Name: microcrystal electron diffraction of small molecules
Uploaded: 2021-03-15T15:00:00
Description: Watch this Scientific Journal Video about Microcrystal Electron Diffraction of Small Molecules at JoVE.com

Gonen实验室得到了霍华德休斯医学研究所的资金支持。这项研究得到了美国国立卫生研究院P41GM136508的支持。

1. 制备小分子样品
<ol><li>将少量（0.01 - 1 mg）粉末、液体或固体转移到小瓶或管中。</li>
	<li>对于已经呈粉末形式的样品，使用盖子密封试管，直到需要样品为止。在尝试方法1（步骤3）或步骤2（步骤4）之前，将液体样品干燥成粉末。 注意：溶解在液体中的样品可以使用下面的方法3（5.X）</li>
</ol>2. 准备透射电镜网格
注意：一些带有自动加载系统的TEM要?...

<ol>
	<li>Shi, D., Nannenga, B. L., Iadanza, M. G., Gonen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+electron+crystallography+of+protein+microcrystals.">Three-dimensional electron crystallography of protein microcrystals.</a> eLife. 2, 01345 (2013).</li><li>Nannenga, B. L., Shi, D., Leslie, A. G. W., Gonen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+structure+determination+by+continuous-rotation+data+collection+in+MicroED.">High-resolution structure determination by continuous-rotation data collection in MicroED.</a> Nature Methods. 11 (9), 927-930 (2014).</li><li>Hattne, J., Martynowycz, M. W., Penczek, P. A., Gonen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroED+with+the+Falcon+III+direct+electron+detector.">MicroED with the Falcon III direct electron detector.</a> IUCrJ. 6 (5), 921-926 (2019).</li><li>Hattne, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroED+data+collection+and+processing.">MicroED data collection and processing.</a> Acta Crystallographica Section A Foundations and Advances. 71 (4), 353-360 (2015).</li><li>Henderson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+and+limitations+of+neutrons,+electrons+and+X-rays+for+atomic+resolution+microscopy+of+unstained+biological+molecules.">The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules.</a> Quarterly Reviews of Biophysics. 28 (2), 171-193 (1995).</li><li>Martynowycz, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroED+structure+of+the+human+adenosine+receptor+determined+from+a+single+nanocrystal+in+LCP.">MicroED structure of the human adenosine receptor determined from a single nanocrystal in LCP.</a> BioRxiv. , 316109 (2020).</li><li>Martynowycz, M. W., Zhao, W., Hattne, J., Jensen, G. J., Gonen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collection+of+continuous+rotation+MicroED+data+from+ion+beam-milled+crystals+of+any+size.">Collection of continuous rotation MicroED data from ion beam-milled crystals of any size.</a> Structure. 27 (3), 545-548 (2019).</li><li>Martynowycz, M. W., Gonen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand+incorporation+into+protein+microcrystals+for+MicroED+by+on-grid+soaking.">Ligand incorporation into protein microcrystals for MicroED by on-grid soaking.</a> Structure. , (2020).</li><li>Martynowycz, M. W., Khan, F., Hattne, J., Abramson, J., Gonen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroED+structure+of+lipid-embedded+mammalian+mitochondrial+voltage-dependent+anion+channel.">MicroED structure of lipid-embedded mammalian mitochondrial voltage-dependent anion channel.</a> Proceedings of the National Academy of Sciences. 117 (51), 32380-32385 (2020).</li><li>Jones, C. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CryoEM+method+MicroED+as+a+powerful+tool+for+small+molecule+structure+determination.">The CryoEM method MicroED as a powerful tool for small molecule structure determination.