Os autores gostariam de agradecer ao pessoal PXRR no NSLS (Brookhaven National Laboratory) para a assistência durante a coleta de dados e Pillon Monica para ajuda com a purificação do DNA. Este trabalho foi financiado pelos Institutos Canadenses de Pesquisa em Saúde (MOP 67.189).

1. Protein Purification O ligante flexível que liga o. N-(oligomerização) e C-terminal (de ligação ao ADN), domínios de SEQA auxilia o reconhecimento de hemimethylated repete GATC separadas por 1-3 voltas sobre o DNA Para este estudo, utilizou-se uma variante de SEQA dimérica (SeqAΔ (41-59)-A25R) com um ponto de mutação no domínio N-terminal que impede ainda mais a oligomerização e um linker encurtado que restringe a ligação a DNA em tandem GATC repete separada exclusivamente pela uma vez no DNA (figura 1) de 2,8. <ol><li> Transformar BL21 (DE3) células com o plasmídeo que codifica SEQA sob o controle do promotor T7, </li><li> Placa da reacção de transformação em placas de agar LB incluindo 100 fig / ml de ampicilina, </li><li> Escolha colónias mistas para inocular uma cultura de pequena escala durante a noite (meio LB com 100 ug / ml de ampicilina), </li><li> Na manhã seguinte, inocula um L 1 de suporte, utilizando uma diluição de 1:100 do overnight cultura, </li><li> Cultivar as células a uma OD 600 de ~ 0,7 e induzir a produção de proteínas por adição de isopropil β-D-1-tiogalactopiranósido (IPTG) para uma concentração final de 1 mM, </li><li> Continuar a incubação durante 3 horas a 37 ° C com agitação orbital e depois a colheita das células por centrifugação (10 min a 3300 g), </li><li> Ressuspender o sedimento de células em tampão de purificação A e lise por sonicação, </li><li> Limpar o lisado por centrifugação (40 min a 39.000 g) e carregar o sobrenadante numa coluna de heparina, equilibrada com tampão de purificação, </li><li> Eluir SEQA utilizando um gradiente linear de NaCl a 1 M (SEQA elui a ~ 0,7 M de NaCl), </li><li> Reunir as fracções contendo SEQA juntos, dilua para reduzir a força iónica da amostra e de carga em uma coluna de cromatograf ia de troca catiónica equilibrada com tampão de purificação, </li><li> Usando um gradiente de sal linear, SEQA pura elui a ~ 0,4 M de NaCl, </li><li> Reunir as fracções contendo SEQA juntos, concentrandoe (3 mg / ml) em tampão de armazenamento e o armazenamento. </li></ol> 2. Purificação de DNA <ol><li> Encomendar complementares oligonucleotídeos unmethylated e metilados de sua empresa favorita, </li><li> Dissolver 1 mol de cada DNA de cadeia única liofilizado em 800 ul de autoclavado DDH 2 O, vórtice e deixe descansar por 10-20 min, </li><li> Adicionar 800 ul de tampão de carga pré-aquecida 2X a cada oligonucleótido, </li><li> Durante 20-30 oligonucleótidos nucleótidos de comprimento, preparar um gel desnaturante a 10% grande (160 x 250 x 1 mm): * Misture bem 80 ml de mistura de PAGE a 10%, 80 ul de TEMED e 800 ul de persulfato de amónio por gel e derrame, * Uma vez polimerizado, retire o pente e lavar os poços com DDH 2 O fundo, * Reúna os géis no elenco gel incluindo placa de arrefecimento e depois preencher os reservatórios superior e inferior com tampão de corrida (1X TBE), * Pré-executar o gel em 700-750 V para aquecer o gel até 55 ° C, * Pare a execução e lavar a thoro poçosughly com tampão de corrida. </li><li> Aquecer os oligonucleótidos a 90 ° C durante 2 min, </li><li> Vortex e girar as amostras e