Name: neutron crystallography data collection and processing for modelling hydrogen atoms in protein structures
Uploaded: 2020-12-01T15:00:00.000+00:00
Duration: 10 min 10 s
Description: Watch this Scientific Journal Video about Neutron Crystallography Data Collection and Processing for Modelling Hydrogen Atoms in Protein Structures at JoVE.com

蛋白质表达，纯化和结晶实验在结构分子生物学中心（CSMB）进行，该中心是美国能源部橡树岭国家实验室生物与环境研究用户设施。中子衍射数据是在BL-11B MaNDi上收集的，位于ORNL的散裂中子源（SNS），由美国能源部基础能源科学办公室科学用户设施司赞助。作者感谢Brendan Sullivan在数据减少方面的帮助。X射线衍射数据是在北卡罗来纳州立大学的分子教育，技术和研究创新中心（METRIC）设施中收集的，该中心由北卡罗来纳州提供支持。GCS感谢南非国家研究基金会（NRF）和ORNL的毕业生机会（GO！）计划的部分支持。FM感谢美国农业部NIFA Hatch 211001的支持。

作者没有什么可透露的。

中子蛋白晶体学是一种高度灵敏的技术，用于探测蛋白质中的质子化状态和水分子取向。这些信息揭示了蛋白质催化机制，因为质子化和氢键相互作用的变化通常是酶化学的核心10。中子蛋白晶体学虽然是一种信息丰富的技术，但在计划进行中子衍射实验之前，应考虑许多因素，即:
<ol><li>需要大蛋白质晶体进行数据收集。</li>
	<li>氢和其他元素的散射性质，如金属离子。</li>
	<li>处理氘代样品时，结构细化和模型构建软件的局限性。</li>
</ol>中子蛋白晶体学是一种通量有限的技术。与X射线衍射数据集相比，由于技术固有的局限性（通量有限，准劳厄，更长波长），中子数据集预计会有更高的R因子和更低的完整性，冗余和信噪比。单个帧的数据收集通常为 12 – 18 小时。实验的成功很大程度上取决于样品大小和质量，0.1 mm3的晶体通常是最低要求3。中子衍射需要产生大量的蛋白质来建立10至800μL的结晶滴。生长足够大晶体的最小体积可以使用体积计算器估计，给定晶体和样品参数（https://neutrons.ornl.gov/imagine）。大晶体的生长最普遍地通过蒸汽扩散3完成。悬挂式滴结晶允许晶体在10-25 μL范围内的大液滴中生长，而使用市售的坐滴设备可以设置高达~50 μL的较大液滴14，54。硅化九孔玻璃板可用于设置非常大的液滴，体积高达800μL。这些玻璃板被放置在汉普顿研究公司市售的"三明治盒"中。进一步的结晶技术包括间歇结晶，其中液滴大小的限制由容器决定。批量结晶实验设置的范围可以从微升到毫升55。也可以使用透析技术进行结晶，其中蛋白质通过透析膜或沿沉淀剂浓度梯度或通过多孔塞（如琼脂糖）的反扩散与沉淀剂平衡56，57。播种提供了另一种获得所需体积的晶体的替代方法。微晶和晶状体已成功用于大晶体生长，包括NcLPMO9D45的大晶体。蛋白质相图的一些知识，包括温度对溶解度的影响，有助于大晶体生长。
在规划中子衍射实验时，必须优化蛋白质制备，以在衍射数据收集过程中最大限度地提高信噪比7。为了规避H原子引起的密度抵消和高不相干散射，可以通过用H原子交换其同位素D来改善中子SLD图谱，D具有正相干散射长度和低不相干散射长度。为此，对氢化蛋白质晶体进行蒸气交换以对抗氘化结晶缓冲液。这确保了溶剂分子和不稳定的可滴定蛋白H原子的H / D交换23。蒸汽交换是通过将氢化晶体安装到具有D2O基氘化结晶缓冲液"塞子"的石英毛细管中来进行的，它代表了一种有效，温和的技术，最常用于14，23，35。更换可能需要数周时间，最好需要频繁更换氘代缓冲液，以确保最大的H / D更换。H/ D交换也可以通过将晶体直接浸泡在氘代缓冲液中来进行。为避免将晶体置于由于D2O暴露而产生的应力下，应通过逐渐增加D2O:H2O比来逐渐进行浸泡过程58。除此之外，氢化蛋白的结晶也可以在氘代缓冲液中进行，用于在不稳定的H位点进行H / D交换22，59。然而，应该注意的是，基于D2O的缓冲液对蛋白质溶解度有影响，需要进一步调整已知的基于H2O的条件3，59。在某些情况下，基于D2O的缓冲液也会导致更小的晶体59。通过在氘化培养基中表达蛋白质以生成氘化样品，可以实现可滴定和碳结合的H原子与D的完全交换20。脱氘样品的中子SLD图谱将得到显着改善，不再显示氢化样品对应物的密度抵消。当表征在蛋白质或辅因子中不可交换位点结合的H / D时，这是有益的。然而，氘代蛋白的表达既高成本又低产量60。橡树岭国家实验室（ORNL）结构分子生物学中心（CSMB）为寻求生成氘化样本（https://www.ornl.gov/facility/csmb）的用户提供了氘传设施。纯化表达通常在1 L尺度上的生物反应器中进行，产生约50mg纯化蛋白61。
