Name: microcrystallography of protein crystals and in cellulo diffraction
Uploaded: 2017-07-21T14:00:00.000+00:00
Duration: 9 min 35 s
Description: Watch this Scientific Journal Video about Microcrystallography of Protein Crystals and In Cellulo Diffraction at JoVE.com

Los autores desean reconocer a Chan-Sien Lay por proporcionar fotografías de microcristales purificados, Daniel Eriksson y Tom Caradoc-Davies para apoyo en la línea de rayos MX2 del sincrotrón australiano y Kathryn Flanagan y Andrew Fryga de la instalación FlowCore de la Universidad Monash por su Ayuda invaluable.

<ol>
	<li>Tari, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+utility+of+structural+biology+in+drug+discovery.">The utility of structural biology in drug discovery.</a> Methods Mol Bio (Clifton, N.J). 841, 1-27 (2012).</li><li>Gati, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+crystallography+on+in+vivo+grown+microcrystals+using+synchrotron+radiation.">Serial crystallography on in vivo grown microcrystals using synchrotron radiation.</a> IUCrJ. 1 (2), (2014).</li><li>Redecke, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natively+inhibited+Trypanosoma+brucei+cathepsin+B+structure+determined+by+using+an+X-ray+laser.">Natively inhibited Trypanosoma brucei cathepsin B structure determined by using an X-ray laser.</a> Science. 339 (2013), 227-230 (2013).</li><li>Shi, D., Nannenga, B. L., Iadanza, M. G., Gonen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+electron+crystallography+of+protein+microcrystals.">Three-dimensional electron crystallography of protein microcrystals.</a> eLife. 2, e01345 (2013).</li><li>Evans, G., Axford, D., Waterman, D., Owen, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecular+microcrystallography.">Macromolecular microcrystallography.</a> Crystallog. Rev. 17 (2), 105-142 (2011).</li><li>Smith, J. L., Fischetti, R. F., Yamamoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-crystallography+comes+of+age.">Micro-crystallography comes of age.</a> Curr Opin Struct Biol. 22 (5), 602-612 (2012).</li><li>Boudes, M., Garriga, D., Coulibaly, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reflections+on+the+Many+Facets+of+Protein+Microcrystallography.">Reflections on the Many Facets of Protein Microcrystallography.</a> Aust J Chem. 67 (12), 1793-1806 (2014).</li><li>Nelson, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+cross-beta+spine+of+amyloid-like+fibrils.">Structure of the cross-beta spine of amyloid-like fibrils.</a> Nature. 435 (7043), 773-778 (2005).</li><li>Coulibaly, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+organization+of+cypovirus+polyhedra.">The molecular organization of cypovirus polyhedra.</a> Nature. 446 (7131), 97-101 (2007).</li><li>Coulibaly, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+atomic+structure+of+baculovirus+polyhedra+reveals+the+independent+emergence+of+infectious+crystals+in+DNA+and+RNA+viruses.">The atomic structure of baculovirus polyhedra reveals the independent emergence of infectious crystals in DNA and RNA viruses.</a> Proc Natl Acad Sci U S A. 106 (52), 22205-22210 (2009).</li><li>Johansson, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+a+photosynthetic+reaction+centre+determined+by+serial+femtosecond+crystallography.">Structure of a photosynthetic reaction centre determined by serial femtosecond crystallography.</a> Nat Comm. 4, (2013).</li><li>Li, D., Boland, C., Aragao, D., Walsh, K., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harvesting+and+Cryo-cooling+Crystals+of+Membrane+Proteins+Grown+in+Lipidic+Mesophases+for+Structure+Determination+by+Macromolecular+Crystallography.">Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography.</a> J Vis Exp. (67), (2012).</li><li>Roedig, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+micro-patterned+silicon+chip+as+sample+holder+for+macromolecular+crystallography+experiments+with+minimal+background+scattering.">A micro-patterned silicon chip as sample holder for macromolecular crystallography experiments with minimal background scattering.</a> Sci Rep. 5, 10451 (2015).</li><li>Koopmann, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+protein+crystallization+opens+new+routes+in+structural+biology.">In vivo protein crystallization opens new routes in structural biology.</a> Nature Methods. 9 (3), 259-262 (2012).</li><li>Gallat, F. -. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+crystallography+at+X-ray+free-electron+lasers:+the+next+generation+of+structural+biology.">In vivo crystallography at X-ray free-electron lasers: the next generation of structural biology.</a> Phil Trans R Soc B. 369 (1647), 20130497 (2014).</li><li>Duszenko, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+protein+crystallization+in+combination+with+highly+brilliant+radiation+sources+offers+novel+opportunities+for+the+structural+analysis+of+post-translationally+modified+eukaryotic+proteins.">In vivo protein crystallization in combination with highly brilliant radiation sources offers novel opportunities for the structural analysis of post-translationally modified eukaryotic proteins.</a> Acta Cryst. F. 71 (8), 929-937 (2015).</li><li>Axford, D., Ji, X., Stuart, D. I., Sutton, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+cellulo+structure+determination+of+a+novel+cypovirus+polyhedrin.">In cellulo structure determination of a novel cypovirus polyhedrin.</a> Acta Cryst D. 70 (5), 1435-1441 (2014).</li><li>Sawaya, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+crystal+structure+obtained+at+2.9+&#197;+resolution+from+injecting+bacterial+cells+into+an+X-ray+free-electron+laser+beam.">Protein crystal structure obtained at 2.9 &#197; resolution from injecting bacterial cells into an X-ray free-electron laser beam.</a> Proc Natl Acad Sci U S A. 111 (35), 12769-12774 (2014).</li><li>Tsutsui, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Diffraction-Quality+Protein+Crystal+Processed+as+an+Autophagic+Cargo.">A Diffraction-Quality Protein Crystal Processed as an Autophagic Cargo.</a> Mol Cell. 58 (1), 186-193 (2015).