imediatamente antes de colocar o gel, </li><li> Execute o gel em ~ 700 V e pará-lo uma vez que seu oligonucleotídeo migrou meio. (Note-se que em um gel de poliacrilamida a 10%, azul de bromofenol a co-migra com oligonucleótidos ~ 20 bases de comprimento e xileno cianol FF com ~ 60 bases de comprimento), </li><li> Pare o gel, desmontá-la da caixa de gel e remover os espaçadores, </li><li> Sobre uma superfície plana, remova uma placa de vidro e cobrir o gel com película de plástico, </li><li> Vire o gel ao redor, retire a placa de outro vidro e cubra com filme plástico, </li><li> Marcar as bandas com luz UV e uma placa fluorescente por trás do gel para ver a sombra DNA, </li><li> Cortar a banda com uma lâmina de barbear em pedaços pequenos e transferi-las para um tubo estéril de 15 ml, </li><li> Adicionar 9 ml de tampão de eluição e eluir a noite a 37 ° C, com agitação, </li><li> Transferir cuidadosamente a soluçãopara um tubo de centrífuga de autoclavado utilizando uma ponta de pipeta de carregamento de gel, para evitar a transferência de peças de acrilamida e adicionar 1 ml de acetato de sódio 3 M, pH 7 (diluição 1:10), mais 25 ml de etanol a 100% gelado (2,5 volumes), </li><li> Incubar a -20 ° C durante pelo menos 3 horas, </li><li> Girar e transferir o sobrenadante para um tubo separado, </li><li> Seque a pelota no speed-vac em fogo médio, </li><li> Ressuspender o sedimento em 400 ul de autoclavado DDH 2 O e transferir para um tubo fresco, </li><li> Adicionar 40 ul de acetato de sódio 3 M, a pH 7 e 1 ml de etanol a 100% e misturar bem (vortex) e incubar 30 min à temperatura ambiente, seguido de 30 min a -20 ° C, </li><li> Girar durante 15 min a 18000 g e descarte do sobrenadante, </li><li> Enxaguar o sedimento com 100 ul de etanol a 70% frio para remover o sal residual a partir do sedimento e centrifugação durante 6 min a 18.000 g. Descartar o etanol e secar o sedimento no speed-vac, </li><li> Ressuspender o sedimento em um total de 100 ul de autoclavado DDH <sub&gt; 2 O. Medir a concentração do oligonucleótido, </li><li> Para emparelhar as cadeias duplas de ADN hemimethylated, misturar concentrações equimolares de as cadeias complementares individuais e aquecer as misturas a 95 ° C num banho de água durante 5 minutos e, em seguida, deixar arrefecer lentamente até à temperatura ambiente dentro do banho de água. </li></ol> 3. Proteína-DNA Formação de Complexos e Análise <ol><li> Mistura de volumes iguais de SeqAΔ purificada (41-59)-A25 (81 uM) e DNA hemimethylated (81 uM), </li><li> Incubar à temperatura ambiente por 15 minutos e armazenar a 4 ° C até que esteja pronto para usá-lo, </li><li> Tela para condições de cristalização usando comerciais esparsas de matriz telas, </li><li> Uma vez que leva a cristalização iniciais terem sido identificados, optimizar as condições de crescimento de cristais de qualidade de difração, </li><li> Cryoprotect os resultantes cristais SEQA-DNA, seja aumentando a quantidade de PEG 400 presente na solução de cristalização para uma concentração final de 25% (v / v) ou a adição de glicerol a 20% (v / v) à solução de cristalização, </li><li> Colher cristais individuais com um laço de nylon, e flash-congelá-los em nitrogênio líquido, </li><li> Teste o limite de difração de cada cristal em 100 K. </li></ol> 4. Resultados representativos Para obter a estrutura de cristal de SeqAΔ (41-59)-A25R ligado ao DNA