作者希望帮助DNA纯化感谢PXRR的工作人员在布鲁克海文国家实验室NSLS（）数据收集过程中的协助和莫妮卡皮隆方面奔去。这项工作是由加拿大卫生研究院（67189澳门元）。

1。蛋白质纯化的柔性连接器连接的N-（低聚化）和C-末端（DNA结合）域的SeqA辅助识别由一至三个匝上的DNA分离的半甲基化的GATC重复。在这项研究中，我们使用了二聚体的变种SeqA（SeqAΔ（41-59）-A25R）的N-端结构域的点突变，防止进一步寡聚化和DNA结合的串联GATC重复分离完全由一个的缩短链接器限制一匝的DNA上（ 图1）2,8。 <ol><li>与质粒编码SeqA的T7启动子的控制下，变换的BL21（DE3）细胞， </li><li>板包括100μg/ ml氨苄青霉素的LB-琼脂平板上的转化反应中， </li><li>任选混合菌落接种到一个小规模的过夜培养物（LB培养基，用100微克/毫升氨苄青霉素）， </li><li>第二天早晨，接种1 L的媒体采用1:100的稀释度的overniGHT文化， </li><li>生长细胞的OD 600〜0.7，通过加入异丙基β-D-1-硫代吡喃半乳糖苷（IPTG）至终浓度为1mM，诱导蛋白质的生产</li><li>与轨道摇动，在37℃下继续培养3小时，然后通过离心收集细胞（10分钟3300克）， </li><li>重悬细胞沉淀，净化缓冲液A中，并通过超声裂解， </li><li>清除的溶胞产物通过离心（40分钟39000克），并加载到肝素柱纯化缓冲液平衡的上清液， </li><li>厄鲁特SeqA 1 M氯化钠（SeqA洗脱在〜0.7 M氯化钠），用线性梯度</li><li>游泳池SeqA含馏分一起，稀释至样品的离子强度和负载降低到纯化缓冲液平衡的阳离子交换层析柱， </li><li>使用的线性盐梯度，纯SeqA〜0.4M的NaCl的洗脱， </li><li>游泳池的SeqA含部分，concentratE（3毫克/毫升），并存储在存储缓冲器中。 </li></ol> 2。 DNA的纯化<ol><li>订购互补的未甲基化和甲基化寡核苷酸从您最喜爱的公司， </li><li> 1μmol的每个冻干的单链DNA溶解于800μl的灭菌DDH 2 O，涡让它坐10-20分钟， </li><li>预热2X上样缓冲液中加入800μl的每个寡核苷酸， </li><li> 20-30个核苷酸的寡核苷酸，准备一个大10％的变性凝胶（160×250×1毫米）: *拌匀，倒80毫升10％PAGE组合，80μLTEMED和800μL过硫酸铵每凝胶， *聚合后，取出梳子和DDH 2 O彻底冲洗井， *组装凝胶凝胶铸造包括冷却板，然后填充有运行缓冲液（1X TBE缓冲液）的顶部和底部的水库， *预运行凝胶在700-750 V温暖的凝胶，高达55°C， *停止运行，冲洗井thoroughly与运行缓冲液。 </li><li>的寡核苷酸加热至90℃，持续2分钟， </li><li>涡街流量计和旋样品，并立即装货前的凝胶， </li><li> 〜700 V和运行的凝胶，一旦你的寡核苷酸迁移中途停止。 （请注意，在10％聚丙烯酰胺凝胶的溴酚蓝共迁移与〜20个碱基长，和二甲苯青FF〜60个碱基长的寡核苷酸）， </li><li>停止凝胶，拆解从凝胶中删除的间隔， </li><li>在一个平坦的表面，除去一块玻璃板，并用保鲜膜覆盖凝胶， </li><li>将周围的凝胶，删除其他的玻璃板，并用保鲜膜覆盖， </li><li>标记带紫外光和荧光板背后的凝胶看到的DNA阴影， </li><li>用剃刀刀片切带切成小块，并将它们传送到一个​​无菌的15毫升的试管中， </li><li>加入9毫升的洗脱缓冲液洗脱，边搅拌边在37℃下过夜， </li><li>小心地将溶液转移蒸压使用凝胶加载的移液管尖，以避免传输的丙烯酰胺件和添加3M乙酸钠（1:10稀释）在pH为7的1毫升，冰鲜100％乙醇（2.5体积）中加25毫升的离心管， </li><li>在-20℃下孵育至少3小时， </li><li>自旋向下，将上清转移到一个单独的管， </li><li>干燥沉淀的速度，真空中火， </li><li>将沉淀重悬于400μl的灭菌DDH 2 O，并转移到一个新的管， </li><li>加入40μl的3M醋酸钠在pH 7和1毫升100％乙醇;拌匀（涡流），并在室温下孵育30分钟，然后在-20℃下30分钟， </li><li>旋转为15分钟18000克和弃去上清液， </li><li>用100微升70％冷乙醇洗涤沉淀，在18000克的6分钟，以除去残留的盐从粒料和自旋。