</li><li>Boudes, M., Garriga, D., Fryga, A., Caradoc-Davies, T., Coulibaly, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pipeline+for+structure+determination+of+in+vivo-grown+crystals+using+in+cellulo+diffraction.">A pipeline for structure determination of in vivo-grown crystals using in cellulo diffraction.</a> Acta Cryst D. 72, 576-585 (2016).</li><li>Doye, J. P. K., Poon, W. C. K. <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+in+vivo.">Protein crystallization in vivo.</a> Curr Opin Colloid Interface Sci. 11 (1), 40-46 (2006).</li><li>Mori, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Bombyx+mori+cytoplasmic+polyhedrosis+virus+polyhedrin+in+insect+cells+by+using+a+baculovirus+expression+vector,+and+its+assembly+into+polyhedra.">Expression of Bombyx mori cytoplasmic polyhedrosis virus polyhedrin in insect cells by using a baculovirus expression vector, and its assembly into polyhedra.</a> J Gen Virol. 74, 99-102 (1993).</li><li>Arevalo, M. T., Wong, T. M., Ross, 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=Expression+and+Purification+of+Virus-like+Particles+for+Vaccination.">Expression and Purification of Virus-like Particles for Vaccination.</a> J Vis Exp. (112), (2016).</li><li>Yates, L. A., Gilbert, R. J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+Production+and+Purification+of+Recombinant+Murine+Kindlin-3+from+Insect+Cells+for+Biophysical+Studies.">Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies.</a> J Vis Exp. (85), (2014).</li><li>Berger, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MultiBac+Protein+Complex+Production+Platform+at+the+EMBL.">The MultiBac Protein Complex Production Platform at the EMBL.</a> J Vis Exp. (77), (2013).</li><li>Margine, I., Palese, P., Krammer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Functional+Recombinant+Hemagglutinin+and+Neuraminidase+Proteins+from+the+Novel+H7N9+Influenza+Virus+Using+the+Baculovirus+Expression+System.">Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System.</a> J Vis Exp. (81), (2013).</li><li>Khurana, A., Kronenberg, 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+Method+For+Production+of+Recombinant+mCD1d+Protein+in+Insect+Cells.">A Method For Production of Recombinant mCD1d Protein in Insect Cells.</a> J Vis Exp. (10), (2007).</li><li>Ricardo, R., Phelan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+and+Determining+the+Viability+of+Cultured+Cells.">Counting and Determining the Viability of Cultured Cells.</a> J Vis Exp. (16), (2008).</li><li>Baskaran, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+cellulo-derived+structure+of+PAK4+in+complex+with+its+inhibitor+Inka1.">An in cellulo-derived structure of PAK4 in complex with its inhibitor Inka1.</a> Nat Comm. 6, 1-11 (2015).</li><li>Armour, B. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-target+Parallel+Processing+Approach+for+Gene-to-structure+Determination+of+the+Influenza+Polymerase+PB2+Subunit.">Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit.</a> J Vis Exp. (76), (2013).</li><li>Otwinowski, Z., Minor, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+of+X-ray+diffraction+data+collected+in+oscillation+mode.">Processing of X-ray diffraction data collected in oscillation mode.</a> Methods Enzymol. 276, 307-326 (1997).</li><li>Battye, T. G. G., Kontogiannis, L., Johnson, O., Powell, H. R., Leslie, A. G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iMOSFLM:+a+new+graphical+interface+for+diffraction-image+processing+with+MOSFLM.">iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM.</a> Acta Cryst D. 67, 271-281 (2011).</li><li>Kabsch, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XDS.">XDS.</a> Acta Cryst D. 66, 125-132 (2010).</li><li>French, S., Wilson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+treatment+of+negative+intensity+observations.">On the treatment of negative intensity observations.</a> Acta Cryst.A. 34, 517-525 (1978).</li><li>Winn, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+the+CCP4+suite+and+current+developments.">Overview of the CCP4 suite and current developments.</a> Acta Cryst D. 67, 235-242 (2011).</li><li>Foadi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clustering+procedures+for+the+optimal+selection+of+data+sets+from+multiple+crystals+in+macromolecular+crystallography.">Clustering procedures for the optimal selection of data sets from multiple crystals in macromolecular crystallography.</a> Acta Cryst D. 69 (8), 1617-1632 (2013).</li><li>Rey, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virology:+holed+up+in+a+natural+crystal.">Virology: holed up in a natural crystal.</a> Nature. 446 (7131), 35-37 (2007).</li><li>Sch&#246;nherr, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+investigation+of+dynamic+protein+crystallization+in+living+cells.">Real-time investigation of dynamic protein crystallization in living cells.</a> Struct Dyn. 2 (4), 041712 (2015).</li><li>Hasegawa, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+crystallization+of+human+IgG+in+the+endoplasmic+reticulum+of+engineered+Chinese+hamster+ovary+(CHO)+cells.">In vivo crystallization of human IgG in the endoplasmic reticulum of engineered Chinese hamster ovary (CHO) cells.</a> J Biol Chem. 286 (22), 19917-19931 (2011).</li><li>Ishchenko, A., Cherezov, V., Liu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+Delivery+of+Protein+Microcrystals+in+Lipidic+Cubic+Phase+for+Serial+Femtosecond+Crystallography.">Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography.</a> J Vis Exp. (115), e54463 (2016).</li><li>Zander, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MeshAndCollect:+an+automated+multi-crystal+data-collection+workflow+for+synchrotron+macromolecular+crystallography+beamlines.">MeshAndCollect: an automated multi-crystal data-collection workflow for synchrotron macromolecular crystallography beamlines.</a> Acta Cryst. D. 71, 2328-2343 (2015).</li><li>Fan, G. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+calcineurin+crystals+formed+using+the+baculovirus+expression+system.">In vivo calcineurin crystals formed using the baculovirus expression system.</a> Micros Res Tech. 34 (1), 77-86 (1996).</li><li>Nass, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Investigation of protein structure determination using X-ray free-electron lasers. , (2013).</li></ol>

