我们感谢员工同步数据采集期间在瑞士光源，保罗谢勒研究所，菲利根，瑞士支持束线X06DA。我们感谢莫妮卡Gompert优秀的技术支持。该项目由科隆大学，并授予由德国研究委员会INST 216 / 682-1 FUGG支持。从国际研究生院在发展健康和疾病给CP一个博士研究生奖学金是公认的。导致这些结果的研究号赠款协议283570（BioStruct-X）下收到的资金来自欧盟第七框架计划（FP7 / 2007-2013）。

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The authors have nothing to disclose.

X射线晶体学仍然是确定的蛋白质28三维近原子分辨率的结构最快，最准确的方法。但是，它需要秩序井然单晶的生长。这些往往难以得到与结晶状态是人为的。然而，通过X射线晶体与用其他方法，测定确定蛋白质结构的比较特别NMR，通常显示出非常良好的一致性。在PPEP-1的情况下，一个NMR结构最近发表29示出良好的协议与我们的晶体结构14，包括S-环路的流动性。 这个协议描述N端His-标记的rPPEP-1蛋白质用于结构研究和未标记rPPEP-1的结晶和结构确定的生产和纯化。单晶难以在这种情况下，发展和需要特殊microseeding过程。在T他以下部分中，我们将讨论的结果PPEP-1和指示协议如何能够适于任何其他蛋白质的生产和结晶。 在结构设计和表达的变化 rPPEP-1被表示为N-末端His-标记的变体（ 图1A），作为用于结晶优选除去凝血酶His标签消化由于对结晶成功和蛋白质结构30可能产生的影响。对于其他蛋白质也可能是可取的附加测试C末端His标记的版本或离开的N末端（ 例如 ，使用相同的限制性位点，载体pET22b和在3&#39;端没有终止密码子的反向引物克隆） His-标签未切割，如在一些情况下，标签可在结晶期间帮助。对于rPPEP-1的C-末端His标签的构建物的产率和稳定性差。此外，对于最佳秒的初始测试oluble蛋白表达应包括在下面的大肠杆菌菌株:BL21（DE3），BL21（DE3）pLysS中或Lemo21（DE3），BL21（DE3）的密码子加RIPL或罗塞塔2（DE3），BL21（DE3）星C41（DE3），在三个不同温度/温育时间（37℃，在30℃下5小时，并在20℃过夜3-4小时）。克隆前，通过使用ExPASy PeptideCutter工具31，以防止感兴趣的蛋白质的裂解检查内部凝血酶切割位点的存在。可替代地，可有向量HRV3C提供的切割位点（ 即 EMBL的PETM-14 32）或TEV蛋白酶（ 即 EMBL的PETM-11）。 蛋白质纯化 对于rPPEP-1构建体是通过在冰/水超声处理进行细胞裂解。对于倾向于聚集或沉淀更"温柔"细胞裂解法涉及细胞破碎可能会使用更敏感蛋白质ð。检查大量不溶性蛋白质的第一次离心步骤后，使用裂解法如化学细胞裂解时它没有检测到。对于rPPEP-1的纯化通常被用来将2ml NiNTA树脂。读出的制造商的选择的树脂的蛋白质结合能力有多大的指令并相应地调整。在rPPEP-1，其中只需1毫升树脂的混合物中使用的，大量的蛋白质不结合在柱上，并在流过（ 图1D）中发现的第一个纯化。使用太多的树脂可能会降低纯化蛋白的纯度。另一方面。调整咪唑浓度所使用的His-标记构建体的NiNTA基质的结合亲和力。的这种逐步洗涤过程此处优选的（ 即 10毫米，30毫米，50毫米，70 mM咪唑），其中，A280被监控并且被允许在每个步骤中，以达到基线。以这种方式没有蛋白质丢失，即使它会在一个洗脱高咪唑清洗步骤（ 例如 ，在50或70毫米）。在的情况下NHIS-rPPEP -1-一些蛋白质已洗脱在30 mM咪唑，但目前还不清楚其代表了什么样的蛋白质的物种。当咪唑透析或稀释了的蛋白质溶液的一些蛋白质沉淀/聚集体。