</a> ACS Central Science. 4 (11), 1587-1592 (2018).</li><li>Dick, M., Sarai, N. S., Martynowycz, M. W., Gonen, T., Arnold, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailoring+tryptophan+synthase+TrpB+for+selective+quaternary+carbon+bond+formation.">Tailoring tryptophan synthase TrpB for selective quaternary carbon bond formation.</a> Journal of the American Chemical Society. 141 (50), 19817-19822 (2019).</li><li>Gallagher-Jones, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-&#229;ngstr&#246;m+cryo-EM+structure+of+a+prion+protofibril+reveals+a+polar+clasp.">Sub-&#229;ngstr&#246;m cryo-EM structure of a prion protofibril reveals a polar clasp.</a> Nature Structural &amp; Molecular Biology. 25 (2), 131-134 (2018).</li><li>Ting, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+scaffold+peptide+in+the+biosynthesis+of+amino+acid-derived+natural+products.">Use of a scaffold peptide in the biosynthesis of amino acid-derived natural products.</a> Science. 365 (6450), 280-284 (2019).</li><li>de la Cruz, M. J., Martynowycz, M. W., Hattne, J., Gonen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroED+data+collection+with+SerialEM.">MicroED data collection with SerialEM.</a> Ultramicroscopy. 201, 77-80 (2019).</li><li>Mastronarde, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+electron+microscope+tomography+using+robust+prediction+of+specimen+movements.">Automated electron microscope tomography using robust prediction of specimen movements.</a> Journal of Structural Biology. 152 (1), 36-51 (2005).</li><li>Schorb, M., Haberbosch, I., Hagen, W. J. H., Schwab, Y., Mastronarde, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Software+tools+for+automated+transmission+electron+microscopy.">Software tools for automated transmission electron microscopy.</a> Nature Methods. 16 (6), 471-477 (2019).</li><li>Kabsch, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XDS.">XDS.</a> Acta Crystallographica Section D Biological Crystallography. 66 (2), 125-132 (2010).</li><li>Winter, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DIALS:+Implementation+and+evaluation+of+a+new+integration+package.">DIALS: Implementation and evaluation of a new integration package.</a> Acta Crystallographica Section D. 74 (2), 85-97 (2018).</li><li>de la Cruz, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic-resolution+structures+from+fragmented+protein+crystals+with+the+cryoEM+method+MicroED.">Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED.</a> Nature Methods. 14 (4), 399-402 (2017).</li><li>Shi, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+collection+of+MicroED+data+for+macromolecular+crystallography.">The collection of MicroED data for macromolecular crystallography.</a> Nature Protocols. 11 (5), 895-904 (2016).</li><li>Nannenga, B. L., Shi, D., Hattne, J., Reyes, F. E., Gonen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+catalase+determined+by+MicroED.">Structure of catalase determined by MicroED.</a> eLife. 3, 03600 (2014).</li><li>Martynowycz, M. W., Zhao, W., Hattne, J., Jensen, G. J., Gonen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Qualitative+Analyses+of+Polishing+and+Precoating+FIB+Milled+Crystals+for+MicroED.">Qualitative Analyses of Polishing and Precoating FIB Milled Crystals for MicroED.</a> Structure. 27 (10), 1594-1600 (2019).</li></ol>