Los autores no tienen nada que revelar.

Este protocolo proporciona dos enfoques para analizar microcristales con el objetivo de facilitar el análisis de cristales muy pequeños que se hubieran pasado por alto en el pasado. Pasos críticos para la purificación de microcristales El protocolo presentado ha sido optimizado utilizando la poliedrina Bombyx mori CPV1 expresada en células Sf9 como sistema modelo. Sin embargo, los microcristales in vivo muestran una gran variabilidad en la resistencia mecánica. Por ejemplo, los cristales de catepsina B similares a agujas crecidos en células de insecto son rígidos y altamente resistentes al estrés mecánico, y podrían purificarse usando un protocolo similar al descrito aquí 14 . Por otra parte, los cristales de luciferasa de la luciérnaga, también crecidos en células de insecto cultivadas y con una morfología análoga similar a aguja, se disuelven inmediatamente tras la lisis celular 38 . Por lo tanto, el protocolo para la extracción y purificación de in vivoLos cristales tendrán que adaptarse en una base de error de ensayo para casos particulares como se describe en los pasos 1.3, 2a.3-4, 2b.4, 3.1.3-4 y 3.2a.5. Para los cristales frágiles, la separación (parcial) de los residuos celulares puede conseguirse mediante centrifugación a baja velocidad ( es decir, 200 xg) o adición de un cojín de sacarosa en la parte inferior del tubo en lugar de sonicación repetida. Como regla general, las muestras de cristal preparadas para la recopilación de datos de difracción no necesitan ser altamente purificadas. Por lo tanto, una purificación rápida es preferible a cryo-enfriar los cristales antes de que pudieran decaer. Aplicación a microcristales in vitro e in vivo Aunque ambos protocolos se ilustran aquí con el análisis de cristales crecidos in vivo , la metodología se puede usar fácilmente para microcristales crecidos en bandejas de cristal a partir de proteína recombinante purificada. En esteSe deben considerar las siguientes modificaciones: i) el soporte de malla puede usarse para recoger los microcristales directamente de la gota de cristalización, en lugar de transferirlos por pipeteo; Ii) puesto que la cantidad de cristales puede ser un factor limitante, se necesitará cuidado especial cuando se borre el exceso de líquido del soporte de malla, para evitar la eliminación excesiva de cristales; Iii) se necesitarán ensayar diferentes tampones crioprotectores, tal como se hace en cristalografía convencional (véase la referencia 30 ). Por otro lado, para in vivo cristales -grown, el enfoque en cellulo es muy recomendable. Debido a que las células son más fáciles de visualizar y manipular que los cristales purificados, este enfoque es más eficiente en términos de uso de la viga y más accesible a los investigadores con poca o ninguna experiencia en microcristalografía. También es más adecuado para cristales frágiles que pueden verse afectados por la extracción y purificación.<html> De la célula debido a cambios en el ambiente químico, dilución de las proteínas circundantes y daño mecánico. Además, las células proporcionan un ambiente protector a los cristales, aliviando la necesidad de la adición de un crioprotector. Se ha demostrado previamente la omisión del tratamiento de crioprotección para prevenir la acumulación de defectos y mejorar el isomorfismo entre cristales individuales, para la recogida de datos a temperatura ambiente 40 . El protocolo in cellulo también elimina el requisito de incubación en solución crioprotectora, al mismo tiempo que permite la recolección de datos a temperatura criogénica. En general, en el celulo la recopilación de datos tiene dos ventajas principales: 1) puede producir datos de mejor calidad y mayor resolución para un período determinado de tiempo de haz 20 ; Y 2) mantiene la proteína cristalizada en un entorno celular aumentando las posibilidades de retener un ligando biológicamente relevante. Ontent &quot;&gt; Limitaciones de la técnica Una complicación intrínseca de la microcristalografía serial es el importante número de conjuntos de datos parciales generados durante la recolección de datos. En consecuencia, el procesamiento de datos se convierte en un proceso cada vez más lento. Sin embargo, el flujo de trabajo básico para el procesamiento de datos puede ser ampliamente automatizado. Por ejemplo, la selección óptima de cristales isomorfos puede determinarse mediante un análisis de agrupamiento jerárquico 28 , por ejemplo implementado en BLEND 36 . Además, los programas desarrollados para el análisis de rayos X de láser de electrones libres (XFEL) también pueden adaptarse para procesar datos de microcristalografía en serie recopilados en líneas de luz de sincrotrón 2 y, en algunas líneas de haz, se han implementado rasterizaciones automatizadas de malla y detección de cristales. Recogida de datos 2 , 41 .