在这种情况下，降低的咪唑洗脱缓冲液至150mM的浓度和暴露于高咪唑浓度的时候， 例如，通过洗脱减少咪唑浓度成与含有缓冲液的5-10倍体积而不咪唑烧杯计划洗脱体积。对于NHIS-rPPEP-1温育，每mg蛋白质2单位凝血酶在4℃下过夜足以His标签切割至几乎100％。通过使用每mg蛋白质1-10单位在4℃至20℃调节凝血酶的感兴趣的蛋白质的量。当使用5-10分钟间隔的离心超滤装置暂停和混合在p浓缩蛋白rotein均匀化浓度梯度在集中建立。否则，高度浓缩蛋白能聚集/沉淀。 结晶 rPPEP -1-不断产生高度共生晶体（单晶生长在彼此的顶部，从而具有多个晶格），它们不适合于X-射线衍射分析。的解释可能是过多的核事件在相图（ 图7）的成核区的发生。 <img alt="图7" src="/files/ftp_upload/55022/55022fig7.jpg" /> 图7:蛋白质结晶实验的相图。晶体只能形成，当该蛋白质是过饱和。成核发生在亚稳区中的成​​核区和晶体生长。当蛋白质是欠，落将保持清晰。 &lt;p系列="jove_content"&gt;蛋白质或沉淀剂浓度的降低并没有使局势则没有rPPEP-1的核不再是观察水珠保持清晰。 Microseeding是选择的方法中，作为微小晶核直接带入稳区，其中rPPEP-1的结晶最终成长（ 图7）。通过的方式，原来的状态（2.4米磷酸铵二元，0.1M的Tris pH值8.5）位于C5位（ 图4）（而pH和沉淀剂浓度的高端）设计优化屏幕最新的条件对应于一个状态与一个高的倾向较低的过饱和来表示亚稳区16（ 图7）的一部分。因此，那些播种期间带来了核有可能成长为大单晶。为了优化该过程（新形成的晶体的数量和大小）的原原种股票可以稀释10 <suP&gt; -2至10 -5。这里的种子储备的几个稀释度可以测试，以确定最佳的种子稀释为生产的大型单晶。或者使用动物晶须或尾毛（兔，猫，灰鼠或马）连胜播种可以采用33。 该过程可用于共结晶产物肽和rPPEP-1底物的肽。在优化过程中250:在空间群P2 1的结晶衍射高达1.25埃从种子种群得到稀释。晶体生长在两个空间群P2 1 2 1 2 1（未结合的蛋白质）和P2 1（复杂的结构），而从最优化过程的磷酸铵画面内非常类似的条件始发。由于在两个R-PPEP-1结晶形式，主要是所有的小分子和肽处理的活性部位的无底涂侧发现的晶体堆积单独可以是soakeD写入晶体。该分子的这一侧是从存在于晶体中的溶剂可及。然而分子寻址两个子站点或底涂侧只需要进行共结晶与rPPEP-1，为S-环开口将被要求容纳它们的活性位点。一个事实，即也限制底物肽的长度以3个残基的 - 此外，rPPEP-1是由邻近的分子在从靠近S3&#39;site（在此领域促进晶体接触的）的底物结合位点的出口接触底涂侧这两晶体形式。 水晶安装和低温保护 蛋白质晶体的安装是需要在体视显微镜下操作一些技巧，因此需要一些练习的方法。选择尼龙环（或选择的其他环， 例如 litholoop）的最佳长度，以防止过量的溶剂，有助于背景和晶体周围ð散射和因此较低的信号的衍射数据的信噪比。最佳环的大小/长度也使得晶体更容易为晶体通过循环不打滑的钓鱼。对于rPPEP-1的结晶，其是在最长尺寸约100-200微米，0.1-0.2毫米大小的尼龙线圈被选择。的冷冻保护也可能有助于数据质量的恶化，当转移到低温条件下的晶体可能会遇到渗透压休克。这可能会阻碍甚至破坏晶体的内部秩序。慎重选择冷冻保护剂的种类和浓度。 rPPEP-1晶体是冷冻保护与任一20％甘油或30％的蔗糖。如果结晶rPPEP-1与另一个配位体的基板肽复合物或复合物，蔗糖应选择作为冷冻保护甘油结合rPPEP-1 14的基板结合位点的引物位点。 结构测定 该解决方案AutoSol获得49.2±18.4贝叶斯-CC，0.41的FOM（品质因数），0.17歪斜和0.85的相关性RMS。模型映射相关性是0.86和ΔR/ R 无因子是0.21 / 0.24。所有这些参数表明一个成功的结构解决方案。