在收集中子衍射数据后，进行细化和交互式模型构建。可以使用多个软件套件运行细化，包括phenix.refine，nCNS或SHELXL28，31，32，33。Phenix套件是最常用的软件，用于与Coot一起优化中子衍射数据，Coot用于从中子SLD图手动构建模型34。虽然Phenix和Coot都允许处理中子衍射数据，但它们可能缺乏处理与中子数据和氘化样品相关的特性所必需的某些特征。例如，Coot 不包含氘代残留物的几何优化，这可能导致模型构建过程中的复杂情况，因为"真实空间细化"特征会导致"爆炸"残留物（补充图 26）62。这可以通过为所有氘代残留物生成约束文件来解决。但是，这是一个密集的过程，此类库目前尚未公开。在Phenix中执行优化时，H和D的可交换H / D站点最初将设置为0.50占用。随着细化，H和D的占用将根据中子SLD图进行细化。在交互式模型构建过程中，差密度 Fo-Fc 图在评估 H/D 占用率方面非常有用。地图可用于确定哪些位点具有高D占用率，这在质子化状态具有催化相关性的活动位点尤其有用63。然而，当H:D占用率接近0.70:0.30，这导致中子SLD图中的信号完全消除64。还应该考虑到，准劳埃中子数据集的完整性通常在80%左右，低于X射线衍射数据的常规观测≥98%。因此，在Phenix中细化中子衍射数据时，从模型中计算出缺失的观测振幅（Fo）以完成反射列表，从而引入模型偏差。为了解释这种潜在的偏差，应在交互式模型构建期间检查"no_fill"地图，而不是"填充"地图。
用户可以选择对其结构进行联合X射线/中子数据细化，或仅对中子数据进行细化。可视化中子SLD图谱，特别是在较低分辨率下，最初可能令人不安，特别是对于氢化蛋白质，尽管H / D蒸汽交换，H仍存在于不可交换的位点。这导致中子密度图取消，给人的印象是不连续的图65，66。收集相应的X射线数据集有利地补充了联合细化中的这些取消（图13A和图13B）。联合细化策略通常涉及根据X射线数据提炼蛋白质骨架坐标，而中子衍射数据用于细化H / D原子在可交换位点的位置和占用28。由于在可交换地点引入联合H/D占用增加了正在改进的参数数量，因此使用X射线数据的联合细化也增加了数据与参数的比率。联合细化需要在相同温度下在同一晶体或相同条件下生长的晶体上收集相应的X射线数据集。对于在室温（300K）下收集的中子衍射数据，应使用低剂量数据收集策略在室温下收集相应的X射线数据集，以限制辐射损伤。相比之下，经过氚化的样品提供改进的连续中子SLD图谱，因为它们不具有相同幅度的H / D信号消除。然而，包括金属和硫在内的某些元素的中子散射长度使它们在中子SLD图中不可见，即使蛋白质已经氘化（图13C-F）18。如果需要表征金属，最好在联合细化中利用X射线衍射或应用光谱技术来补充衍射实验。仅中子数据优化通常在中子数据集具有高分辨率或使用过氘蛋白质时执行。此外，如果正在研究对辐射损伤高度敏感的蛋白质，仅中子数据改进特别有用，因为X射线衍生的结构可能具有辐射诱导的伪影。如果要进行仅中子数据的细化，则必须确定相应的中子数据集是否具有足够的完整性和分辨率。
ORNL提供了两个用于收集中子衍射数据的设施:HFIR的IMAGINE光束线以及SNS36，67的MaNDi光束线。虽然这两种仪器都为使用类似原理收集中子衍射数据集提供了有效的手段，但每种仪器都有独特的规格，在应用光束时间时应予以考虑。IMAGINE 收集准劳厄数据，并针对晶胞高达 ~100 Å 的晶体的室温数据收集进行了优化。MaNDi可用于收集室温和低温数据，采用TOF-Laue收集方法收集晶胞高达约300 Å的晶体。在收集完整的数据集之前，对晶体进行测试以评估获得的衍射图的质量，其中晶体暴露于中子束中单帧。如果晶体质量足够高，将收集完整的中子衍射数据集，将其索引，集成，缩放和合并，该过程类似于X射线数据处理。IMAGINE利用Lauegen和Lscale，MaNDi利用Mantid封装并采用三维轮廓拟合48，50，51，68，69，70。成为这些设施用户的科学家将获得MTZ或HDL格式的数据集，以进行进一步分析。
中子衍射是一种非破坏性、高度灵敏的技术，用于探测生物大分子的质子化状态和氢键相互作用。它对光敏蛋白和金属蛋白特别有用。在进行实验之前，必须考虑有关该技术以及数据处理的几个考虑因素，但是结果产生的结果可能会对感兴趣蛋白质的催化机制提供有价值的见解。中子蛋白晶体学是对计算、结构、生化和光谱研究的补充，使其成为生物学家用于表征生物大分子的技术工具箱中的宝贵工具。