hemimethylated, nós consecutivamente três parâmetros optimizados no DNA: (i) a separação entre hemimethylated sequências GATC, (ii) o comprimento do duplex e (iii) o ausência / presença de 5 &#39;saliências. Os ensaios de deslocamento da mobilidade electro-indicam que SeqAΔ (41-59)-A25R liga preferencialmente repete GATC separadas por 9-10 pares de bases (Figura 1). Portanto, inicialmente peneirado duplex 23-24 pares de base (bps) de comprimento contendo duas seqüências hemimethylated GATC separados por 9 ou 10 bps. Três duplexes produziu cristais bem perfilada (Figura 2). Alembora nenhum dos cristais difractados de alta resolução, o 23 bps duplex longo com os dois sítios GATC separados por 9 bps difractado raios X melhor do que o resto, o que indica que a separação GATC de 9 pb foi preferida para a cristalização. Por isso, fixo o espaçamento inter-GATC a 9 bps para todas as telas subseqüentes. Geralmente, a cristalização do DNA é favorecido para comprimentos exactos correspondentes a duplex voltas helicoidais, porque as moléculas de ADN múltiplas pode empilhar cabeça-a-cauda, ​​para formar uma contínua B-DNA dentro do cristal 9. Portanto, é reduzido o comprimento total dos duplexes a 21 bps (ou seja, duas voltas helicoidais). Enquanto um 21 bps duplex com extremidades rombas não produziram cristais de difração de qualidade (dados não mostrados), um 21 bps duplex com uma saliência 5 &#39;de um único nucleótido em cada extremidade deu cristais que difractado a 5 Å em nossa fonte de origem. A melhoria em relação ao limite de difracção sugerido que end-to-end duplex associação foi efectivamente favorecering empacotamento cristalino. Desde SEQA interage com sequências GATC que estão na mesma face do DNA, a face oposta do duplex de ADN devem ser expostos ao solvente. Para melhorar ainda mais os cristais do complexo, então a sequência modificada de modo a incluir o duplex um dinucleótido CG entre os dois sítios GATC promover ranhura backbone interacções com moléculas de DNA adjacentes no cristal por meio da face oposta do duplex, um método que tem sido utilizado para melhorar a cristalização do DNA entre os 10 passado. No entanto, o limite de difracção dos cristais crescidos com o duplex contendo CG era idêntica às cultivadas com um duplex de ADN similares que não contêm o dinucleótido CG (Figura 3). Este resultado indicou que a ranhura backbone interacções não são importantes neste caso. Nós posteriormente optimizada do comprimento das saliências, comparando os cristais crescidos com um duplex de ADN que tinha dois nucleótidos adicionais em cadaExtremidade 5 &#39;. Esta alteração não teve um efeito drástico sobre a morfologia do cristal, bem como, o limite de difracção, indicando que as sequências de nucleótidos adicionais mudou dramaticamente os contactos moleculares e organização de cristal aumentada (Figura 3). A estrutura cristalina da SeqAΔ (41-59)-A25R ligado a este último duplex de ADN confirmou que a face livre do duplex de ADN não se envolve em contactos de cristais, tal como esperado a partir do efeito limitado da introdução de um dinucleotídeo CG. Apesar do tamanho relativo das proteínas e de DNA, a maioria das interacções entre os companheiros de simetria são mediados por interacções proteína-proteína e proteína-DNA (Figura 4). É interessante notar que, neste caso particular, o efeito benéfico de uma saliência 5 &#39;dinucleótido não é