El uso de la cristalografía de rayos X para la determinación de estructuras de alta resolución de macromoléculas biológicas ha experimentado una progresión constante en las últimas dos décadas. La creciente aceptación de la cristalografía de rayos X por investigadores no expertos ejemplifica la democratización de este enfoque en muchos campos de las ciencias de la vida 1 . Históricamente, los cristales con dimensiones inferiores a ~ 10 μm han sido considerados como desafiantes, si no inutilizables, para la determinación de la estructura. La creciente disponibilidad de rayos de luz de microfoco dedicados a fuentes de radiación de sincrotrón en todo el mundo y los avances tecnológicos, tales como el desarrollo de herramientas para manipular microcristales, han eliminado gran parte de estas barreras que obstaculizan el amplio uso de la microcristalografía de rayos X. Avances en microcristalografía de rayos X serie 2 , 3 y micro difracción de electrones 4 haHemos demostrado que el uso de micro y nanocristales para la determinación de la estructura no sólo es factible, sino que a veces es preferible al uso de grandes cristales 5 , 6 , 7 . Estos avances se aplicaron por primera vez al estudio de péptidos [ 8] y cristales naturales producidos por virus de insectos [ 9 , 10] . Ahora se utilizan para una amplia gama de macromoléculas biológicas, incluyendo los sistemas más difíciles, como proteínas de membrana y grandes complejos [ 11] . Para facilitar el análisis de estos microcristales, se han analizado en meso , en particular proteínas de membrana 12 y en chips microfluídicos 13 . La disponibilidad de estas nuevas metodologías microcrystallography ha planteado la posibilidad de utilizar enComo una nueva ruta para la biología estructural 14 , 15 , 16 que ofrece una alternativa a la cristalogénesis clásica in vitro . Desafortunadamente, incluso cuando se pueden producir cristales in vivo , quedan varios obstáculos tales como la degradación o pérdida de ligandos durante la purificación de las células, dificultad en la manipulación y visualización de los cristales en la línea de luz del sincrotrón y tediosos experimentos de difracción de rayos X. Como una alternativa cristales también se han analizado directamente dentro de la célula sin ningún paso de purificación 17 , 18 , 19 . Un análisis comparativo sugiere que tal en los enfoques cellulo puede ser más eficiente que el análisis de cristales purificados y los datos de rendimiento de mayor resolución [ 20] . Este protocolo está enTendieron a ayudar a los investigadores nuevos a la microcristalografía de proteínas. Proporciona metodologías centradas en la preparación y manipulación de muestras para experimentos de difracción de rayos X en una línea de luz de sincrotrón. Se proponen dos opciones utilizando cristales aislados para microcristalografía clásica o células que contienen cristal ordenadas por citometría de flujo para análisis de celulo ( Figura 1 ).

<table><tbody><tr><td>Sf9 cells</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>SF900-SFM insect medium</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>1 L cell culture flask</td><td>Thermofisher Scientific</td><td></td><td></td></tr><tr><td>Shaking incubator for insect cell culture</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>50 mL conical tubes</td><td>Falcon</td><td></td><td></td></tr><tr><td>Centrifuge with swing buckets for 50 mL tubes</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Sonicator equiped with a 19 mm probe</td><td>MSE Soniprep 150&amp;nbsp;</td><td></td><td></td></tr><tr><td>Glass slides</td><td>Hampton Research</td><td></td><td></td></tr><tr><td>Hemacytometer</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Propidium iodide</td><td>Thermofisher Scientific</td><td></td><td></td></tr><tr><td>BD Influx cell sorter&amp;nbsp;</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Hampton Heavy atom screens</td><td>Hampton Research</td><td></td><td></td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Micromesh</td><td>Mitigen</td><td></td><td>700/25 meshes offer a larger surface. Indexed meshes can be purchased for systematic studies.</td></tr><tr><td>Paper wick</td><td>Mitigen</td><td></td><td>The size of the paper wick can be varied for optimal flow. This will largely depend on the nature of the crystals and cryoprotectant used.</td></tr><tr><td>Ethylene glycol</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Trypan blue</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>MX2 microfocus beamline</td><td>Australian Synchrotron</td><td></td><td>A list of available microfocus beamlines can be found in Boudes &lt;em&gt;et al.&lt;/em&gt; (2014) &lt;em&gt;Reflections on the Many Facets of Protein&lt;br/&gt; Microcrystallography.&lt;/em&gt; Australian Journal of Chemistry 67 (12), 1793&amp;ndash;1806,&lt;br/&gt; doi:10.1071/CH14455.</td></tr></tbody></table>

microcrystallography of protein crystals and in cellulo diffraction

El advenimiento de líneas de luz de microfoco de alta calidad en muchas instalaciones de sincrotrón ha permitido el análisis de rutina de cristales menores de 10 μm en su dimensión más grande, lo que representaba un desafío. Presentamos dos flujos de trabajo alternativos para la determinación de la estructura de microcristales de proteínas por cristalografía de rayos X con un enfoque particular en los cristales crecidos in vivo . Los microcristales se extraen de las células mediante sonicación y se purifican mediante centrifugación diferencial o se analizan en celulo después de la clasificación celular mediante citometría de flujo de células que contienen cristal. Opcionalmente, los cristales purificados o las células que contienen cristal se empapan en soluciones de átomos pesados ​​para el phasing experimental. Estas muestras se preparan entonces para experimentos de difracción de una manera similar por aplicación sobre un soporte de micromesh y enfriamiento rápido en nitrógeno líquido. Describimos brevemente y comparar los experimentos de difracción en serie de microcristales aislados y cristal-Que contienen células que utilizan una línea de luz de sincrotrón microfoco para producir conjuntos de datos adecuados para la fase, la construcción de modelos y el refinamiento. Estos flujos de trabajo se ejemplifican con cristales de la poliedrina Bombyx mori cypovirus 1 (BmCPV1) producida por la infección de células de insecto con un baculovirus recombinante. En este caso de estudio, en el análisis de celulo es más eficiente que el análisis de cristales purificados y produce una estructura en ~ 8 días desde la expresión hasta el refinamiento.