中子大分子晶体学是一种技术，为蛋白质的结构和潜在化学提供了一个独特的窗口。在概念上与X射线衍射相似，中子衍射提供了大分子结构的原子细节，然而，中子与原子核的相互作用使轻原子的定位成为可能，通常很难用X射线衍射检测到1。在 X 射线衍射过程中，X 射线从电子云中散射，使得氢 （H） 等轻原子在没有接近亚 Ångström 分辨率的电子密度图中不可见2。相反，中子的散射强度取决于与原子核的复杂相互作用，同一元素的同位素显示出不同的散射长度。因此，轻原子及其同位素，如氢（1H）和氘（2H或D），在中子散射长度密度（SLD）图中具有与骨架碳，氮和氧原子相当的可见性。此外，由于中子散射的大小与电子的数量无关，因此当轻元素彼此靠近时，轻元素的散射不会被重元素遮挡，正如在X射线散射中观察到的那样。H及其同位素D在采用中子衍射时的能见度增强，提供了有关催化重要残基、辅因子和配体质子化状态的宝贵信息，并有助于水分子的取向，揭示了有关催化机理和蛋白质化学的重要信息3。中子衍射还具有非破坏性技术的优点，特别适用于对电离敏感的生物样品，例如具有金属中心的蛋白质或光敏氧化还原辅因子2。本文的主要重点是概述获得高质量中子蛋白晶体结构的工作流程。我们将感兴趣的读者推荐给Podjarny et al.4，Blakeley5，Blakeley et al.6和O'Dell et al.3，以很好地概述中子蛋白衍射和Ashkar等人7对中子散射的进一步应用。
中子主要在核反应过程中产生，采用两个过程之一:反应堆源的核裂变或加速器源的散裂8。反应堆源通过利用 235U同位素的核裂变来提供连续的中子束，而散裂中子源通过用质子轰击目标（例如液态金属，如汞）来产生脉冲中子束9。位于田纳西州橡树岭的橡树岭国家实验室（ORNL）在高通量同位素反应堆（HFIR）拥有稳态中子源，在散裂中子源（SNS）拥有60 Hz脉冲源。位于HFIR的IMAGINE光束线是针对生物大分子优化的中子衍射仪（补充图1）10。IMAGINE采用中子成像板探测器，使用2.8 – 4.5 Å范围内的窄带通测量准Laue数据，该带通来自具有单元单元边缘<150 Å的单晶。位于SNS的高分子中子衍射仪（MaNDi）是一种飞行时间（TOF）劳埃中子衍射仪，配有球形探测器阵列框架（DAF）（补充图2）11。MaNDi 采用 2.0 – 6.0 Å12 之间的可调 2 Å 波长带宽，测量来自具有 10 – 300 Å 范围内单元边缘的单晶的数据。
产生中子的过程是高度能量密集型的，与同步子源处的X射线束通量相比，导致中子束通量相对较弱13。为了确保在数据收集过程中具有足够的信噪比，有必要生长合适尺寸和质量的晶体14。通常，需要体积>0.1 mm3的晶体来收集具有足够统计数据的数据15。除了较低的通量外，还必须考虑中子与样品核之间相互作用的固有特性16。对于同一元素的同位素，中子的散射长度不同，这一特性可以在小角度中子散射（SANS）中得到有利利用，以掩盖或突出显示样品的区域 - 这一过程称为对比度匹配17。在衍射实验中，H的负相干中子散射长度（-3.741 fm表示1H）可导致中子散射密度图特征的抵消，因为其他生物相关原子的相干中子散射长度包括碳（12C为6.6511 fm），氮（14N为9.37 fm），氧（16O为5.803 fm）， 磷（31P为5.13 fm）和硫（32S为2.804 fm）为正（表1）12，14。此外，H （25.274 fm） 的大不相干散射长度增加了数据收集过程中的背景，影响了数据集的质量并影响了数据分辨率7。为了规避H引入的这些限制，对于中子衍射，有必要将H换成其同位素氘2H（D），其具有正相干中子散射长度（6.671 fm）和显着降低的非相干散射长度（4.04 fm）19。这可以通过氘散来实现，在氘透过程中，蛋白质由在完全氘代培养基中生长的生物体表达，确保在H位点20完全掺入D。也可以通过仅在可交换位点（可滴定基团）处用D代替H来部分氘化蛋白质，而不可交换的碳结合位点保持氢化21。这可以通过在氘代母液中生长氢化蛋白质晶体来实现22。然而，最常见的是，氢化蛋白的H / D交换是在基于H2O的缓冲液中生长适当大的晶体23之后通过蒸汽交换进行的。在这种情况下，晶体安装在石英毛细管中，并与基于D20的母液进行蒸汽平衡。
中子源的中子通量有限，导致数据收集时间更长，从几天到几周不等24。在ORNL，IMAGINE和MaNDi都采用2-6 Å范围内的窄波长带通来优化数据收集25。数据可以在室温或低温下收集。冷冻数据收集可以潜在地提高数据质量，并为冷冻捕获催化中间体开辟可能性。在收集中子衍射数据之后，通常在相同温度下在同一晶体上或在相同条件下生长的晶体上收集X射线数据集26。在相同温度下收集数据允许针对X射线和中子数据进行结构细化，防止任何潜在的温度引起的伪影，例如水的能见度和位置的变化或具有交替构象的残留物的占用27。联合X射线中子数据细化增加了数据与参数的比率，并提供了允许根据X射线数据细化蛋白质骨架坐标的优势，而中子衍射数据用于细化H / D原子的位置28。当使用部分氘代样品时，这特别有用，其中由于蛋白质上不可交换位点处的H原子存在密度抵消。虽然X射线结构的数量远远超过蛋白质数据库（PDB）中沉积的中子结构的数量，但最初设计用于改进X射线数据的软件包已经扩展到包括中子数据3，29，30。在数据收集之后，可以使用phenix.refine，CNSsolve（nCNS）或SHELXL28，31，32，33等细化包来优化模型。在细化过程中，可以使用COOT34可视化中子散射密度图，以便手动拟合。按照结构解决方案，坐标以及中子和/或X射线衍射数据文件可以提交给PDB，PDB将验证并存放模型，使其可供公众访问18，29，30。
蛋白质的结构分析是一种多方面的方法，其中许多技术用于探测其功能和机制35。中子蛋白晶体学提供了有价值的化学见解，以扩展和补充其他研究的发现，如X射线衍射，光谱学，核磁共振（NMR）或微晶电子衍射（microED）36。中子蛋白衍射具有独特的优势，可以提供对酶学机制的见解，因为H原子是其化学的核心。没有中子诱导的辐射损伤使它们成为特别适合研究金属蛋白的探针37。我们在这里展示了从样品制备到数据收集，细化和分析的中子蛋白衍射过程的代表性示例（图1）。已经生长了足够大小的晶体，用于中子衍射实验的金属蛋白神经孢子菌LPMO9D（NcLPMO9D）。数控LPMO9D是一种含铜的金属蛋白，通过在糖苷键处插入氧原子来参与顽固纤维素的降解38，39。NcLPMO9D活性位点在由N端组氨酸和第二保守组氨酸组成的特征性"组氨酸支撑"内包含一个单核铜中心（补充图3）40。真菌LPMO的N端被甲基化，但在酵母的重组表达过程中不会发生过渡后修饰。在NcLPMO9D静息状态下，铜中心以Cu2+氧化态存在，并通过单电子还原为Cu1 +被激活，允许分子氧结合并通过迅速还原为超氧化物物种而被激活41，42。整个NcLPMO9D反应需要进一步添加一个电子和两个质子以形成羟基化多糖产物43。负责从多糖底物中提取氢原子（HAA）的活性氧物种的身份尚未确定，目前正在进行密集的结构和计算研究44，45。鉴于NcLPMO9D活性位点的氧化还原化学性质，减轻辐射损伤尤其重要。我们在这里说明在NcLPMO9D晶体上的室温和低温数据收集，以确定NcLPMO9D结构在静止状态和活化还原形式中分别46。重点将放在蛋白质晶体安装，用于数据收集的光束线仪器设置，数据和坐标文件的准备以及建模全原子中子结构所需的改进步骤。