难辨梭状芽孢杆菌是院内抗生素相关性腹泻感染1的主要原因之一。这种革兰氏阳性厌氧细菌通过其孢子形式通过粪 - 口途径传播。在过去的十年，新的&#39;流行&#39;&#39;或&#39;&#39;超毒&#39;株（ 如 BI / NAP1 / 027）造成的在北美和欧洲的2个新的感染和死亡率急剧增加。 艰难梭菌病机相关性（CDAD）是一种威胁生命结肠炎的高死亡率3。症状的范围从腹泻4至伪膜性肠炎5和经常致命的中毒性巨结肠6。 CDAD治疗是困难的，因为该毒株是耐多药，复发率高7。此刻疗法包括抗生素甲硝唑，fidaxomicin或万古霉素，或者在repetitively复发病例粪便菌群移植。新的治疗策略，迫切需要8。一些进展记录为治疗性单克隆抗体Bezlotoxumab，瞄准艰难梭菌毒素B 9，日前顺利通过III期临床试验，并申请了与FDA和EMA的批准。此外，新的抗生素被此刻测试在临床试验10的不同阶段。 要制定有效的治疗新的治疗靶点必须进行标识。最近发现的艰难梭菌蛋白酶脯氨酸脯氨酸内肽酶-1（PPEP-1; CD2830 / Zmp1; EC 3.4.24.89）是这样一种有前途的靶，如在敲除菌株缺乏PPEP-1的降低的C毒力。难辨体内 11。 PPEP-1是分泌金属蛋白酶12,13在它们的C-末端裂解2 梭状粘附13从而释放粘合杆菌IA从人体肠道上皮细胞。因此，参与维持艰难梭菌的固着和能动表型之间的平衡。为了开发选择性抑制剂对PPEP-1，了解它是如何认识它的底物的三维结构的深入了解是必不可少的。我们已经解决PPEP-1单独和复合物的第一晶体结构的衬底的肽14。 PPEP-1是第一个已知的蛋白酶的两个脯氨酸残基的15之间选择性裂解肽键。其结合在双扭结方式在基片和通过位于在S环覆盖蛋白酶活性位点14个残基的延伸脂族-芳族的网络稳定它。此底物结合模式是唯一的PPEP-1和人蛋白酶迄今未找到。这使得它有前途的药物靶标，以及脱靶酶抑制剂不太可能的影响。 为了发展和屏幕选择PPEP-1 INHibitors在将来需要大量的纯净，单分散PPEP-1蛋白。此外，为了确定与PPEP-1第一抑制剂，共晶体结构的结合将要确定的模式。在我们的手中PPEP-1不断推出共生晶体。因此，我们已开发出一种优化过程，以产生PPEP-1的单衍射质量的晶体。在这个协议中，我们详细描述了PPEP-1 14的生产，纯化，结晶和结构的解决方案。我们在缺乏分泌信号序列，亲和层析和大小排阻色谱法除去纯化标记的一个PPEP-1变体的大肠杆菌用胞内表达，随后通过锌单波长异常色散microseeding 16成优化屏幕和结构确定（锌- SAD）17。这个协议可以适于生产和结构确定的其他蛋白质（ 例如&lt;/ em&gt;的金属蛋白酶），特别是用于生产共生晶体蛋白质。根据要求，构建体的质粒DNA（质粒pET28a-NHIS-rPPEP-1）和衍射数据可以提供用于教育目的。