Una presentación general de ambos métodos alternativos para la determinación de la estructura utilizando microcristales in vivo se presenta ( Figura 1 ]. Los poliedros se pueden purificar fácilmente mediante sonicación y centrifugación. Debido a su densidad, forman una capa en la parte inferior del tubo por debajo de una capa de desechos que se pueden eliminar por pipeteado ( Figura 3a y 3b ). La muestra se somete luego a varias rondas de sonicación y se lava hasta que se alcanza un nivel de pureza suficiente como se juzga a partir del aspecto blanco y calcáreo del gránulo ( Figura 3c ). Para el enfoque en celulo , el perfil de citometría de flujo de las células no infectadas se compara con el perfil de las células que contienen cristal y se utiliza para determinar qué población de células debe seleccionarse para clasificar las células que contienen cristal alejadas deLas otras células ( Figura 4 ). En este ejemplo, las células que contienen poliedros tienen una mayor dispersión lateral que las células no infectadas. Pueden observarse otras diferencias en los patrones de dispersión para otros tipos de cristales y la modificación debe modificarse en consecuencia. Los microcristales purificados o las células que contienen cristal se pipetean sobre una micromesh ( Figura 5 ). Las células teñidas con azul de tripano se pueden visualizar fácilmente usando cámaras disponibles en la mayoría de líneas de luz de sincrotrón (Figura 5b), mientras que los cristales purificados son laboriosos para identificar y alinear con el haz de rayos X debido a su pequeño tamaño (Figura 5c). Cuando se recopilan datos, lo mejor es proceder en un patrón de rejilla para minimizar los procedimientos de centrado, y para evitar exponer el doble de la misma celda o cristal, o faltar alguno ( Figura 5a ). Colección de datosDeben ser procesados ​​cuidadosamente para tener en cuenta las diferencias en los límites de difracción, el efecto del daño por radiación y una posible falta de isomorfismo. <img alt="Figura 1" class="xfigimg" src="/files/ftp_upload/55793/55793fig1.jpg" /> Figura 1 : Visión general de dos métodos para la determinación de estructuras utilizando microcristales in vivo . Los microcristales se producen en cultivo celular mediante sobreexpresión de la proteína de interés utilizando un sistema de expresión recombinante. En casos específicos, los microcristales también pueden producirse naturalmente por las células en condiciones particulares. La fila superior muestra el enfoque clásico en el que las células se lisan y los cristales se purifican mediante centrifugación diferencial. Los microcristales purificados se pipetean sobre una micromesh. Después de la eliminación del exceso de líquido, se añade una solución crioprotectora, el líquido sobrante se vuelve a secar y la malla se enfría por enfriamiento rápido. Para los rayos X dif<html> Los cristales se alinean cuidadosamente con el haz de rayos X, que puede ser el paso limitante en la recolección de datos. La fila inferior presenta el enfoque en celulo . Las células se clasifican por citometría de flujo para seleccionar específicamente células que contienen cristal. Las células se tiñen con azul trypan para facilitar la visualización y propagación sobre una micromesh. En este caso, la adición de un crioprotector es opcional. Para los experimentos de difracción de rayos X, las células se visualizan fácilmente usando cámaras típicas disponibles en las líneas de luz de sincrotrón facilitando el proceso de centrado. Imagen del sistema automatizado de cromatografía líquida cortesía de GE Healthcare AB, Uppsala, Suecia. <a href="http://ecsource.jove.com/files/ftp_upload/55793/55793fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 2" class="xfigimg" src="/files/ftp_upload/55793/55793fig2.jpg" /> = &quot;Xfig&quot;&gt; Figura 2 : Ejemplos de Microcristales In Vivo . Ejemplos seleccionados de cristales in vivo: ( a ) células Sf9 con poliedros de cipovirus; ( B ) células LD652 con esferoides de entomopoxvirus; ( C ) Bacillus thuringiensis con cristales Cry3A (flecha); ( D ) células Sf9 con cristales de catepsina B de Trypanosoma brucei ; ( E ) células Sf21 con cristales de calcineurina (flechas); ( F ) células COS7 monocapa con PKA: cristales Inka1; ( G ) células CHO con cristales de IgG. Reproducido de 14 , 18 , 29 , 37 , 39 , 42 , con permiso. Barra de escala = 10 μm. <a href="http://ecsource.jove.com/files/ftp_upload/55793/55793fig2large.jpg" target="_blank">Por favor, haga clic aquí para ver una</a>De esta cifra. <img alt="tabla 1" src="/files/ftp_upload/55793/55793table1.jpg" /> Tabla 1: Resumen de cristales in vivo cultivados en células cultivadas que expresan proteína recombinante. <a href="http://ecsource.jove.com/files/ftp_upload/55793/55793table1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>

Watch this Scientific Journal Video about Microcrystallography of Protein Crystals and In Cellulo Diffraction at JoVE.com

Microcrystallography of Protein Crystals and In Cellulo Diffraction

Se presenta un protocolo para la cristalografía de rayos X utilizando microcristales de proteínas. Se comparan dos ejemplos que analizan microcristales de crecimiento in vivo después de la purificación o en celulo .