弃乙醇和速度真空干燥沉淀， </li><li>重悬沉淀共100μl的灭菌DDH <sub&gt; 2 O。测量的寡核苷酸的浓度， </li><li>要退火的半甲基化的DNA双链，混合等摩尔浓度的互补单链，并加热该混合物至95℃，在水浴中5分钟，然后让它们慢慢冷却至室温的内部温度的水浴。 </li></ol> 3。蛋白质-DNA复合物的形成与分析<ol><li>混合纯化SeqAΔ相等体积的（41-59）-A25（81μM）和半甲基化的DNA（81μM）， </li><li>在室温下孵育15分钟并存储在4°C，直到您准备使用它， </li><li>屏幕的结晶条件下使用商业稀疏矩阵屏幕， </li><li>一旦初始结晶线索已经确定，优化的条件下成长衍射晶体， </li><li> Cryoprotect所得SeqA-DNA的晶体通过增加的量的结晶溶液中​​至最终浓度为25％的PEG 400本（体积/体积）或到的结晶溶液中​​加入20％甘油（V / V）， </li><li>炒到单独的晶体用尼龙的循环，和闪存的在液氮中冻结， </li><li>测试，在100 K，每个晶体的衍射极限</li></ol> 4。代表性的成果要获得的晶体结构SeqAΔ（41-59）-A25R绑定到半甲基化的DNA，我们连续优化的DNA上的三个参数:（ⅰ）分离半甲基化的GATC序列之间，（ii）长度的双面;及（iii）的没有/ 5&#39;突出存在的。 电迁移率变动分析表明SeqAΔ（41-59）-A25R优先结合GATC重复由9-10个碱基对（ 图1）隔开。因此，我们初步筛选复式月23-24个碱基对（BPS）long，它包含两个分离的半甲基化的GATC序列由9或10个基点。三复式取得了很好的板状结晶（ 图2）。铝虽然没有高分辨率的晶体衍射，23个基点长全双工与两个GATC网站，相隔9个基点衍射的X射线比其他人更好，这表明，GATC​​首选的结晶分离的9个基点。因此，我们固定的间GATC的间距为9个基点，随后的所有画面。 一般来说，DNA结晶为双面的长度对应于精确的螺旋匝的青睐，因为可以堆叠多个DNA分子的头-尾，以形成连续的B-DNA内的晶体9。因此，我们缩短的双链体的总长度，以21个基点（ 即两个螺旋圈）。虽然21个基点复式两端钝圆并没有产生衍射晶体（数据未显示），21个基点复式一个5&#39;突出核苷酸的每一端，并产生晶体的衍射5Å在我们的主场源。上的衍射极限的改进建议，端至端的双工关联确实利于ING水晶包装。 由于SeqA与GATC序列的DNA，在同一面上，交互的DNA双链的相反面应暴露于溶剂。为了进一步提高复合物的结晶的，我们再修改的序列包括两个GATC站点之间的CG二核苷酸与相邻的DNA分子在晶体促进槽骨干的相互作用，通过相反侧的面的双面，方法的双工已被用于提高DNA在过去10结晶。然而，含CG-双工与生长的晶体的衍射极限是具有类似的DNA双链中不包含的CG二核苷酸（ 图3）生长的那些相同。这一结果表明，槽骨干相互作用是在这种情况下，并不重要。其后，我们通过比较，在每个有两个额外的核苷酸与DNA双链的晶体生长优化的悬伸长度5&#39;末端。这种变化有重大影响的晶体形态，以及表明的额外核苷酸极大地改变了分子接触和增强的晶体组织（ 图3），衍射极限。 这最后的确认，不从事晶体接触的自由面的DNA双螺旋的DNA双螺旋结合的晶体结构SeqAΔ（41-59）A25R，如预期推出的CG二核苷酸的效果有限。