<table border="0" cellpadding="0" cellspacing="0" width="657">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Genes / Vectors / cell strains</td>
			<td style="width:83px;"></td>
			<td style="width:136px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pET28a vector</td>
			<td>Merck-Millipore</td>
			<td>69864</td>
			<td>Thrombin cleavable N-terminal His-tag</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">E. coli strain BL21 (DE3) Star</td>
			<td>ThermoFisher Scientific</td>
			<td>C601003</td>
			<td>RNase H deficient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Codon-optimized gene (for E. coli) of PPEP-1 (CD630_28300)</td>
			<td>Geneart (Thermo Fisher Scientific)</td>
			<td>custom</td>
			<td>amino acids 27-220</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Name</td>
			<td style="width:83px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Yeast extract</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tryptone</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antifoam B</td>
			<td>Sigma-Aldrich</td>
			<td>A5757</td>
			<td>aqueous-silicone emulsion</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agar</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kanamycin</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IPTG</td>
			<td>AppliChem</td>
			<td>A1008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris-HCl</td>
			<td>AppliChem</td>
			<td>A1087</td>
			<td>Buffer grade</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>any</td>
			<td></td>
			<td>Buffer grade</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNaseI</td>
			<td>AppliChem</td>
			<td>A3778</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imidazole</td>
			<td>AppliChem</td>
			<td>A1073</td>
			<td>Buffer grade</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thrombin</td>
			<td>Sigma-Aldrich</td>
			<td>T4648</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium phosphate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>215996</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol 100%</td>
			<td>any</td>
			<td></td>
			<td>purest grade</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>84097</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Liquid nitrogen</td>
			<td>any</td>
			<td></td>
			<td>for storage and cryocooling of crystals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Name</td>
			<td style="width:83px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment (general)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Shaking incubator</td>
			<td>any</td>
			<td></td>
			<td>providing temperatures of 20 &#176;C - 37 &#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glassware</td>
			<td>any</td>
			<td></td>
			<td>baffled Erlenmeyer flasks (50 ml - 2.8L)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge for large culture volumes</td>
			<td>any</td>
			<td></td>
			<td>centrifuge for processing volumes up to 12 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sonicator Vibra-Cell VCX500</td>
			<td>Sonics</td>
			<td>SO-VCX500</td>
			<td>or any other sonicator / cell disruptor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultracentrifuge</td>
			<td>any</td>
			<td></td>
			<td>centrifuge providing speeds up to 150.000 x g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NiNTA Superflow resin</td>
			<td>Qiagen</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Empty Glass Econo-Column</td>
			<td>Bio-Rad</td>
			<td>7371007</td>
			<td>or any other empty glass or plastic column</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Size exclusion chromatography column HiLoad Superdex 200 16/600</td>
			<td>GE Healthcare</td>
			<td style="width:136px;">28989335</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chromatography system &#196;kta Purifier</td>