<table><tbody><tr><td>Absorbent Paper Points Size #30-#40, 60 mm length</td><td>DiaDent/DiaVet</td><td>218-292</td><td></td></tr><tr><td>Capillary wax</td><td>Hampton</td><td>HR4-328</td><td></td></tr><tr><td>CCP4</td><td></td><td>Version 7.0.077</td><td></td></tr><tr><td>Conical Centrifuge Tubes (15 mL)</td><td>Corning</td><td>CLS430790</td><td></td></tr><tr><td>Conical Centrifuge Tubes (50mL)</td><td>Corning</td><td>CLS430828</td><td></td></tr><tr><td>Coot</td><td></td><td>Version 0.8.9.2</td><td></td></tr><tr><td>CrystalCap ALS</td><td>Hampton</td><td>HR4-779</td><td></td></tr><tr><td>Curved-Tip Forceps</td><td>Mitegen</td><td>TW-CTF-1</td><td></td></tr><tr><td>Deuterium chloride solution, 35 wt. % in D2O, &amp;ge;99 atom % D</td><td>Sigma-Aldrich</td><td>543047</td><td></td></tr><tr><td>Deuterium oxide 99.9 atom % D</td><td>Sigma-Aldrich</td><td>151882</td><td></td></tr><tr><td>Dual Thickness MicroLoops 1000 &amp;micro;m</td><td>Mitegen</td><td>M5-L18SP-1000</td><td></td></tr><tr><td>FiveEasy pH meter F20-Std-Kit</td><td>Mettler Toledo</td><td>30266626</td><td></td></tr><tr><td>Foam Dewars Standard Vessel 800 ml</td><td>Spearlab</td><td>M-FD-800</td><td></td></tr><tr><td>Four Color Mounting Clay</td><td>Hampton</td><td>HR4-326</td><td></td></tr><tr><td>HEPES, BioUltra, for molecular biology, &amp;ge;99.5% (T),</td><td>Sigma-Aldrich</td><td>54457</td><td></td></tr><tr><td>High flux rotating anode X-ray diffractomemeter with EIGER 4M detector</td><td>Rigaku, Oxford Cryostream and Dectris</td><td>XtaLAB Synergy-R</td><td>Home source X-ray diffractometer</td></tr><tr><td>Magnetic Wand Straight</td><td>Mitegen</td><td>M-R-1013198</td><td></td></tr><tr><td>Microloader, tip for filling Femtotips and other glass microcapillaries (for research use only), 0.5 &amp;ndash; 20 &amp;micro;L, 100 mm, light gray, 192 pcs. (2 racks &amp;times; 96 pcs.)</td><td>Eppendorf</td><td>930001007</td><td></td></tr><tr><td>Microtubes volume 1.5 mL</td><td>Eppendorf</td><td>Z606340</td><td></td></tr><tr><td>Petri Dishes with Clear Lid 100 mm diameter</td><td>Fischerbrand</td><td>FB0875713</td><td></td></tr><tr><td>Phenix</td><td></td><td>Version 1.14-3260</td><td></td></tr><tr><td>Pin Tong 18 mm</td><td>Mitegen</td><td>M-R-1013196</td><td></td></tr><tr><td>Pipette Volume 0.1-2.5 &amp;mu;L</td><td>Eppendorf Research</td><td>Z683779</td><td></td></tr><tr><td>Pipette Volume 100-1000 &amp;mu;L</td><td>Eppendorf Research</td><td>Z683825</td><td></td></tr><tr><td>Pipette Volume 10-100 &amp;mu;L</td><td>Eppendorf Research</td><td>Z683809</td><td></td></tr><tr><td>Pipette Volume 20-200 &amp;mu;L</td><td>Eppendorf Research</td><td>Z683817</td><td></td></tr><tr><td>Poly(ethylene glycol) BioXtra, average mol wt 3,350, powder</td><td>Sigma-Aldrich</td><td>P4338</td><td></td></tr><tr><td>Quartz Capillary , 1.00 mm inner diameter, 80 mm length</td><td>Hampton</td><td>HR6-146</td><td>Thin-walled capillary</td></tr><tr><td>Research Stereomicroscope System</td><td>Olympus</td><td>SZX16</td><td></td></tr><tr><td>Reusable B3 (SSRL/SAM Style) Goniometer Bases</td><td>Mitegen</td><td>GB-B3-R</td><td></td></tr><tr><td>Round - Miniature Hollow Glass Tubing (VitroTubes) Clear Fused Quartz / 1.00 mm inner diameter, 100 mm length</td><td>VitroCom</td><td>CV1012</td><td>Thick-walled capillary</td></tr><tr><td>Sandwich Box with cover</td><td>Hampton</td><td>HR3-132</td><td></td></tr><tr><td>Siliconized 9 Well Glass Plate</td><td>Hampton</td><td>HR3-134</td><td></td></tr><tr><td>Sitting Drop Crystallization Plate (24 Big Well)</td><td>Mitegen</td><td>XQ-P-24S-A</td><td></td></tr><tr><td>Sodium deuteroxide solution, 40 wt. % in D2O, 99 atom % D</td><td>Sigma-Aldrich</td><td>176788</td><td></td></tr><tr><td>Thick Siliconized circle cover slides (22 mm x 0.96 mm)</td><td>Hampton</td><td>HR3-247</td><td></td></tr><tr><td>Universal Pipet Tips, 0.1 - 10 &amp;micro;L</td><td>VWR</td><td>76322-528</td><td></td></tr><tr><td>Universal Pipet Tips, 1 - 100 &amp;micro;L</td><td>VWR</td><td>76322-136</td><td></td></tr><tr><td>Universal Pipet Tips, 100 - 1000 &amp;micro;L</td><td>VWR</td><td>76322-154</td><td></td></tr><tr><td>Universal Pipet Tips, 20 - 200 &amp;micro;L</td><td>VWR</td><td>76322-150</td><td></td></tr><tr><td>Universal Pipet Tips, 1 - 20 &amp;micro;L</td><td>VWR</td><td>76322-134</td><td></td></tr><tr><td>Wax pen</td><td>Hampton</td><td>HR4-342</td><td></td></tr></tbody></table>