尽管蛋白质和DNA的相对大小，对称配合的大部分之间的相互作用介导的蛋白质-蛋白质，蛋白质-DNA相互作用（ 图4）。有趣的是，在这个特殊的情况下，一个5&#39;二核苷酸悬的有益效果是没有由于形成伪连续DNA。相反，在5&#39;末端的甲基化链项目远离的DNA轴并与之交互的复杂的近端SeqA分子，解释为什么两个核苷酸瓦特<html> ERE严格要求改变晶体包装8,11。 <img alt="图1" src="/files/ftp_upload/4266/4266fig1.jpg" /> 图1。半甲基化DNA结合SeqAΔ（41-59）A25R。 （A）示意图的领域SeqA，介导蛋白质寡聚化和DNA结合。（B）电泳迁移率变动分析受到越来越多的碱基对分开的两个半甲基化的GATC序列的DNA片段包含的SeqAΔ（41-59）A25R（ X）。最左侧的车道（标记为空白）包含与5的等摩尔混合物的DNA，7，12，21，25和34个碱基对没有SeqAΔ（41-59）之间的两个GATC序列A25R。（C）的图中描绘了三个变量的DNA双链上进行了优化，以获得衍射晶体。 upload/4266/4266fig2.jpg"佛:内容的宽度="6英寸的FO:src ="/ files/ftp_upload/4266/4266fig2highres.jpg"/&gt; 图2。间GATC的距离。摘要23日至24日bp的复式含9个或10个碱基对分开的两个半甲基化的GATC网站获得的寡核苷酸和晶体不同的影响 。所有图片晶体在相同的放大倍率和规模栏显示为100微米。分辨率极限每个SeqAΔ（41-59）A25R-DNA晶体在Rigaku RU-300 X-射线发生器系统的基础上收集衍射图像。 <a href="http://www.jove.com/files/ftp_upload/4266/4266fig2large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图3" src="/files/ftp_upload/4266/4266fig3.jpg" fo:content-width="6in" fo:src="/files/ftp_upload/4266/4266fig3highres.jpg" /> 图3。改变DNA的影响结束。摘要吨他用寡核苷酸，获得的晶体，用21个基点长双链含有分离两个半甲基化的GATC网站，由9个碱基对，并包括0，1或2个核苷酸的5&#39;末端出挑。所有图片晶体在相同的放大倍率和规模栏显示为100微米。分辨率极限每个SeqAΔ（41-59）-A25R-DNA晶体在光束线X12C和X29（NSLS，BNL）的基础上收集衍射图像。 <a href="http://www.jove.com/files/ftp_upload/4266/4266fig3large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图4" src="/files/ftp_upload/4266/4266fig4.jpg" /> 图4。水晶包装SeqAΔ（41-59）-A25R绑定到DNA核苷酸悬:从（A）顶部和侧面（B）。的不对称单位包含两个SeqAΔ（41-59）-A25R-DNA复合物，其中的蛋白质以灰色显示，而该DNA以橙色显示。对称Ry的:相关SeqAΔ（41-59）-A25R-DNA分子以白色表示的蛋白质和黄色的DNA。本图是使用PyMOL 12。这个数字与动画1。 <a href="http://www.jove.com/files/ftp_upload/4266/4266fig4large.jpg" target="_blank">点击此处查看大图</a> 。 动画1。 <a href="http://www.jove.com/files/ftp_upload/4266/4266movie1.avi" target="_blank">点击这里观看电影</a> 。