devido à formação de um pseudo-DNA contínua. Em vez disso, a extremidade 5 &#39;dos ​​projectos de partículas metiladas distância dos eixos de DNA e interage com a molécula SEQA proximal do complexo, o que explica os nucleótidos dois w<html> ere estritamente necessária para mudar a embalagem cristal de 8,11. <img alt="Figura 1" src="/files/ftp_upload/4266/4266fig1.jpg" /> Figura 1. A ligação do SeqAΔ (41-59)-A25R ao DNA hemimethylated. (A) Representação esquemática dos domínios do SEQA que medeiam a oligomerização de proteínas e ligação ao ADN. (B) ensaio de desvio da mobilidade electroforética de SeqAΔ (41-59)-A25R com DNAs contendo dois hemimethylated sequências GATC, separados por um número crescente de pares de bases ( X). A pista mais à esquerda (rotulada em branco) contém uma mistura equimolar de DNAs com 5, 7, 12, 21, 25 e 34 pares de bases entre as duas sequências GATC, na ausência de SeqAΔ (41-59)-A25R. (C) diagrama representando as três variáveis ​​optimizados sobre os dúplices de ADN para se obter cristais de qualidade de difração. upload/4266/4266fig2.jpg &quot;fo: conteúdo largura =&quot; 6in &quot;fo: src =&quot; files/ftp_upload/4266/4266fig2highres.jpg / &quot;/&gt; Figura 2. Efeito da variação da inter-GATC Resumo distância. Dos oligonucleótidos utilizados e os cristais obtidos com 23-24 pb de comprimento duplexes contendo dois sítios GATC hemimethylated separados por 9 ou 10 pares de bases. Todas as imagens de cristais foram tiradas com a mesma ampliação e a barra de escala indica 100 mm. O limite de resolução de cada SeqAΔ (41-59)-A25R-DNA de cristal é baseada em imagens de difração coletados em um RU-300 Rigaku sistema gerador de raios-X. <a href="http://www.jove.com/files/ftp_upload/4266/4266fig2large.jpg" target="_blank">Clique aqui para ver maior figura</a> . <img alt="Figura 3" src="/files/ftp_upload/4266/4266fig3.jpg" fo:content-width="6in" fo:src="/files/ftp_upload/4266/4266fig3highres.jpg" /> Figura 3. Efeito da variação do DNA termina Resumo. De tele oligonucleótidos utilizados e os cristais obtidos com 21 duplexes bps longos contendo dois sítios GATC hemimethylated separados por nove pares de bases, incluindo 0, 1 ou 2 nucleótidos saliências na extremidade 5 &#39;. Todas as imagens de cristais foram tiradas com a mesma ampliação e a barra de escala indica 100 mm. O limite de resolução de cada SeqAΔ (41-59)-A25R-DNA de cristal é baseada em imagens de difração coletados em linhas de luz X12C e X29 (NSLS, BNL). <a href="http://www.jove.com/files/ftp_upload/4266/4266fig3large.jpg" target="_blank">Clique aqui para ver maior figura</a> . <img alt="Figura 4" src="/files/ftp_upload/4266/4266fig4.jpg" /> Figura 4. Empacotamento cristalino de SeqAΔ (41-59) A25R-ligado ao DNA com saliência dinucleótido: Vista de cima (A) e lateral (B). A unidade assimétrica contém dois SeqAΔ (41-59)-A25R-DNA complexos onde a proteína é mostrado a cinzento, enquanto o DNA é mostrada na laranja. Symmetry relacionado SeqAΔ (41-59)-A25R-ADN moléculas são mostrados em branco para a proteína e amarelo para o DNA. Esta figura foi preparada utilizando PyMOL 12. Este valor está relacionado ao Filme 1. <a href="http://www.jove.com/files/ftp_upload/4266/4266fig4large.jpg" target="_blank">Clique aqui para ver maior figura</a> . Filme 1. <a href="http://www.jove.com/files/ftp_upload/4266/4266movie1.avi" target="_blank">Clique aqui para ver filme</a> .