neutron crystallography data collection and processing for modelling hydrogen atoms in protein structures

中子晶体学是一种结构技术，可以确定生物大分子内的氢原子位置，产生有关质子化和水合状态的机械重要信息，同时不会引起辐射损伤。相比之下，X射线衍射仅提供有关光原子位置的有限信息，并且X射线束会迅速引起光敏辅助因子和金属中心的辐射损伤。这里介绍的是橡树岭国家实验室（ORNL）的IMAGINE和MaNDi光束线所采用的工作流程，一旦生长出合适尺寸（>0.1 mm3）的蛋白质晶体，就可以获得中子衍射结构。我们展示了在石英毛细管中安装氢化蛋白质晶体，用于中子衍射数据收集。还介绍了用含D2O的缓冲液安装的晶体的蒸汽交换过程，以确保用氘替换可交换位点的氢原子。氘的掺入减少了氢原子不相干散射产生的背景，并防止了由其负相干散射长度引起的密度抵消。在高通量同位素反应器（HFIR）的IMAGINE上使用准Laue数据收集来说明样品对准和室温数据收集策略。此外，在散裂中子源（SNS）的MaNDi飞行时间仪器上演示了晶体镶样和液氮中的快速冷冻，用于冷冻数据收集以捕获不稳定的反应中间体。还将处理模型坐标和衍射数据文件的准备以及中子散射长度密度（SLD）图的可视化。最后将讨论针对中子数据或针对联合X射线/中子数据的结构细化，以获得目标蛋白质的全原子结构。确定中子结构的过程将使用裂解多糖单加氧酶 Neurospora crassa LPMO9D的晶体进行演示，LPMO9D是一种含铜的金属蛋白， 通过 糖苷键的氧化切割参与顽固性多糖的降解。

Watch this Scientific Journal Video about Neutron Crystallography Data Collection and Processing for Modelling Hydrogen Atoms in Protein Structures at JoVE.com

Neutron Crystallography Data Collection and Processing for Modelling Hydrogen Atoms in Protein Structures