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没有利益冲突的声明。

高分子X射线晶体学面临的最大挑战之一是获得衍射晶体。在蛋白质或蛋白质-DNA复合物的情况下，这一挑战，加剧了由于必须进行优化的附加变量。人们普遍相信，相邻的DNA分子，在更长的伪全双工的主要参数进行优化以提高关联的DNA的长度和存在的粘出挑。然而，我们已经表明，这些悬伸的性质及长度可以有额外的影响的蛋白质-DNA复合物的晶体堆积。虽然这个协议设计的合理设计的寡核苷酸为?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录＃ </td><td> 评论（可选） </td></tr><tr><td> TRIS </td><td> Bioshop </td><td> TRS003.5 </td><td></td></tr><tr><td>乙二胺四乙酸（EDTA） </td><td> Fisher Scientific则</td><td> E478-500 </td><td></td></tr><tr><td> Dithiotheitrol（DTT） </td><td>生物技术有限责任公司</td><td> DB0058 </td><td></td></tr><tr><td>氯化钠</td><td> Bioshop </td><td> SOD002.10 </td><td></td></tr><tr><td>甘油</td><td>卡利登</td><td> 5350-1 </td><td></td></tr><tr><td>蔗糖</td><td> Sigma-Aldrich公司</td><td> S5016-500G </td><td></td></tr><tr><td>十二烷基硫酸钠（SDS） </td><td> Bioshop </td><td> SDS001.500 </td><td></td></tr><tr><td>尿素</td><td> Bioshop </td><td> URE001.5 </td><td></td></tr><tr><td> 40％的29:1双/丙烯酰胺</td><td>生物技术有限责任公司</td><td> A0007-500ML </td><td>储存于4°C </td></tr><tr><td>硼酸</td><td> EMD </td><td> BX0865-1 </td><td></td></tr><tr><td>二甲苯蓝FF </td><td> Bio-Rad公司</td><td> 161-0423 </td><td></td></tr><tr><td>溴酚蓝</td><td> Bioshop </td><td> BR0222 </td><td></td></tr><tr><td>可调式双垂直凝胶体系</td><td> CBC科技公司公司</td><td> DASG-250 </td><td></td></tr><tr><td>指数结晶屏幕</td><td>汉普顿研究</td><td> HR2-144 </td><td>储存于4°C </td></tr><tr><td>精灵我结晶屏</td><td>翡翠生物系统公司</td><td> EBS-WIZ-1 </td><td>储存于4°C </td></tr><tr><td>精灵II结晶屏</td><td&gt;绿生物系统公司</td><td> EBS-WIZ-2 </td><td>储存于4°C </td></tr><tr><td>经典结晶画面</td><td> Qiagen公司</td><td> 130701 </td><td>储存于4°C </td></tr><tr><td> Intelliplate盘</td><td>艺术罗宾斯工具</td><td> 102-0001-00 </td><td></td></tr><tr><td></td><td></td><td></td><td>解决方案 蛋白质纯化用缓冲液:100mM的TRIS pH值为8，2mM的EDTA，1mM DTT和5％甘油。 蛋白储存缓冲液:20mM的TRIS pH值为8，150mM的NaCl，5mM的DTT，0.5mM EDTA和5％甘油。 凝胶装载组合:20克蔗糖，25毫克的溴酚蓝，二甲苯青FF 25毫克，1毫升10％w / v的SDS，10毫升10×TBE缓冲液加入到第70毫升蒸压DDH 2 O中温和加热搅拌直到溶解蔗糖和调整最终体积为100毫升与蒸压DDH 2 O存放于4℃ 2X上样缓冲液:加入11克尿素凝胶上样混合至10毫升。热板搅拌，直到尿素溶解。分装在2 mL管和存储在4°C。 10X PAGE混合:混合420.4克尿素，100毫升10×TBE缓冲液（蒸压），250毫升40％在DDH 2 O 29:1的双/丙烯酰胺搅拌，直至完全溶解并调节音量，以1公升。在黑暗的瓶子存放于4℃ 10X TBE:溶解 108克 TRIS，55克硼酸和9.3克EDTA DDH 2 O在1升在室温下的高压灭菌器和存储。 洗脱缓冲液:稀8毫升DDH 2 O200毫升上的5M NaCl，2毫升1 M TRIS液pH7.5，0.4毫升的0.5M EDTA pH值为8在室温下的高压灭菌器和存储。 </td></tr></tbody></tabLE&gt;