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Um dos maiores desafios em cristalografia macromolecular de raios-X é a obtenção de cristais de qualidade de difração. No caso de complexos de proteínas ou proteína-DNA, este desafio é agravado devido às variáveis ​​adicionais que devem ser optimizadas. Acredita-se que o comprimento do DNA e a presença de saliências pegajosas para melhorar associação de moléculas de DNA adjacentes a mais longo pseudo-duplex são os principais parâmetros para optimizar. No entanto, mostraram que a natureza e extensão...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Catálogo # </td><td> Comentários (opcional) </td></tr><tr><td> TRIS </td><td> Bioshop </td><td> TRS003.5 </td><td></td></tr><tr><td> Ácido etilenodiaminotetra-acético (EDTA) </td><td> Fisher Scientific </td><td> E478-500 </td><td></td></tr><tr><td> Dithiotheitrol (TDT) </td><td> Bio Básico Inc. </td><td> DB0058 </td><td></td></tr><tr><td> NaCl </td><td> Bioshop </td><td> SOD002.10 </td><td></td></tr><tr><td> Glicerina </td><td> Caledon </td><td> 5350-1 </td><td></td></tr><tr><td> Sacarose </td><td> Sigma-Aldrich </td><td> S5016-500G </td><td></td></tr><tr><td> Dodecilsulfato de sódio (SDS) </td><td> Bioshop </td><td> SDS001.500 </td><td></td></tr><tr><td> Uréia </td><td> Bioshop </td><td> URE001.5 </td><td></td></tr><tr><td> 40% Bis 29:1 / acrilamida </td><td> Bio Básico Inc. </td><td> A0007-500ml </td><td> Armazenar a 4 ° C </td></tr><tr><td> Ácido bórico </td><td> EMD </td><td> BX0865-1 </td><td></td></tr><tr><td> Xileno cianol FF </td><td> Bio-Rad </td><td> 161-0423 </td><td></td></tr><tr><td> Azul de bromofenol </td><td> Bioshop </td><td> BR0222 </td><td></td></tr><tr><td> Sistema de Gel dupla ajustável Vertical </td><td> CBC Scientific Company Inc. </td><td> DASG-250 </td><td></td></tr><tr><td> Tela de cristalização índice </td><td> Hampton Research </td><td> HR2-144 </td><td> Armazenar a 4 ° C </td></tr><tr><td> Assistente de tela cristalização I </td><td> Esmeralda BioSystems </td><td> EBS WIZ-1 </td><td> Armazenar a 4 ° C </td></tr><tr><td> Assistente de tela cristalização II </td><td&gt; Emerald BioSystems </td><td> EBS WIZ-2 </td><td> Armazenar a 4 ° C </td></tr><tr><td> Clássicos tela cristalização </td><td> Qiagen </td><td> 130701 </td><td> Armazenar a 4 ° C </td></tr><tr><td> Bandejas Intelliplate </td><td> Arte Robbins Instruments </td><td> 102-0001-00 </td><td></td></tr><tr><td></td><td></td><td></td><td> Soluções Tampão de purificação de proteína: 100 mM de TRIS, pH 8, EDTA 2 mM, DTT 2 mM e glicerol a 5%. Tampão de proteínas de armazenamento: Tris 20 mM pH 8, NaCl 150 mM, DTT 5 mM, EDTA 0,5 mM e glicerol a 5%. Gel mistura de carga: Adicionam-se 20 g de sacarose, 25 mg de azul de bromofenol, 25 mg de xileno cianol FF, 1 ml de 10% w / v de SDS e 10 ml de TBE 10X e 70 ml de DDQ autoclavado 2 O. Agita-se com aquecimento suave até que a sacarose é dissolvido e ajustar a finalo volume a 100 ml com autoclavado DDH 2 O. Armazenar a 4 ° C. 2X tampão de carregamento: Adicionar 11 g de ureia em 10 ml de mistura de carregamento de gel. Agita-se sobre uma placa quente até que a ureia dissolve-se. Alíquota em tubos de 2 ml e armazenar a 4 ° C. Mistura PAGE 10X: Misturar 420,4 g de ureia, 100 ml de TBE 10X (autoclavada), 250 ml de 40% de 29:1 Bis / Acrilamida em DDH 2 O. Agitar até totalmente dissolvido e ajustar o volume para 1 litro. Armazenar em frascos escuros a 4 ° C. TBE 10X: Dissolver 108 g de TRIS, 55 g de ácido bórico e 9,3 g de EDTA em 1 litro de DDH 2 O. Autoclave e armazenar à temperatura ambiente. Tampão de eluição: Diluir 8 ml de NaCl 5 M, 2 mL de 1 M de Tris pH 7,5, 0,4 ml de EDTA 0,5 M pH 8 em 200 ml de DDQ 2 O. Autoclave e armazenar à temperatura ambiente. </td></tr></tbody></table&gt;