Microcristalografía de Protein Crystals y<em&gt; En Cellulo</em&gt; Difracción

The advent of high-quality microfocus beamlines at many synchrotron facilities has permitted the routine analysis of crystals smaller than 10 &#181;m in ...

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JoVE Journal

Biochemistry

Microcrystallography of Protein Crystals and In Cellulo Diffraction

9.0K vistas.  Monash University.  Se presenta un protocolo para la cristalografía de rayos X utilizando microcristales de proteínas. Se comparan dos ejemplos que analizan microcristales de crecimiento in vivo después de la purificación o en celulo . 

Video: Microcrystallography of Protein Crystals and In Cellulo Diffraction

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to precisely maneuver the nucleation and crystal growth towards desired sizes of protein crystals. The controlled crystallization is based on sample investigation with in situ Dynamic Light Scattering (DLS) while all visual changes in the droplet are monitored online with the help of a microscope coupled to a CCD camera, thus enabling a full investigation of the protein droplet during all stages of crystallization. The use of in situ DLS measurements throughout the entire experiment allows a precise identification of the highly supersaturated protein solution transitioning to a new phase &#8211; the formation of crystal nuclei. By identifying the protein nucleation stage, the crystallization can be optimized from large protein crystals to the production of protein microcrystals. The experimental protocol shows an interactive crystallization approach based on precise automated steps such as precipitant addition, water evaporation for inducing high supersaturation, and sample dilution for slowing induced homogeneous nucleation or reversing phase transitions.

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Here we present a protocol for controlled production of protein microcrystals. The process uses an automated device allowing controlled manipulation of several crystallization parameters. The protein crystallization is carried-out by controlled and automated addition of crystallization solutions while monitoring and investigating the radius distribution of particles in the crystallization droplet.

Cristales de proteína creciente con distintas dimensiones usando automatizado de cristalización juntada con In Situ dispersión ligera dinámica

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to ...

Investigación

Bioquímica

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The devices are patterned from epoxy glue using soft lithography and are suitable for in situ X-ray diffraction experiments at room temperature. The sample wells are lidded on both sides with polymeric polyimide foil windows that allow diffraction data collection with low X-ray background. This fabrication method is undemanding and inexpensive. After the sourcing of a SU-8 master wafer, all fabrication can be completed outside of a cleanroom in a typical research lab environment. The chip design and fabrication protocol utilize capillary valving to microfluidically split an aqueous reaction into defined nanoliter sized droplets. This loading mechanism avoids the sample loss from channel dead-volume and can easily be performed manually without using pumps or other equipment for fluid actuation. We describe how isolated nanoliter sized drops of protein solution can be monitored in situ by dynamic light scattering to control protein crystal nucleation and growth. After suitable crystals are grown, complete X-ray diffraction datasets can be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. The protocol provides custom scripts to process diffraction datasets using a suite of software tools to solve and refine the protein crystal structure. This approach avoids the artefacts possibly induced during cryo-preservation or manual crystal handling in conventional crystallography experiments. We present and compare three protein structures that were solved using small crystals with dimensions of approximately 10-20 &#181;m grown in chip. By crystallizing and diffracting in situ, handling and hence mechanical disturbances of fragile crystals is minimized. The protocol details how to fabricate a custom X-ray transparent microfluidic chip suitable for in situ serial crystallography. As almost every crystal can be used for diffraction data collection, these microfluidic chips are a very efficient crystal delivery method.

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes in detail how to fabricate and operate microfluidic devices for X-ray diffraction data collection at room temperature. Additionally, it describes how to monitor protein crystallization by dynamic light scattering and how to process and analyze obtained diffraction data.

Chips de microfluídica para dispersión ligera dinámica In Situ mediante difracción de rayos x de cristal y In Situ para la cristalografía Serial

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The ...

Química

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We describe a versatile microfluidic chip that enables the production of crystals by the efficient method of counter-diffusion. The convection-free environment provided by the microfluidic channels is ideal for crystal growth and useful to diffuse a substrate into the active site of the crystalline enzyme. Here we applied this approach to the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus in the presented example. After crystallization and substrate diffusion/soaking, the crystal structure of the enzyme:substrate complex was determined at room temperature by serial crystallography and the analysis of multiple crystals directly inside the chip. The whole procedure preserves the genuine diffraction properties of the samples because it requires no crystal handling.

A versatile microfluidic device is described that enables the crystallization of an enzyme using the counter-diffusion method, the introduction of a substrate in the crystals by soaking, and the 3D structure determination of the enzyme:substrate complex by a serial analysis of crystals inside the chip at room temperature.

Cristalización y determinación estructural de una enzima: complejo de sustratos por cristalografía serie en un chip microfluídico versátil

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We ...