中子蛋白晶体学是一种结构技术，允许氢原子的定位，从而提供蛋白质功能的重要机制细节。我们在这里介绍了用于安装蛋白质晶体，中子衍射数据收集，结构细化以及中子散射长度密度图分析的工作流程。

中子晶体学数据收集和处理，用于蛋白质结构中氢原子的建模

Neutron crystallography is a structural technique that allows determination of hydrogen atom positions within biological macromolecules, yielding ...

neutron-crystallography-data-collection-processing-for-modelling

Nanyang Technological University

Research

JoVE Journal

Chemistry

4.9K 次观看.  North Carolina State University. 中子蛋白晶体学是一种结构技术，允许氢原子的定位，从而提供蛋白质功能的重要机制细节。我们在这里介绍了用于安装蛋白质晶体，中子衍射数据收集，结构细化以及中子散射长度密度图分析的工作流程。

视频: Neutron Crystallography Data Collection and Processing for Modelling Hydrogen Atoms in Protein Structures

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the propagation of neurodegenerative disorders. Such assemblies consist in multiple protein subunits organized in a non-covalent way to form large macromolecular objects that can execute a variety of cellular functions or cause detrimental consequences. Atomic insights into the assembly mechanisms and the functioning of those macromolecular assemblies remain often scarce since their inherent insolubility and non-crystallinity often drastically reduces the quality of the data obtained from most techniques used in structural biology, such as X-ray crystallography and solution Nuclear Magnetic Resonance (NMR). We here present magic-angle spinning solid-state NMR spectroscopy (SSNMR) as a powerful method to investigate structures of macromolecular assemblies at atomic resolution. SSNMR can reveal atomic details on the assembled complex without size and solubility limitations. The protocol presented here describes the essential steps from the production of 13C/15N isotope-labeled macromolecular protein assemblies to the acquisition of standard SSNMR spectra and their analysis and interpretation. As an example, we show the pipeline of a SSNMR structural analysis of a filamentous protein assembly.

Structures of supramolecular protein assemblies at atomic resolution are of high relevance because of their crucial roles in a variety of biological phenomena. Herein, we present a protocol to perform high-resolution structural studies on insoluble and non-crystalline macromolecular protein assemblies by magic-angle spinning solid-state nuclear magnetic resonance spectroscopy (MAS SSNMR).

固体核磁共振波谱法研究高分子组件的原子尺度结构

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the ...

科研

生物化学

Microcrystallography of Protein Crystals and In Cellulo Diffraction

The advent of high-quality microfocus beamlines at many synchrotron facilities has permitted the routine analysis of crystals smaller than 10 &#181;m in their largest dimension, which used to represent a challenge. We present two alternative workflows for the structure determination of protein microcrystals by X-ray crystallography with a particular focus on crystals grown in vivo. The microcrystals are either extracted from cells by sonication and purified by differential centrifugation, or analyzed in cellulo after cell sorting by flow cytometry of crystal-containing cells. Optionally, purified crystals or crystal-containing cells are soaked in heavy atom solutions for experimental phasing. These samples are then prepared for diffraction experiments in a similar way by application onto a micromesh support and flash cooling in liquid nitrogen. We briefly describe and compare serial diffraction experiments of isolated microcrystals and crystal-containing cells using a microfocus synchrotron beamline to produce datasets suitable for phasing, model building and refinement.
These workflows are exemplified with crystals of the Bombyx mori cypovirus 1 (BmCPV1) polyhedrin produced by infection of insect cells with a recombinant baculovirus. In this case study, in cellulo analysis is more efficient than analysis of purified crystals and yields a structure in ~8 days from expression to refinement.

Microcrystallography of Protein Crystals and In Cellulo Diffraction

A protocol is presented for X-ray crystallography using protein microcrystals. Two examples analyzing in vivo-grown microcrystals after purification or in cellulo are compared.

蛋白晶体的微结晶和<em&gt;在Cellulo</em&gt;衍射

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

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials because of their extracellular localization and the centrality of carbohydrate import for pneumococcal metabolism, respectively. Described here is a rationalized integrated protocol to carry out a comprehensive characterization of SP0092, which can be extended to other carbohydrate SBPs from the pneumococcus and other bacteria. This procedure can aid the structure-based design of inhibitors for this class of proteins. Presented in the first part of this manuscript are protocols for biochemical analysis by thermal shift assay, multi angle light scattering (MALS), and size exclusion chromatography (SEC), which optimize the stability and homogeneity of the sample directed to crystallization trials and so enhance the probability of success. The second part of this procedure describes the characterization of the SBP crystals using a tunable wavelength anomalous diffraction synchrotron beamline, and data collection protocols for measuring data that can be used to resolve the crystallized protein structure.

A streamlined protocol for performing an extensive biochemical and structural characterization of a carbohydrate substrate binding protein from Streptococcus pneumoniae is presented.