样品制备通常是一个迭代过程，在筛选和数据收集后进行优化。对于小分子样品，通常谨慎的做法是首先尝试网格制备而不对网格进行辉光放电，因为许多药物往往是疏水性的10，11。如果网格的纳米晶沉积物太少，最好在第一次对网格进行辉光放电后重试。可能是冻干粉末的晶体太大太厚，无法收集良好的数据。在这些情况下，可以从较大晶体的边缘?...

微晶电子衍射（MicroED）是一种电子冷冻显微镜（cryo-EM）方法，用于从亚微米尺寸的晶体1，2中确定原子分辨率结构。将晶体应用于标准透射电子显微镜（TEM）网格，并通过投入液态乙烷或液氮进行冷冻。然后将网格加载到在低温下运行的TEM中。晶体位于网格上并筛选初始衍射质量。连续旋转 MicroED数据是从筛选晶体的子集中收集的，其中数据使用快速相机保存为电影3。这些电影被转换为标准的晶体学格式，并且处理方式几乎与X射线晶体学实验4相同。
MicroED最初是为了研究蛋白质微晶1，2而开发的。蛋白质晶体学的一个瓶颈是为传统的同步加速器X射线衍射实验生长出大而有序的晶体。由于电子与比X射线强几个数量级的物质相互作用，产生可检测衍射所需的晶体尺寸的限制要小得多5。此外，弹性与非弹性散射事件的比例对电子更有利，这表明可以在较小的整体暴露5下收集更多有用的数据。不断发展使得MicroED数据可以从最具挑战性的微晶6，7，8，9中收集。
最近，MicroED已被证明是从明显无定形的材料10，11，12，13确定小分子药物结构的有力工具。这些粉末可以直接来自一瓶购买的试剂、纯化柱，甚至可以来自将药丸粉碎成细粉10。这些粉末肉眼看起来是无定形的，但可以完全由纳米晶体组成，或者仅以更大的非结晶、无定形部分含有微量的纳米晶体沉积物。将材料应用于网格是容易的，晶体鉴定、筛选和数据收集的后续步骤甚至可能在不久的将来实现自动化14。虽然其他人可能使用不同的方法来制备样品和数据收集，但这里详细介绍了 Gonen 实验室开发和使用的用于制备 MicroED 的小分子样品和数据收集的协议。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.1-1.5mL Eppendorf tubes</td>
			<td>Fisher Scientific</td>
			<td>14-282-300</td>
			<td>Any vial or tube will do.</td>
		</tr>
		<tr>
			<td>Autogrid clips</td>
			<td>Thermo-Fisher</td>
			<td>1036173</td>
			<td>Clipped grids are not required for MicroED. They are required for Thermo-Fisher TEMs equipped with an autoloader system.</td>
		</tr>
		<tr>
			<td>Autogrid C-rings</td>
			<td>Thermo-Fisher</td>
			<td>1036171</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbamazapine</td>
			<td>Sigma</td>
			<td>C4024-1G</td>
			<td>Any amount will suffice for these experiments</td>
		</tr>
		<tr>
			<td>CMOS based detector</td>
			<td>Thermo-Fisher</td>
			<td>CetaD 16M</td>
			<td>We used a CetaD 16M, but any detector with rolling shutter mode or sufficiently fast readout is acceptable.&#160;</td>
		</tr>
		<tr>
			<td>Delphi software</td>
			<td>Thermo-Fisher</td>
			<td>N/A</td>
			<td>Software on Thermo-Fisher TEM systems that allows for manual rotation of the sample stage</td>
		</tr>
		<tr>
			<td>EPU-D software</td>
			<td>Thermo-Fisher</td>
			<td>N/A</td>
			<td>Commercial software for the acquisition of MicroED data</td>
		</tr>
		<tr>
			<td>Glass cover slides</td>
			<td>Hampton</td>
			<td>HR3-231</td>
			<td></td>
		</tr>
		<tr>
			<td>Glow discharger</td>
			<td>Pelco</td>
			<td>easiGlow</td>
			<td></td>
		</tr>
		<tr>
			<td>High PrecisionTweezers</td>
			<td>EMS</td>
			<td>78325-AC</td>
			<td>Any high precision tweezer will do</td>
		</tr>
		<tr>
			<td>Liquid nitrogen vessel</td>
			<td>Spear Lab</td>
			<td>FD-800</td>
			<td>A standard foam vessel for handling specimens under liquid nitrogen - 800mL</td>
		</tr>
		<tr>
			<td>SerialEM software</td>
			<td>UC Boulder</td>
			<td>N/A</td>
			<td>Free software distributed by D. Mastronarde. Department of Molecular, Cellular, and Developmental Biology</td>
		</tr>
		<tr>
			<td>TEM grids</td>
			<td>Quantifoil/EMS</td>
			<td>Q310CMA</td>
			<td>Multi-A 300 mesh grids were used here, but any thin carbon grids will work. For these small molecules, we suggest starting with continuous carbon.&#160;</td>
		</tr>
		<tr>
			<td>transmission electron microscope (TEM)</td>
			<td>Thermo-Fisher</td>
			<td>Talos Arctica</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman circular filter paper</td>
			<td>Millipore-Sigma</td>
			<td>WHA1001090</td>
			<td>90mm or larger</td>
		</tr>
	</tbody>
</table>