Microcrystal Electron Diffraction of Small Molecules

A detailed protocol for preparing small molecule samples for microcrystal electron diffraction (MicroED) experiments is described. MicroED has been developed to solve structures of proteins and small molecules using standard electron cryo-microscopy (cryo-EM) equipment. In this way, small molecules, peptides, soluble proteins, and membrane proteins have recently been determined to high resolutions. Protocols are presented here for preparing grids of small-molecule pharmaceuticals using the drug carbamazepine as an example. Protocols for screening and collecting data are presented. Additional steps in the overall process, such as data integration, structure determination, and refinement are presented elsewhere. The time required to prepare the small-molecule grids is estimated to be less than 30 min.

Here, we describe the procedures developed in our laboratory for preparing powders of small molecule crystals for microcrystal electron diffraction (MicroED) experiments.

Difracción de electrones de microcristales de moléculas pequeñas

A detailed protocol for preparing small molecule samples for microcrystal electron diffraction (MicroED) experiments is described. MicroED has been ...

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins on-chip with the dialysis method and allowing in situ single-crystal or serial crystallography experiments at room temperature. The protocol details the fabrication process of the microchips, the manipulation of the on-chip crystallization experiments and the treatment of the in situ collected X-ray diffraction data for the structural elucidation of the protein sample. The main feature of this microfabrication procedure lies on the integration of a commercially available, semipermeable regenerated cellulose dialysis membrane in between two layers of the chip. The molecular weight cut-off of the embedded membrane varies depending on the molecular weight of the macromolecule and the precipitants. The device exploits the advantages of microfluidic technology, such as the use of minute volumes of samples (&#60;1 &#181;L) and fine tuning over transport phenomena. The chip coupled them with the dialysis method, providing precise and reversible control over the crystallization process and can be used for investigating phase diagrams of proteins at the microliter scale. The device is patterned using a photocurable thiolene-based resin with soft imprint lithography on an optically transparent polymeric substrate. Moreover, the background scattering of the materials composing the microchips and generating background noise was evaluated rendering the chip compatible for in situ X-ray diffraction experiments. Once protein crystals are grown on-chip up to an adequate size and population uniformity, the microchips can be directly mounted in front of the X-ray beam with the aid of a 3D printed holder. This approach addresses the challenges rising from the use of cryoprotectants and manual harvesting in conventional protein crystallography experiments through an easy and inexpensive manner. Complete X-ray diffraction data sets from multiple, isomorphous lysozyme crystals grown on-chip were collected at room temperature for structure determination.

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

This paper details the fabrication protocol of microfluidic chips developed for on-chip protein crystallization with the dialysis method and in situ X-ray diffraction experiments. The microfabrication process makes it possible to integrate a semipermeable regenerated cellulose dialysis membrane with any molecular weight cut-off, between two layers of the chip.

Cristalización de proteínas en chip por microdiálisis para estudios de difracción de rayos X in situ

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins ...

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 ...

Biología

On-Chip Crystallization and Large-Scale Serial Diffraction at Room Temperature

Biochemical reactions and biological processes can be best understood by demonstrating how proteins transition among their functional states. Since cryogenic temperatures are non-physiological and may prevent, deter, or even alter protein structural dynamics, a robust method for routine X-ray diffraction experiments at room temperature is highly desirable. The crystal-on-crystal device and its accompanying hardware and software used in this protocol are designed to enable in situ X-ray diffraction at room temperature for protein crystals of different sizes without any sample manipulation. Here we present the protocols for the key steps from device assembly, on-chip crystallization, optical scanning, crystal recognition to X-ray shot planning and automated data collection. Since this platform requires no crystal harvesting nor any other sample manipulation, hundreds to thousands of protein crystals grown on chip can be introduced into an X-ray beam in a programmable and high-throughput manner.

This contribution describes how to set up protein crystallization on crystal-on-crystal devices and how to perform automated serial data collection at room temperature using the on-chip crystallization platform.

Cristalización en chip y difracción en serie a gran escala a temperatura ambiente

Biochemical reactions and biological processes can be best understood by demonstrating how proteins transition among their functional states. Since ...

High-Throughput Protein Crystallization via Microdialysis

Understanding the structure-function relationships of macromolecules, such as proteins, at the molecular level is vital for biomedicine and modern drug discovery. To date, X-ray crystallography remains the most successful method for solving three-dimensional protein structures at atomic resolution. With recent advances in serial crystallography, either using X-ray free electron lasers (XFELs) or synchrotron light sources, protein crystallography has progressed to the next frontier, where the ability to acquire time-resolved data provides important mechanistic insights into the behavior of biological molecules at room temperature. This protocol describes a straightforward high-throughput (HTP) workflow for screening crystallization conditions through the use of a 96-well dialysis plate. These plates follow the Society for Biomolecular Screening (SBS) standard and can be easily set up using any standard crystallization laboratory. Once optimal conditions are identified, large quantities of crystals (hundreds of microcrystals) can be produced using the dialyzer. To validate the robustness and versatility of this approach, four different proteins were crystallized, including two membrane proteins.

High-Throughput Protein Crystallization via Microdialysis

The presented protocol describes a straightforward approach for screening protein crystallization conditions and crystal growth using a 96-well high-throughput dialysis plate. The use of dialyzer tubes for the large-scale growth of microcrystals is also demonstrated for serial crystallography and MicroED applications.

Cristalización de proteínas de alto rendimiento mediante microdiálisis

Understanding the structure-function relationships of macromolecules, such as proteins, at the molecular level is vital for biomedicine and modern drug ...

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 ...