碳水化合物转运基质结合蛋白 SP0092 的生化和结构表征

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the disordered and protease labile C-terminus of the protein. This manuscript describes the methods to purify recombinant Nsa1 from S. cerevisiae for structural analysis by both X-ray crystallography and SAXS. X-ray crystallography was utilized to solve the structure of the well-ordered N-terminal WD40 domain of Nsa1, and then SAXS was used to resolve the structure of the C-terminus of Nsa1 in solution. Solution scattering data was collected from full-length Nsa1 in solution. The theoretical scattering amplitudes were calculated from the high-resolution crystal structure of the WD40 domain, and then a combination of rigid body and ab initio modeling revealed the C-terminus of Nsa1. Through this hybrid approach the quaternary structure of the entire protein was reconstructed. The methods presented here should be generally applicable for the hybrid structural determination of other proteins composed of a mix of structured and unstructured domains.

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

This method describes the cloning, expression, and purification of recombinant Nsa1 for structural determination by X-ray crystallography and small-angle X-ray scattering (SAXS), and is applicable for the hybrid structural analysis of other proteins containing both ordered and disordered domains.

结合 x 射线晶体学与小角度 x 射线散射到模型的非结构化区域的 Nsa1 从s 酿酒酵母

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the ...

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the investigation of biological materials, biofuel processing, and bio-inspired materials covering nanometer to micrometer length scales. The methods presented here for investigating physical properties (i.e., size and shape) of membrane proteins (here, MmIAP, an intramembrane aspartyl protease from Methanoculleus marisnigri) in solutions of micelle-forming detergents are well-suited for this small-angle neutron scattering instrument, among others. Other biophysical characterization techniques are hindered by their inability to address the detergent contributions in a protein-detergent complex structure. Additionally, access to the Bio-Deuteration Lab provides unique capabilities for preparing large-scale cultivations and expressing deuterium-labeled proteins for enhanced scattering signal from the protein. While this technique does not provide structural details at high-resolution, the structural knowledge gap for membrane proteins contains many addressable areas of research without requiring near-atomic resolution. For example, these areas include determination of oligomeric states, complex formation, conformational changes during perturbation, and folding/unfolding events. These investigations can be readily accomplished through applications of this method.

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

This protocol demonstrates how to obtain a low-resolution ab initio model and structural details of a detergent-solubilized membrane protein in solution using small-angle neutron scattering with contrast-matching of the detergent.

用于膜蛋白结构分析和从头建模的小角中子散射实验中的对比匹配洗涤剂

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the ...

Fully Autonomous Characterization and Data Collection from Crystals of Biological Macromolecules

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for even the most challenging projects in structural biology. However, most facilities still require the presence of a scientist on site to perform the experiments. A new generation of automated beamlines dedicated to the fully automatic characterization of, and data collection from, crystals of biological macromolecules has recently been developed. These beamlines represent a new tool for structural biologists to screen the results of initial crystallization trials and/or the collection of large numbers of diffraction data sets, without users having to control the beamline themselves. Here we show how to set up an experiment for automatic screening and data collection, how an experiment is performed at the beamline, how the resulting data sets are processed, and how, when possible, the crystal structure of the biological macromolecule is solved.

Here, we describe how to use the automated screening and data collection options available at some synchrotron beamlines. Scientists send cryocooled samples to the synchrotron, and the diffraction properties are screened, the data sets are collected and processed and, where possible, a structure solution is carried out&#8212;all without human intervention.

生物大分子晶体的完全自主表征及数据采集

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for ...

Workflow and Tools for Crystallographic Fragment Screening at the Helmholtz-Zentrum Berlin

Fragment screening is a technique that helps to identify promising starting points for ligand design. Given that crystals of the target protein are available and display reproducibly high-resolution X-ray diffraction properties, crystallography is among the most preferred methods for fragment screening because of its sensitivity. Additionally, it is the only method providing detailed 3D information of the binding mode of the fragment, which is vital for subsequent rational compound evolution. The routine use of the method depends on the availability of suitable fragment libraries, dedicated means to handle large numbers of samples, state-of-the-art synchrotron beamlines for fast diffraction measurements and largely automated solutions for the analysis of the results.
Here, the complete practical workflow and the included tools on how to conduct crystallographic fragment screening (CFS) at the Helmholtz-Zentrum Berlin (HZB) are presented. Preceding this workflow, crystal soaking conditions as well as data collection strategies are optimized for reproducible crystallographic experiments. Then, typically in a one to two-day procedure, a 96-membered CFS-focused library provided as dried ready-to-use plates is employed to soak 192 crystals, which are then flash-cooled individually. The final diffraction experiments can be performed within one day at the robot-mounting supported beamlines BL14.1 and BL14.2 at the BESSY&#160; II electron storage ring operated by the HZB in Berlin-Adlershof (Germany). Processing of the crystallographic data, refinement of the protein structures, and hit identification is fast and largely automated using specialized software pipelines on dedicated servers, requiring little user input.
Using the CFS workflow at the HZB enables routine screening experiments. It increases the chances for successful identification of fragment hits as starting points to develop more potent binders, useful for pharmacological or biochemical applications.

Crystallographic fragment screening at the Helmholtz-Zentrum Berlin is performed using a workflow with dedicated compound libraries, crystal handling tools, fast data collection facilities&#160;and largely automated data analysis. The presented protocol intends to maximize the output of such experiments to provide promising starting points for downstream structure-based ligand design.

赫尔姆霍尔茨-曾特鲁姆柏林水晶碎片筛选工作流程和工具

Fragment screening is a technique that helps to identify promising starting points for ligand design. Given that crystals of the target protein are ...