			<td>GE Healthcare</td>
			<td>28406264</td>
			<td>or any other chromatography system</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dialysis tubing Spectra/Por 3</td>
			<td>Spectrum Labs</td>
			<td>132724</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dialysis tubing closures</td>
			<td>Spectrum Labs</td>
			<td>132738</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultrafiltration units (concentrators) 10.000 NWCO</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UV-Vis spectrophotometer</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Name</td>
			<td style="width:83px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment (crystallography)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Low volume pipette 0.1-10 &#181;l</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Positive displacement pipette Microman M10</td>
			<td>Gilson</td>
			<td>F148501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Crystallization robot</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96-well crystallization plates TTP IQ with three protein wells</td>
			<td>TTP</td>
			<td style="width:136px;">4150-05810</td>
			<td>or any other 96-well crystallization plate&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">24-well CombiClover Junior Plate</td>
			<td>Jena Bioscience</td>
			<td>EB-CJR</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Crystal Clear Sealing Tape</td>
			<td>Hampton Research</td>
			<td>HR3-511</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Siliconized Glass Cover Slides</td>
			<td>Hampton Research</td>
			<td>HR3-225</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Commercial crystallization screens: SaltRx, Index, PEG/Ion, Crystal</td>
			<td>Hampton Research</td>
			<td>diverse</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Commercial crystallization screens: Wizard, PACT++, JCSG++</td>
			<td>Jena Bioscience</td>
			<td>diverse</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">JBS Beads-for-Seeds</td>
			<td>Jena Bioscience</td>
			<td>CO-501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CrystalCap SPINE HT (nylon loops)</td>
			<td>Hampton Research</td>
			<td>diverse</td>
			<td>loop sizes 0.025 mm - 0.5 mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CrystalCap Vial</td>
			<td>Hampton Research</td>
			<td>HR4-904</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Cryogenic Foam Dewar 800 ml</td>
			<td>Hampton Research</td>
			<td>HR4-673</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Cryogenic Foam Dewar 2L</td>
			<td>Hampton Research</td>
			<td>HR4-675</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vial Clamp, Straight</td>
			<td>Hampton Research</td>
			<td>HR4-670</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CrystalWand Magnetic, Straight</td>
			<td>Hampton Research</td>
			<td>HR4-729</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CryoCane 6 Vial Holder</td>
			<td>Hampton Research</td>
			<td>HR4-711</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CryoSleeve</td>
			<td>Hampton Research</td>
			<td>HR4-708</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CryoCane Color Coder - White</td>
			<td>Hampton Research</td>
			<td>HR4-713</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scalpel</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Straight microforcep</td>
			<td>any</td>
			<td></td>
			<td>for manipulation of sealing tape. etc.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acupuncture needle</td>
			<td>any</td>
			<td></td>
			<td>e.g. from a pharmacy</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;">Stereo microscope</td>
			<td>any</td>
			<td></td>
			<td style="width:191px;">for inspection of crystallization plates and crystal mounting, magnification up to 160X</td>
		</tr>
	</tbody>
</table>