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

大肠杆菌 SeqA是一个负调节DNA复制，以防止过 ​​早封存半甲基化的GATC集群内再引发事件的起源，复制1。除了 ​​原点，SeqA被发现在复制叉，它举办新复制的DNA到更高的有序结构。 SeqA联营公司只有微弱的单GATC序列，但它形成高亲和力DNA双链包含多个GATC网站的配合。功能和结构的最小单元SeqA一个二聚体，由此说明的要求中的至少两个的GATC序列与半甲基化DNA的3形成的高亲和性的络合物。此外，SeqA的体系结构，与低聚和由柔性连接器分离的DNA结合结构域，允许绑定GATC重复分离最多三螺旋匝。因此，在分子水平上理解的功能SeqA需要的结构模拟裂解SeqA绑定到多个GATC序列。在蛋白质-DNA结晶，DNA可以没有一个特殊的效果取决于蛋白质和DNA的相对大小和体系结构在包装上的相互作用。如果蛋白质的DNA或脚印大多数的DNA的比大时，晶体堆积主要是介导的蛋白质 - 蛋白质相互作用。相反，当该蛋白质是相同的尺寸或小于该DNA，或者它仅覆盖的一小部分的DNA，DNA-DNA和DNA-蛋白质相互作用支配晶体包装。因此，蛋白质-DNA复合物的结晶化需要的DNA长度为4的系统的筛选和DNA末端（钝或悬）5-7。在这份报告中，我们将介绍如何设计，优化，净化，结晶半甲基化DNA双链串联GATC重复复杂的二聚体的变种SeqA（SeqAΔ（41-59）-A25R），以获得合适的晶体结构的决心。

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

蛋白质-DNA复合物的晶体结构，可以提供蛋白质的功能，机制，以及特定的相互作用的性质的洞察。这里，我们报告如何优化共结晶与双链DNA的长度，序列和两端<em&gt;大肠杆菌</emSeqA，复制起始的负调控因子。

迭代优化的DNA双链的SeqA-DNA复合物的结晶

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 次观看.  McMaster University. 蛋白质-DNA复合物的晶体结构，可以提供蛋白质的功能，机制，以及特定的相互作用的性质的洞察。这里，我们报告如何优化共结晶与双链DNA的长度，序列和两端大肠杆菌

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

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.

X射线晶体学的蛋白质结晶

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

科研

生物学

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.

在油脂介孔膜蛋白的结晶

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.

使用油脂全线Bicelle方法的膜蛋白的高通量结晶

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.

使用机器人在油脂中间相高通量膜蛋白的结晶

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

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.

优化和利用<em&gt;农杆菌</em&gt;介导的瞬态蛋白生产中<em&gt;烟草</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.

细胞壁的弹性性能的原子力显微镜为基础的映射:在组织，细胞，亚细胞和决议

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.

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

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.

产生和人类INO80染色质重塑复合物及其亚纯化

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

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

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

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

高效的哺乳动物细胞表达和细胞外糖蛋白的结晶一步纯化

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

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

逆转录病毒整合位点的扩增，新一代测序和基因组DNA制图

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...