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

Escherichia coli SEQA é um regulador negativo da replicação de ADN que impede eventos reinício prematuras por sequestrantes hemimethylated GATC aglomerados dentro da origem de replicação 1. Para além da origem, SEQA é encontrado nos garfos de replicação, onde organiza DNA recentemente replicado em maiores estruturas ordenadas 2. SEQA associados apenas fracamente com únicas seqüências GATC, mas forma complexos de alta afinidade com duplex de DNA contendo vários sites GATC. A unidade estrutural e funcional mínimo de SEQA é um dímero, explicando assim a exigência de pelo menos duas sequências GATC, para formar um complexo de alta afinidade com o DNA hemimethylated 3. Além disso, a arquitectura SEQA, com a oligomerização de ligação de ADN e domínios separados por um ligante flexível, permite a ligação de repetições GATC separados por até três voltas helicoidais. Portanto, a compreensão da função de SEQA em um nível molecular requer a ana estruturallise de SEQA ligada a várias sequências GATC. Na cristalização da proteína-DNA, o DNA pode ter nenhum efeito para uma excepcional sobre as interacções de embalagem, dependendo dos tamanhos relativos e da arquitectura da proteína e DNA. Se a proteína é maior do que o DNA ou pegadas maioria do DNA, o empacotamento cristalino é primariamente mediada por interacções proteína-proteína. Por outro lado, quando a proteína é do mesmo tamanho ou menor do que o DNA ou que abrange apenas uma fracção das interacções de DNA, DNA-DNA e DNA-proteína dominar empacotamento cristalino. Por conseguinte, a cristalização do DNA-proteína complexos requer o rastreio sistemático de 4 ADN de comprimento e as extremidades de ADN (sem corte ou saliência) 5-7. Neste relatório, nós descrevemos como projetar, otimizar, purificar e cristalizar duplex de DNA hemimethylated contendo repetições em tandem GATC em complexo com uma variante dimérica SEQA (SeqAΔ (41-59)-A25R) para obter cristais adequados para a determinação da estrutura.

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

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

Estrutura cristalina de complexos DNA-proteína pode fornecer informações sobre a função da proteína, mecanismo, assim como, a natureza da interacção específica. Aqui, nós relatamos como otimizar o comprimento, a sequência e as extremidades do DNA duplex para co-cristalização com<em&gt; Escherichia coli</em&gt; SEQA, um regulador negativo da iniciação da replicação.

Iterativo Otimização de Duplexos de DNA para Cristalização de SEQA-Complexos de DNA

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

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

Research

JoVE Journal

Biology

9.9K visualizações.  McMaster University. Estrutura cristalina de complexos DNA-proteína pode fornecer informações sobre a função da proteína, mecanismo, assim como, a natureza da interacção específica. Aqui, nós relatamos como otimizar o comprimento, a sequência e as extremidades do DNA duplex para co-cristalização com Escherichia coli SEQA, um regulador negativo da iniciação da replicação.

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

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

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

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

Avaliar Trials bidimensional cristalização de proteínas da membrana Pequenas de Estudos Biologia Estrutural por Cristalografia Electron

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

Biologia

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.

Cristalização de proteínas para cristalografia de raios 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.

Cristalização de proteínas da membrana lipídica em mesofases

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 rendimento cristalização de proteínas de membrana Usando o método Bicelle lipídico

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.

A utilização de um robô para alta capacidade de cristalização de proteínas de membrana em mesofases lipídicas

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

Identification of Post-translational Modifications of Plant Protein Complexes

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

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

Identificação de modificações pós-translacionais de Complexos de proteína vegetal

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

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

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

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

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

Otimização e Utilização de<em&gt; Agrobacterium</em&gt; Mediada produção de proteína transiente em<em&gt; Nicotiana</em

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

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

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

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

Mapeamento baseado em AFM das propriedades elásticas de paredes celulares: em tecido, os celulares, e as Resoluções Subcelulares

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

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

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

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

Imagens em tempo real de uma única Engineered RNA transcritos em células vivas usando raciométrica Bimolecular Beacons

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

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

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

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

Geração e Purificação de INO80 cromatina remodelação Complexos Humanos e subcomplexos

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