microcrystal electron diffraction of small molecules

描述了制备用于微晶电子衍射（MicroED）实验的小分子样品的详细方案。MicroED已被开发用于使用标准电子冷冻显微镜（cryo-EM）设备解决蛋白质和小分子的结构。通过这种方式，小分子、肽、可溶性蛋白和膜蛋白最近被测定到高分辨率。这里介绍了以药物卡马西平为例制备小分子药物网格的方案。提出了筛选和收集数据的协议。整个过程中的其他步骤（如数据集成、结构确定和优化）将在别处介绍。准备小分子网格所需的时间估计不到30分钟。

MicroED是一种冷冻电镜方法，它利用电子和物质之间的强相互作用，可以研究消失的小晶体12，13。在这些步骤之后，有望从微晶中收集晶体学格式的衍射电影（视频1）。在这里，使用卡马西平12演示该技术。结果显示了来自在TEM网格上鉴定的卡马西平微晶的连续旋转MicroED数据集（视频1）。一个好的数据集具有...

Watch this Scientific Journal Video about Microcrystal Electron Diffraction of Small Molecules at JoVE.com

Microcrystal Electron Diffraction of Small Molecules

在这里，我们描述了我们实验室开发的用于制备用于微晶电子衍射（MicroED）实验的小分子晶体粉末的程序。

小分子的微晶电子衍射

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

在这个过程中，我们展示了如何在几分钟内从小分子粉末变成三维结构。这是化学界的一项变革性技术。许多化合物不容易长成大晶体。使用MicroED，我们可以从纳米尺寸的晶体中确定原子分辨率结构。演示该程序的将是加州大学洛杉矶分校教授Mike Martynowycz博士。首先，将少量粉末，液体或固体转移到一个小瓶或管中。对于已经呈粉末形式的样品，使用盖子密封试管，直到需要样品为止。同样，将液体样品干燥成粉末。将塑料薄膜缠绕在玻璃盖玻片的一端。将TEM网格放在盖玻片顶部的薄膜上，棕色碳面朝上。然后，将带有网格的载玻片放入辉光放电室，并使用 15 皮安的负设置对盖玻片进行辉光放电约 30 秒。在将样品添加到网格之前，将网格存储在盖玻片上的盖玻片内衬有滤纸的玻璃培养皿内。使用镊子从覆盖的培养皿中取出辉光放电TEM网格，并将其放在圆形滤纸上，碳面朝上。使用小刮刀取出一小勺粉末，然后将其放在滤纸上TEM网格旁边的小方形玻璃盖玻片上。将另一个小的方形载玻片或盖玻片放在粉末上。用手指将两个载玻片轻轻摩擦在一起，制成细粉。将盖玻片倾斜并将它们放置在滤纸上的TEM网格上方，然后继续将盖玻片摩擦在一起，仅比发光放电TEM网格高出几厘米。观察粉末是否朝网格下落。通过取下两个玻璃盖玻片中的一个来盖住细磨的粉末。然后用一张滤纸轻轻地将盖玻片上的细粉刷到TEM网格上。使用一组镊子抓住TEM网格的边缘，确保尖端不会刺穿任何网格方块。将网格抬到滤纸上方一到两厘米处，并将网格与下面的纸张成 90 度角。轻轻敲击镊子，同时保持网格牢固地镊子以去除任何松散的粉末。用手将带有网格的镊子尖端直接移动到液氮容器中来冷冻网格，然后等待网格和镊子停止沸腾。在液氮下，将网格放置在样品架中，碳侧朝向，使样品在碳支撑膜之前被光束击中。将样品架装入TEM中，确保网格始终保持在液氮温度。对于自动装载机系统，将夹紧的网格放入液氮冷却容器中的盒中，这允许自动装载机机器人接受盒式磁带，同时保持样品安全，以便在自动装载机和色谱柱之间穿梭。打开TEM柱阀，使用手面板将放大倍率调节到尽可能低的放大倍率。通过调整手面板上的强度旋钮来找到光束，以便在荧光屏上可以看到圆形明亮区域。在收集高分辨率MicroED数据之前，使用适当的软件在低放大倍率下获取全网格图谱，确保显微镜在低倍和高放大倍率成像中都对准良好。识别网格方块，薄膜上没有破碎的碳和可见的黑色或深色颗粒。使用手持面板上的操纵杆或在收集的图集上虚拟地在网格周围导航，以搜索未损坏且包含微晶体的网格方块。将这些方块的中心添加到网格位置列表中以进行研究，该位置可以添加到显微镜用户界面或显微镜自动化软件中的笔记本中。将放大倍率设置为 500 到 1300 倍。调整每个存储的格网位置的中心高度，并将保存的 z 值更新为记下的位置。在荧光屏或平板相机上搜索网格上的小黑点或颗粒。一个好的样品在高放大倍率下通常会有锋利的边缘，表明晶体是有序的。将定位的电位晶体移动到屏幕中心并增加放大倍率，使TEM进入高放大倍率模式。插入所选区域孔径并将孔径更改为更大或更小的尺寸，以确保所选区域大于晶体。通过按下TEM手板上的折射按钮切换到折射模式，确保荧光屏已插入。