000-8000 rpm durante 1-3 minutos) de las muestras con un muestreo posterior de la parte inferior del tubo. Muestras en condiciones óptimas se contienen un gran porcentaje de cristales 2D. No es necesario apuntar a una apariencia más homogénea de las membranas, como la colección más grande y bien ordenada cristales 2D son seleccionados visualmente los datos. Estos tipos de muestras se reconoce fácilmente cuando los ensayos de cristalización se repiten, así como cuando las muestras se utilizan paracrio-ME de recopilación de datos, resultando en un número máximo de imágenes de alta resolución. <img src="/files/ftp_upload/1846/1846fig1.jpg" alt="Figura 1" /> Figura 1. Esta figura muestra borrar el borde de la parrilla con un trozo de papel Whatman # 4 filtro.

<ol>
	<li>Johansen, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bright+field+electron+microscopy+of+biological+specimens+V.+A+low+dose+pre-irradiation+procedure+reducing+beam+damage.">Bright field electron microscopy of biological specimens V. A low dose pre-irradiation procedure reducing beam damage.</a> Micron. 7, 145-156 (1976).</li><li>Schmidt-Krey, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+crystallography+of+membrane+proteins:+Two-dimensional+crystallization+and+screening+by+electron+microscopy.">Electron crystallography of membrane proteins: Two-dimensional crystallization and screening by electron microscopy.</a> Methods. 41, 417-426 (2007).</li><li>Wang, D. N., K&#252;hlbrandt, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+electron+crystallography+of+light-harvesting+chlorophyll+a/b-protein+complex+in+three+different+media.">High-resolution electron crystallography of light-harvesting chlorophyll a/b-protein complex in three different media.</a> J Mol Biol. 217, 691-699 (1991).</li><li>Schmidt-Krey, I., Rubinstein, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+cryomicroscopy+of+membrane+proteins:+specimen+preparation+for+two-dimensional+crystals+and+single+particles.">Electron cryomicroscopy of membrane proteins: specimen preparation for two-dimensional crystals and single particles.</a> Micron. , (2010).</li><li>Schmidt-Krey, I., Mutucumarana, V., Haase, W., Stafford, D. W., K&#252;hlbrandt, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+crystallization+of+human+vitamin+K-dependent+&amp;gamma;-glutamyl+carboxylase.">Two-dimensional crystallization of human vitamin K-dependent &amp;gamma;-glutamyl carboxylase.</a> J Struct Biol. 157, 437-442 (2007).</li><li>Trachtenberg, S., DeRosier, D. J., Zemlin, F., Beckmann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-helical+perturbations+of+the+flagellar+filament:+Salmonella+typhimurium+SJW117+at+9.6+&amp;Aring;+resolution.">Non-helical perturbations of the flagellar filament: Salmonella typhimurium SJW117 at 9.6 &amp;Aring; resolution.</a> J Mol Biol. 276, 759-773 (1998).</li><li>Zhao, G., Johnson, M. C., Schnell, J. R., Kanaoka, Y., Irikura, D., Lam, B. K., Austen, K. F., Schmidt-Krey, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+crystallization+conditions+of+human+leukotriene+C4+synthase+requiring+a+particularly+large+combination+of+specific+parameters.">Two-dimensional crystallization conditions of human leukotriene C4 synthase requiring a particularly large combination of specific parameters.</a> J Struct Biol. 169, 450-454 (2010).</li><li>Cheng, A., Leung, A., Fellmann, D., Quispe, J., Suloway, C., Pulokas, J., Abeyrathne, P. D., Lam, J. S., Carragher, B., Potter, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+automated+screening+of+two-dimensional+crystals.">Towards automated screening of two-dimensional crystals.</a> J Struct Biol. 160, 324-331 (2007).</li><li>Vink, M., Derr, K. D., Love, J., Stokes, D. L., Ubarretxena-Belandia, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+strategy+to+screen+2D+crystallization+trials+of+membrane+proteins.">A high-throughput strategy to screen 2D crystallization trials of membrane proteins.</a> J Struct Biol. 160, 295-304 (2007).</li></ol>

Evaluación adecuada de las muestras requiere una evaluación cuidadosa de un número suficiente de las membranas. Por ejemplo, las muestras con precios tan bajos como 2% de las matrices cristalinas de más de 180 proteoliposomas imágenes dieron la información crítica para la optimización rápida de las condiciones de cristalización 2D 7. Cuando la precipitación de la proteína se produce, una red puede ser abandonada a partir de una mayor búsqueda después de la inspecció...

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<td>400-mesh copper TEM grids coated with carbon film</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>forceps: regular and anti-capillary </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Dumont #5 and Dumont N5AC or similar</td>
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<td>Micropipette and pipette tips</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Whatman #4 filter paper</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>1% uranyl acetate</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Dialysis sample to be screened for 2D crystals</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Glycerol/sucrose-free dialysis buffer</td>
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<td>Optional</td>
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<td>JEOL-1400 transmission electron microscope (TEM)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>similar 80 &#x2013; 120kV TEM equipped with an Lab6 or tungsten filament and film and/or CCD cameras (Gatan Orius SC1000 and/or UltraScan1000 CCD cameras and Gatan Digitial Micrograph software package or Tietz cameras (TVIPS)) </td>
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assessing two-dimensional crystallization trials of small membrane proteins for structural biology studies by electron crystallography