production, crystallization and structure determination of c. difficile ppep-1 via microseeding and zinc-sad

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a target for future development of inhibitors to decrease the virulence of the pathogen. To perform biophysical and structural characterization as well as inhibitor screening, large amounts of pure and active protein will be needed. We have developed a protocol for efficient production and purification of PPEP-1 by the use of E. coli as the expression host yielding sufficient amounts and purity of protein for crystallization and structure determination. Additionally, using microseeding, highly intergrown crystals of PPEP-1 can be grown to well-ordered crystals suitable for X-ray diffraction analysis. The methods could also be used to produce other recombinant proteins and to study the structures of other proteins producing intergrown crystals.

rPPEP-1过表达的几个大肠杆菌菌株，在大肠杆菌 BL21（DE3）中星（ 图1C）的最高产率。第一NiNTA亲和层析步骤之后的6xHis标签可以从大多数蛋白质的成功切除和在第二NiNTA步骤未消化的蛋白质可以从凝血酶消化蛋白质（ 图1D）完全分离。上的S200六百分之十六柱未标记rPPEP-1迁移与偶尔面向最有可能对应于二聚体物种（ 图2A）的单体。蛋白质是纯的（ 图2B），收率从1升大肠杆菌培养物的约50mg蛋白质。 rPPEP-1结晶在各种条件下（ 图3），常含有磷酸盐。该晶体是高度共生在所有条件下，使结晶的优化不得不进行。 图4描述了舍姆用于从条件商业屏幕SaltRx E4（52）（ 图3B-C）的始发晶体执行的最优化过程的即再次共生结晶出现在井中A6-D 6（ 图4）。一个microseed库存从在优化屏幕的状态C6（2.55米磷酸铵二元，0.1M的Tris pH值8.5）中生长的晶体制备并稀释1:在母液1000。与稀释的种子的库存播种成其他剩余的20的条件明确滴中观察到的高的衍射质量大单晶格的晶体（ 图5）在最下降后两天到七天。晶体成尼龙圈（ 图5E）衍射数据的安装到1.67的决议后通过锌SAD（ 表1）收集结构测定，具有异常的信号延伸到2。此外，本机衍射数据，以1.4的决议收集用于测定的HIGH-分辨率结构（ 见表1）。在1.4埃分辨率rPPEP-1的晶体结构可以解决（ 图6），并细化到R上的天然结构模型/ R的0.156 / 0.182 自由因子（PDB ID:5A0P），14（进一步细化统计见表1）。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 结构/数据集 </td><td> 高峰期锌SAD </td><td> 本地人 </td></tr><tr><td>数据采集</td><td></td><td></td></tr><tr><td>空间群</td><td> 为 P2 1 2 1 2 1 </td><td> 为 P2 1 2 1 2 1 </td></tr><tr><td> A，B，C（） </td><td> 43.17，71.68，117.70 </td><td> 43.17，71.77，117.80 </td></tr><tr><td> α，β，γ（°） </td><td> 90，90,90 </tD&gt; <td> 90，90,90 </td></tr><tr><td>波长（埃） </td><td> 1.28254 </td><td> 1 </td></tr><tr><td>决议（A） </td><td> 45.5-1.67（1.72-1.67） </td><td> 45.5-1.40（1.49-1.40） </td></tr><tr><td>观测号</td><td> 779717（34025） </td><td> 310074（45851） </td></tr><tr><td>独特的反射号</td><td> 80960（5597） </td><td> 71874（11172） </td></tr><tr><td>多重</td><td> 9.6（6.1） </td><td> 4.3（4.1） </td></tr><tr><td>完备性（％） </td><td> 99.2（93.0） </td><td> 98.2（95.6） </td></tr><tr><td> Rmerge（％） </td><td> 6.8（56.5） </td><td> 5.2（52.6） </td></tr><tr><td> &lt;I /σ（I）&gt; </td><td> 21.86（2.81） </td><td> 15.66（2.48） </td></tr><tr><td> CC（1/2）（％） </td><td> 100（85.1） </td><td> 99.9（85.5） </td></tr><tr><td>细化统计</td><td></td><td></td></tr><tr><td> - [RW¯¯ORK / R 免费 D（％） </td><td></td><td> 15.60 / 18.17 </td></tr><tr><td>非氢蛋白原子的号</td><td></td><td> 3109 </td></tr><tr><td>水分子的第</td><td></td><td> 532 </td></tr><tr><td>离子/重原子的号</td><td></td><td> 2锌</td></tr><tr><td>其它分子的号</td><td></td><td> - </td></tr><tr><td> TLS组号/链</td><td></td><td> 9 </td></tr><tr><td>根均方误差</td><td></td><td></td></tr><tr><td>键长（埃） </td><td></td><td> 0.006 </td></tr><tr><td>键角（°） </td><td></td><td> 1.022 </td></tr><tr><td>数据b系数（2） </td><td></td><td></td></tr><tr><td>所有的蛋白质原子</td><td></td><td> 16.12 </td></tr><tr><td>水</td><td></td><td> 29.74 </td></tr><tr><td>其他原子</td><td></td><td> 10.12 </td></tr><tr><td> Ramachandran图E（％） </td><td></td><td></td></tr><tr><td>最受宠</td><td></td><td> 98.72 </td></tr><tr><td>此外允许</td><td></td><td> 1.28 </td></tr><tr><td>不允许</td><td></td><td> 0 </td></tr><tr><td> PDB进入</td><td></td><td> 5a0p </td></tr></tbody></table> 表1:数据收集和统计细化。

Watch this Scientific Journal Video about Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD at JoVE.com

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Proline-proline endopeptidase-1 (PPEP-1) is a secreted metalloprotease and promising drug-target from the human pathogen Clostridium difficile. Here we describe all methods necessary for the production and structure determination of this protein.