Sample Preparation and Transfer Protocol for In-Vacuum Long-Wavelength Crystallography on Beamline I23 at Diamond Light Source

Long-wavelength macromolecular crystallography (MX) exploits the anomalous scattering properties of elements, such as sulfur, phosphorus, potassium, chlorine, or calcium, that are often natively present in macromolecules. This enables the direct structure solution of proteins and nucleic acids via experimental phasing without the need of additional labelling. To eliminate the significant air absorption of X-rays in this wavelength regime, these experiments are performed in a vacuum environment. Beamline I23 at Diamond Light Source, UK, is the first synchrotron instrument of its kind, designed and optimized for MX experiments in the long wavelength range towards 5 &#197;. 
To make this possible, a large vacuum vessel encloses all endstation components of the sample environment. The necessity to maintain samples at cryogenic temperatures during storage and data collection in vacuum requires the use of thermally conductive sample holders. This facilitates efficient heat removal to ensure sample cooling to approximately 50 K. The current protocol describes the procedures used for sample preparation and transfer of samples into vacuum on beamline I23. Ensuring uniformity in practices and methods already established within the macromolecular crystallography community, sample cooling to liquid nitrogen temperature can be performed in any laboratory setting equipped with standard MX tools. 
Cryogenic storage and transport of samples only require standard commercially available equipment. Specialized equipment is required for the transfer of cryogenically cooled crystals from liquid nitrogen into the vacuum endstation. Bespoke sample handling tools and a dedicated Cryogenic Transfer System (CTS) have been developed in house. Diffraction data collected on samples prepared using this protocol show excellent merging statistics, indicating that the quality of samples is unaltered during the procedure. This opens unique opportunities for in-vacuum MX in a wavelength range beyond standard synchrotron beamlines.

Here, we present a protocol for cryogenic sample preparation and transfer of crystals into the vacuum endstation on beamline I23 at Diamond Light Source, for long-wavelength macromolecular X-ray crystallography experiments.

金刚石光源下光束线I23真空长波长晶体学的样品制备和转移实验方案

Long-wavelength macromolecular crystallography (MX) exploits the anomalous scattering properties of elements, such as sulfur, phosphorus, potassium, ...

High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without labeling other than deuterium. In particular, neutron backscattering spectroscopy records the scattering signals at multiple scattering angles simultaneously and is well suited to study the dynamics of biological systems on the ps-ns timescale. By employing D2O-and possibly deuterated buffer components-the method allows monitoring of both center-of-mass diffusion and backbone and side-chain motions (internal dynamics) of proteins in liquid state.
Additionally, hydration water dynamics can be studied by employing powders of perdeuterated proteins hydrated with H2O. This paper presents the workflow employed on the instrument IN16B at the Institut Laue-Langevin (ILL) to investigate protein and hydration water dynamics. The preparation of solution samples and hydrated protein powder samples using vapor exchange is explained. The data analysis procedure for both protein and hydration water dynamics is described for different types of datasets (quasielastic spectra or fixed-window scans) that can be obtained on a neutron backscattering spectrometer.
The method is illustrated with two studies involving amyloid proteins. The aggregation of lysozyme into &#181;m sized spherical aggregates-denoted particulates-is shown to occur in a one-step process on the space and time range probed on IN16B, while the internal dynamics remains unchanged. Further, the dynamics of hydration water of tau was studied on hydrated powders of perdeuterated protein. It is shown that translational motions of water are activated upon the formation of amyloid fibers. Finally, critical steps in the protocol are discussed as to how neutron scattering is positioned regarding the study of dynamics with respect to other experimental biophysical methods.

Neutron backscattering spectroscopy offers a nondestructive and label-free access to the ps-ns dynamics of proteins and their hydration water. The workflow is presented with two studies on amyloid proteins: on the time-resolved dynamics of lysozyme during aggregation and on the hydration water dynamics of tau upon fiber formation.

高分辨率中子光谱研究蛋白质和水合水的皮秒纳秒动力学

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without ...

Optimization of Crystal Growth for Neutron Macromolecular Crystallography

The use of neutron macromolecular crystallography (NMX) is expanding rapidly with most structures determined in the last decade thanks to new NMX beamlines having been built and increased availability of structure refinement software. However, the neutron sources currently available for NMX are significantly weaker than equivalent sources for X-ray crystallography. Despite advances in this field, significantly larger crystals will always be required for neutron diffraction studies, particularly with the tendency to study ever-larger macromolecules and complexes. Further improvements in methods and instrumentation suited to growing larger crystals are therefore necessary for the use of NMX to expand.
In this work, we introduce rational strategies and a crystal growth bench (OptiCrys) developed in our laboratory that combines real-time observation through a microscope-mounted video camera with precise automated control of crystallization solutions (e.g., precipitant concentration, pH, additive, temperature). We then demonstrate how this control of temperature and chemical composition facilitates the search for optimal crystallization conditions using model soluble proteins. Thorough knowledge of the crystallization phase diagram is crucial for selecting the starting position and the kinetic path for any crystallization experiment. We show how a rational approach can control the size and number of crystals generated based on knowledge of multidimensional phase diagrams.

Structural studies of biomacromolecules by crystallography require high-quality crystals. Here we demonstrate a protocol that can be used by OptiCrys (a fully automated instrument developed in our lab) and/or microdialysis buttons for growing large high-quality crystals based on knowledge of the crystallization phase diagram.

中子晶体学数据收集和处理，用于蛋白质结构中氢原子的建模

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