Cristalografía de electrones se ha desarrollado como un método que puede ser utilizado como alternativa o en combinación con tres dimensiones de cristalización y cristalografía de rayos X para estudiar la estructura-función de las preguntas de proteínas de membrana, así como proteínas solubles. La detección de dos dimensiones (2D) los cristales mediante microscopía electrónica de transmisión (EM) es el paso crítico en la búsqueda, la optimización y selección de muestras de alta resolución de la recopilación de datos de la crio-ME. A continuación se describen los pasos fundamentales en la identificación de grandes y ordenadas, así como pequeños arreglos en 2D, que potencialmente puede proveer información crítica para la optimización de las condiciones de cristalización. Al trabajar con diferentes aumentos en la EM, los datos sobre una serie de parámetros críticos se obtiene. Menor aumento suministra datos valiosos sobre la morfología y el tamaño de la membrana. A mayores aumentos, las posibles dimensiones de cristal para 2D y se determinan. En este contexto, se describe cómo las cámaras CCD y se transforma en línea de Fourier se utilizan en mayores aumentos para evaluar proteoliposomas por el orden y el tamaño. Mientras que los cristales 2D de proteínas de membrana son más cultivadas por la reconstitución por diálisis, la técnica de detección es igualmente aplicable para los cristales producidos con la ayuda de las monocapas, nativo de cristales 2D, y ordenó a las matrices de proteínas solubles. Además, los métodos descritos aquí son aplicables a la detección de cristales 2D de la aún más pequeña, así como proteínas de membrana más grandes, donde los pequeños las proteínas requieren la misma cantidad de atención en la identificación de los ejemplos y la red de proteínas más grandes pueden ser más fácilmente identificables en las primeras etapas de la selección.

Watch this Scientific Journal Video about Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography at JoVE.com

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

La evaluación de dos dimensiones (2D) ensayos de cristalización para la formación de matrices de orden de proteínas de membrana es una tarea muy difícil y crítica de la cristalografía de electrones. A continuación se describe el enfoque en la detección e identificación de cristales 2D de proteínas de la membrana en su mayoría pequeñas en el rango de 15 - 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 ...

assessing-two-dimensional-crystallization-trials-small-membrane

Research

JoVE Journal

Biology

11.2K vistas.  Georgia Institute of Technology. La evaluación de dos dimensiones (2D) ensayos de cristalización para la formación de matrices de orden de proteínas de membrana es una tarea muy difícil y crítica de la cristalografía de electrones. A continuación se describe el enfoque en la detección e identificación de cristales 2D de proteínas de la membrana en su mayoría pequeñas en el rango de 15 - 90kDa.

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

Microcontact Printing of Proteins for Cell Biology

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. Development of microcontact printing by the Whitesides group (<a href="http://gmwgroup.harvard.edu/">http://gmwgroup.harvard.edu/</a>) in the mid-1990s revolutionalized this field by making microelectronics/microfabrication techniques accessible to laboratories focused on the life sciences. Initial implementations of this method used polydimethylsiloxane (PDMS) stamps to create patterns of functionalized chemicals on material surfaces1. Since then, a range of innovative approaches have been developed to pattern other molecules, including proteins2. This video demonstrates the basic process of creating PDMS stamps and uses them to pattern proteins, as these steps are difficult to accurately express in words. We focus on patterning the extracellular matrix protein fibronectin onto glass coverslips as a specific example of patterning.
An important component of the microcontact printing process is a topological master, from which the stamps are cast; the raised and lowered regions of the master are mirrored into the stamp and define the final pattern. Typically, a master consists of a silicon wafer coated with photoresist and then patterned by photolithography, as is done here. Creation of masters containing a specific pattern requires specialized equipment, and is best approached in consultation with a fabrication center or facility. However, almost any substrate with topology can be used as a master, such as plastic diffraction gratings (see Reagents for one example), and such serendipitous masters provide readily available, simple patterns. This protocol begins at the point of having a master in hand.

Microcontact printing is used extensively to pattern proteins and other molecules on material surfaces. We demonstrate the basic steps of this process, stamping patterns of fibronectin onto glass.

Microcontacto de impresión de las proteínas de Biología Celular

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. ...

Investigación

Biología

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

Cristalización de las proteínas de membrana para la Determinación de la estructura con mesofases lipídico

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

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 ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

Recolección y enfriamiento Cryo-cristales de proteínas de membrana cultivadas en mesofases lipídicas para la Determinación de la estructura por cristalografía macromolecular

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Producción eficiente y purificación de recombinante murino kindlin-3 a partir de células de insecto para estudios biofísicos

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as cells, organelles, membrane vesicles, or viruses at molecular detail. To achieve this, the aqueous sample is rapidly vitrified in liquid ethane, which preserves it in a close-to-native, frozen-hydrated state. In the electron microscope, tilt series are recorded at liquid nitrogen temperature, from which 3D tomograms are reconstructed. The signal-to-noise ratio of the tomographic volume is inherently low. Recognizable, recurring features are enhanced by subtomogram averaging, by which individual subvolumes are cut out, aligned and averaged to reduce noise. In this way, 3D maps with a resolution of 2 nm or better can be obtained. A fit of available high-resolution structures to the 3D volume then produces atomic models of protein complexes in their native environment. Here we show how we use electron cryo-tomography to study the in situ organization of large membrane protein complexes in mitochondria. We find that ATP synthases are organized in rows of dimers along highly curved apices of the inner membrane cristae, whereas complex I is randomly distributed in the membrane regions on either side of the rows. By subtomogram averaging we obtained a structure of the mitochondrial ATP synthase dimer within the cristae membrane.

We present a protocol of how to collect and process electron cryo-tomograms of whole mitochondria. The technique provides detailed insights into the structure, function, and organization of large membrane protein complexes in native biological membranes.

La visualización de la ATP sintasa dímeros en mitocondrias por Electron Cryo-tomografía

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as ...

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.