使用放大旋钮调整相机长度，使边缘至少为一个埃分辨率。调整衍射焦点，使中心光斑尽可能清晰和小。使用衍射移位旋钮，将中心光束移动到荧光屏的中心。插入光束挡块，确保光束在其后面。提起荧光屏并在相机上短暂曝光。检查相应的折射图案，确保图案具有按列和行规则排列的尖锐斑点。将晶体坐标保存在TEM用户界面中或将其写下来，然后对当前网格方块上所有感兴趣的潜在晶体重复衍射筛选。将筛分的晶体以大于 1000 倍的放大倍数居中，并使用自动程序或手动调整晶体的中心高度。插入最适合晶体尺寸和形状的选定区域孔径。沿负方向和正方向倾斜舞台，直到图像被网格条遮挡。并记下这些角度以进行数据收集，方法是在具有卷帘快门读出模式的现代相机上每秒读取数据。通过在显微镜用户界面或专用软件中指定最大倾斜速度的百分比和停止时间来设置旋转速率。将数据保存为各种格式，例如单个帧或作为图像堆栈。从微晶体收集的晶体学格式的折射视频显示了来自TEM网格上鉴定的卡马西平微晶体的连续旋转MicroED数据集。在数据收集、整合和结构求解后，确定高分辨率结构，其清晰度取决于数据的质量和完整性。将样本应用于网格对于获取良好数据至关重要。按照这种方法，可以使用标准晶体学软件进一步处理数据。证明MicroED是一种强大的小分子方法，已经在全球范围内用于解决至关重要分子的新结构。

Research

JoVE Journal

Biochemistry

Microcrystallography of Protein Crystals and In Cellulo Diffraction

Microcrystallography of Protein Crystals and In Cellulo Diffraction

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 ...

科研

生物化学

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

原子力显微镜对单蛋白质分子的力谱

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. ...

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

细菌显示文库的半自动化筛选肽亲和试剂的发现与分离结果分析

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay

用生物层干涉法捕获小分子离子通道蛋白的相互作用动力学

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe ...

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

Analysis of ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

β-淀粉脂质诱导纤维蛋白斑点结构异常的光谱分析

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

单分子跟踪显微镜 - 确定细胞分子扩散状态的工具

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and ...

Preparing Lamellae from Vitreous Biological Samples Using a Dual-Beam Scanning Electron Microscope for Cryo-Electron Tomography

使用双光束扫描电子显微镜从玻璃体生物样品制备薄片进行冷冻电子断层扫描

Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected&#160;human erythrocytes, which could ...

Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows

微图案透射电子显微镜网格，用于在全细胞冷冻电子断层扫描工作流程中指导细胞定位

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules ...

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 ...

The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are ...