生产，结晶和结构测定<em&gt;℃。难辨</em&gt;通过Microseeding和锌SAD PPEP-1

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a ...

production-crystallization-structure-determination-c-difficile-ppep-1

Nanyang Technological University

Research

JoVE Journal

Biochemistry

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

11.5K 次观看.  University of Cologne. Proline-proline endopeptidase-1 (PPEP-1) is a secreted metalloprotease and promising drug-target from the human pathogen Clostridium difficile. Here we describe all methods necessary for the production and structure determination of this protein. 

视频: Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists1. Here we discuss a fully integrated automation solution for non-contact acoustic preparation of peptide serial dilutions in microtiter plates utilizing the example of screening for peptide agonists at the mouse glucagon-like peptide-1 receptor (GLP-1R). Our methods allow for high-throughput cell-based assays to screen for agonists and are easily scalable to support increased sample throughput, or to allow for increased numbers of assay plate copies (e.g., for a panel of more target cell lines).

Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described.

自动声胶肽受体激动剂的效价测定测定的连续稀释

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

科研

生物化学

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

单分子水平的高精度FRET用于生物分子结构测定

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

A Guide to Production, Crystallization, and Structure Determination of Human IKK1/&#945;

A class of extracellular stimuli requires activation of IKK1/&#945; to induce generation of an NF-&#954;B subunit, p52, through processing of its precursor p100. p52 functions as a homodimer or heterodimer with another NF-&#954;B subunit, RelB. These dimers in turn regulate the expression of hundreds of genes involved in inflammation, cell survival, and cell cycle. IKK1/&#945; primarily remains associated with IKK2/&#946; and NEMO as a ternary complex. However, a small pool of it is also observed as a low molecular weight complex(es). It is unknown if the p100 processing activity is triggered by activation of IKK1/&#945; within the larger or the smaller complex pool. Constitutive activity of IKK1/&#945; has been detected in several cancers and inflammatory diseases. To understand the mechanism of activation of IKK1/&#945;, and enable its use as a drug target, we expressed recombinant IKK1/&#945; in different host systems, such as E. coli, insect, and mammalian cells. We succeeded in expressing soluble IKK1/&#945; in baculovirus infected insect cells, obtaining mg quantities of highly pure protein, crystallizing it in the presence of inhibitors, and determining its X-ray crystal structure. Here, we describe the detailed steps to produce the recombinant protein, its crystallization, and its X-ray crystal structure determination.

I&#954;B Kinase 1/&#945; (IKK1/&#945; CHUK) is a Ser/Thr protein kinase that is involved in a myriad of cellular activities primarily through activation of NF-&#954;B transcription factors. Here, we describe the main steps necessary for the production and crystal structure determination of this protein.

人的 ikkknα的生产、结晶和结构测定指南

A class of extracellular stimuli requires activation of IKK1/&#945; to induce generation of an NF-&#954;B subunit, p52, through processing of its ...

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.

采用自动结晶耦合原位动态光散射的不同尺寸生长蛋白晶体

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

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

富玛莱酸氢酶含含蛋白质的表达、纯化、结晶和酶测定

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO

NEMO is a scaffolding protein which plays an essential role in the NF-&#954;B pathway by assembling the IKK-complex with the kinases IKK&#945; and IKK&#946;. Upon activation, the IKK complex phosphorylates the I&#954;B molecules leading to NF-&#954;B nuclear translocation and activation of target genes. Inhibition of the NEMO/IKK interaction is an attractive therapeutic paradigm for the modulation of NF-&#954;B pathway activity, making NEMO a target for inhibitors design and discovery. To facilitate the process of discovery and optimization of NEMO inhibitors, we engineered an improved construct of the IKK-binding domain of NEMO that would allow for structure determination of the protein in the apo form and while bound to small molecular weight inhibitors. Here, we present the strategy utilized for the design, expression and structural characterization of the IKK-binding domain of NEMO. The protein is expressed in E. coli cells, solubilized under denaturing conditions and purified through three chromatographic steps. We discuss the protocols for obtaining crystals for structure determination and describe data acquisition and analysis strategies. The protocols will find wide applicability to the structure determination of complexes of NEMO and small molecule inhibitors.