Protein Crystallization

Protein crystallization, obtaining a solid lattice of biomolecules, elucidates protein structure and enables the study of protein function. Crystallization involves drying purified protein under a combination of many factors, including pH, temperature, ionic strength, and protein concentration. Once crystals are obtained, the protein structure can be elucidated by x-ray diffraction and computation of an electron density model.
This video introduces protein crystallization and shows a general procedure. Protein expression and purification, crystallization, and x-ray diffraction are covered in the procedure. Applications of protein crystallization include in silico drug design, binding site determination, and membrane protein structure analysis.
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Protein crystallization is the process of obtaining a latticed solid form of a protein. These crystals are especially valuable to structural biologists, assisting in the study of protein function. Other techniques, such as mass spec or SDS-PAGE, can only provide information on the one-dimensional structure of proteins. Protein crystallization is complemented by the techniques of recombinant protein expression and x-ray diffraction. This video will show the principles of protein crystallization, a general laboratory procedure, and several of its applications in the biochemical field.
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The first step required in the process is to obtain milligram quantities of very pure protein, typically using recombinant protein expression. The gene corresponding to the protein of interest is cloned into an expression vector, and the expressed protein is fused to an affinity tag, such as poly-histidine, to assist in the purification by affinity chromatography. To learn more, see this collection&#39;s video on affinity chromatography.
Formation of the purified protein into crystals is dependent on the proper combination of many factors, including pH, ionic strength, concentrations of precipitant and protein, temperature, and rate of equilibration. The most common method used is vapor diffusion, of which there are two categories: hanging drop and sitting drop. A droplet containing pure protein, buffer, and precipitant, which is an ionic solid that binds water molecules, reducing water availability for the protein and mimicking higher protein concentration, is in an enclosed microwell with a reservoir with a more highly concentrated mixture of the same buffer and precipitant. At the beginning, the concentrations of protein and precipitant are too low to cause crystallization. During the course of the experiment, water vaporizes from the droplet and collects in the reservoir; a decrease in the amount of water in the droplet causes the system to become supersaturated, and nucleation, followed by crystallization, can occur. The net transfer of water from the droplet is in equilibrium, and the system is maintained until the process is complete.
To visualize the 3D structure, x-ray diffraction is used. To obtain x-ray data from a crystal, it is placed in a monochromatic x-ray beam, where it is exposed to the beam at all angles. Each exposure provides an image, where each spot is a diffracted x-ray, which emerges from the crystal and is registered by a detector. The data are combined to produce a model of the arrangement of atoms within the crystal. The resulting crystal structure demonstrates the 3-dimensional placement of the atoms, with a typical resolution of 2 angstroms.

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Now that we have covered the principles of protein crystallization, let us look at a generalized protocol.
To begin the procedure an expression vector containing the gene of interest is transformed into cells. The cells are incubated and at mid-log phase, expression is initiated by adding an inducer, such as IPTG, which triggers transcription of the gene&#39;s mRNA. After protein expression, the crude material is suspended in lysis buffer, and then clarified by centrifugation.
The clarified lysate is then loaded onto a nickel column, and the polyhistidine-tagged protein binds to the column while all other biomolecules are washed away.
Once several milligrams of pure protein have been obtained, it is ready for crystallization by vapor diffusion. A 24-well hanging/sitting drop tray is filled with varying concentrations of sodium chloride and sodium acetate buffer solutions. For the sitting drop method, equal volumes of protein and reservoir solution are pipetted onto the shelf above each well, and then the tray is covered with transparent tape. The tray is then placed in an incubation chamber, and the wells are monitored for growth the following day, then every few days.
Once a proper crystal has been obtained it is ready for x-ray diffraction analysis. The crystal is mounted on a goniometer to position the crystal at selected orientations. The crystal is illuminated with a monochromatic beam of x-rays at all angles, producing a diffraction pattern. The software converts the two-dimensional images, taken at different orientations, to a three-dimensional model of the density of electrons within the crystal by determining the positions of the atoms in the crystal.
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Now that we have reviewed a procedure, let&#39;s review some useful applications of protein crystallization, and another crystallization technique.
Protein crystallization may be used for in silico drug design. The three-dimensional structure of Influenza virus&#39;s polymerase basic protein 2, which has been linked to viral infection in mammals, was determined by crystallization and x-ray diffraction. Potential binding sites in the protein are visualized, and with the use of a docking program, a three-dimensional molecule was designed that would insert into a cleft in the protein.
Co-crystallization of protein-DNA complexes is also a useful technique. DNA-binding proteins modulate a wide variety of biological functions such as transcription and DNA polymerization and DNA repair; and crystal structures of these complexes can provide insight into protein function, mechanism, and the nature of the specific interaction. The E. coli protein SeqA, a negative regulator of DNA replication, was co-crystallized with hemimethylated DNA.
Integral membrane proteins such as G-protein coupled receptors, or GCPRs, are difficult to crystallize due to their limited amount of polar surface area available for forming crystal lattice contacts, which has led to the development of fusion-protein-assisted protein crystallization. Genes encoding &#946;2 adrenergic receptor, a GCPR, and a lysozyme were inserted into an expression vector. The crystallization of the &#946;2AR- lysozyme fusion protein was achieved due to the increased extracellular hydrophilic surface over the naturally hydrophobic &#946;2AR, provided by the lysozyme, necessary for forming packing interactions in the crystal lattice.
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You&#39;ve just watched JoVE&#39;s video on protein crystallization. This video described its principles, a generalized protocol, and some its uses in the biomedical field. Thanks for watching!
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Microcristalografía de Protein Crystals y

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