We describe protocols for the structure determination of the IKK-binding domain of NEMO by X-ray crystallography. The methods include protein expression, purification and characterization as well as strategies for successful crystal optimization and structure determination of the protein in its unbound form.

NEMO IKK绑定域的生产、结晶和结构确定

NEMO is a scaffolding protein which plays an essential role in the NF-&#954;B pathway by assembling the IKK-complex with the kinases IKK&#945; and ...

Strategic Screening and Characterization of the Visual GPCR-mini-G Protein Signaling Complex for Successful Crystallization

The key to determining crystal structures of membrane protein complexes is the quality of the sample prior to crystallization. In particular, the choice of detergent is critical, because it affects both the stability and monodispersity of the complex. We recently determined the crystal structure of an active state of bovine rhodopsin coupled to an engineered G protein, mini-Go, at 3.1 &#197; resolution. Here, we detail the procedure for optimizing the preparation of the rhodopsin&#8211;mini-Go complex. Dark-state rhodopsin was prepared in classical and neopentyl glycol (NPG) detergents, followed by complex formation with mini-Go under light exposure. The stability of the rhodopsin was assessed by ultraviolet-visible (UV-VIS) spectroscopy, which monitors the reconstitution into rhodopsin of the light-sensitive ligand, 9-cis retinal. Automated size-exclusion chromatography (SEC) was used to characterize the monodispersity of rhodopsin and the rhodopsin&#8211;mini-Go complex. SDS-polyacrylamide electrophoresis (SDS-PAGE) confirmed the formation of the complex by identifying a 1:1 molar ratio between rhodopsin and mini-Go after staining the gel with Coomassie blue. After cross-validating all this analytical data, we eliminated unsuitable detergents and continued with the best candidate detergent for large-scale preparation and crystallization. An additional problem arose from the heterogeneity of N-glycosylation. Heterologously-expressed rhodopsin was observed on SDS-PAGE to have two different N-glycosylated populations, which would probably have hindered crystallogenesis. Therefore, different deglycosylation enzymes were tested, and endoglycosidase F1 (EndoF1) produced rhodopsin with a single species of N-glycosylation. With this strategic pipeline for characterizing protein quality, preparation of the rhodopsin&#8211;mini-Go complex was optimized to deliver the crystal structure. This was only the third crystal structure of a GPCR&#8211;G protein signaling complex. This approach can also be generalized for other membrane proteins and their complexes to facilitate sample preparation and structure determination.

This report describes screening of different detergents for preparing the visual GPCR, rhodopsin, and its complex with mini-Go. Biochemical methods characterizing the quality of the complex at different stages during purification are demonstrated. This protocol can be generalized to other membrane protein complexes for their future structural studies.

视觉GPCR-迷你G蛋白信号复合物的战略筛选与表征，成功结晶

The key to determining crystal structures of membrane protein complexes is the quality of the sample prior to crystallization. In particular, the choice ...

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.

酶的结晶和结构确定:多功能微流体芯片中串行晶体学的基板复合物

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

Single Particle Cryo-Electron Microscopy: From Sample to Structure

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The overall process involves i) vitrifying the specimen in a thin film supported on a cryoEM grid; ii) screening the specimen to assess particle distribution and ice quality; iii) if the grid is suitable, collecting a single particle dataset for analysis; and iv) image processing to yield an EM density map. In this protocol, an overview for each of these steps is provided, with a focus on the variables which a user can modify during the workflow and the troubleshooting of common issues. With remote microscope operation becoming standard in many facilities, variations on imaging protocols to assist users in efficient operation and imaging when physical access to the microscope is limited will be described.

Structure determination of macromolecular complexes using cryoEM has become routine for certain classes of proteins and complexes. Here, this pipeline is summarized (sample preparation, screening, data acquisition and processing) and readers are directed towards further detailed resources and variables that may be altered in the case of more challenging specimens.

单颗粒冷冻电子显微镜:从样品到结构

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The ...

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.

室温下的片上结晶和大规模连续衍射

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