SJ 是 DAE-TIFR 博士生奖学金的获得者，并且资金得到认可。KRV 感谢 DBT B-Life 赠款 DBT/PR12422/MED/31/287/2014 以及印度政府原子能部在项目识别号下的支持。RTI4006.

在大分子的 CryoEM 图中模拟小分子配体

作者没有什么可透露的。

近年来，显微镜硬件和软件的改进导致冷冻电子显微镜结构数量的增加。尽管目前在单颗粒冷冻EM中实现的最高分辨率为1.2 Å 57,58,59，但大多数结构的确定分辨率约为3-4 Å。在中低分辨率图谱中对配体进行建模可能很棘手，而且往往充满歧义。鉴于冷冻电镜在学术界和制药行业广泛用于转化研究和药物发现，确保配体建模正确?...

细胞通过同时独立地进行无数的化学反应来实现其功能，每个化学反应都经过精心调节，以确保它们的生存和对环境线索的适应性。这是通过分子识别实现的，它使生物分子（尤其是蛋白质）能够与其他大分子以及小分子或配体形成瞬时或稳定的复合物1。因此，蛋白质-配体相互作用是生物学中所有过程的基础，包括蛋白质表达和活性的调节、酶对底物和辅因子的识别，以及细胞如何感知和传递信号 1,2。更好地了解蛋白质-配体复合物的动力学、热力学和结构特性揭示了配体相互作用的分子基础，并通过优化药物相互作用和特异性促进了合理的药物设计。研究蛋白质-配体相互作用的一种经济且更快的方法是使用分子对接，这是一种计算方法，可以虚拟筛选各种小分子并预测这些配体与靶蛋白的结合模式和亲和力3。然而，通过 X 射线衍射 （XRD）、核磁共振 （NMR） 或电子冷冻显微镜 （cryoEM） 确定的高分辨率结构的实验证据为此类预测提供了必要的证据，并有助于开发针对给定靶点的更新、更有效的激活剂或抑制剂。本文使用缩写“cryoEM”，因为该技术通常被称为“cryoEM”。然而，关于选择正确的命名法一直存在争议，最近，有人提出了术语冷冻基因样本 Electron Microscopy （cryoEM） 来表示样本处于低温并用电子成像4。同样，从冷冻电镜得出的图被称为电子电位、静电势或库仑电位，为了简单起见，这里我们使用冷冻电镜图5,6,7,8,9,10。
尽管 XRD 一直是蛋白质-配体复合物高分辨率结构测定的金标准技术，但分辨率革命后的11 cryoEM 已经获得了动力，正如过去几年中沉积在电子显微镜数据库 （EMDB） 12 中的库仑电位图或冷冻 EM 图的激增所表明的那样 14。由于样品制备、成像和数据处理方法的进步，2010 年至 2020 年间，使用冷冻电镜的蛋白质数据库 （PDB）14 沉积数量从 0.7% 增加到 17%，2020 年报告的结构中约有 50% 是以 3.5 Å 或更高的分辨率确定的 15,16。CryoEM 已迅速被包括制药行业在内的结构生物学界采用，因为它允许以近原子分辨率研究柔性和非结晶性生物大分子，尤其是膜蛋白和多蛋白复合物，克服了结晶过程并获得通过 XRD 进行高分辨率结构测定所需的衍射良好的晶体。
在cryoEM图谱中准确建模配体至关重要，因为它在分子水平上是蛋白质-配体复合物的蓝图。X 射线晶体学中使用了几种自动配体构建工具，这些工具依赖于配体密度的形状和拓扑结构，以便将配体拟合或构建到电子密度中 17,18,19,20。然而，如果分辨率低于 3 Å，这些方法往往会产生不太理想的结果，因为它们识别和构建所依赖的拓扑特征变得不那么明确。在许多情况下，这些方法已被证明在将配体准确建模到 cryoEM 图谱中是无效的，因为这些图谱是在中低分辨率范围内确定的，通常在 3.5 Å-5 Å17 之间。
使用 cryoEM 测定蛋白质-配体复合物 3D 结构的第一步包括将配体与蛋白质共纯化（当配体与蛋白质具有高结合亲和力时）或在网格制备之前将蛋白质溶液与配体孵育特定持续时间。随后，将少量样品放置在等离子体清洁的多孔透射电镜网格上，然后在液态乙烷中快速冷冻，最后使用冷冻透射电镜成像。对数十万到数百万个单个粒子的 2D 投影图像进行平均，以重建大分子的 3D （3D） 库仑势图。在许多情况下，由于图谱上的各向异性分辨率（即大分子之间的分辨率不均匀）、配体结合区域的灵活性以及数据中的噪声，这些图谱中的配体和溶剂分子的识别和建模带来了重大挑战。许多为 XRD 开发的建模、改进和可视化工具现在正被用于冷冻电子市场，用于相同的目的 18,19,20,21。在本文中，概述了目前用于识别配体、构建模型和优化从 cryoEM 得出的坐标的各种方法和软件。已经提供了一个分步方案来说明使用具有不同分辨率和复杂性的特定蛋白质-配体复合物对配体进行建模所涉及的过程。
在冷冻EM图谱中对配体进行建模的第一步包括识别图谱中的配体密度（非蛋白质）。如果配体结合不会引起蛋白质的任何构象变化，那么计算蛋白质-配体复合物和载脂蛋白之间的简单差异图基本上突出了额外密度的区域，表明配体的存在。这种差异可以立即观察到，因为它只需要两张图谱，甚至在3D细化过程中的中间图也可以用来检查配体是否存在。此外，如果分辨率足够高 （<3.0 Å），那么差异图还可以深入了解水分子的位置以及与配体和蛋白质残基相互作用的离子。
在没有 apo-protein maps 的情况下，现在可以使用 Servalcat22，它可作为独立工具使用，并且作为 Refmac 改进的一部分也已集成到 CCP-EM 软件套件 23,24 和 CCP4 8.0 版本25,26 中。Servalcat 允许使用未锐化的半图和载脂蛋白模型作为输入来计算 FSC 加权差 （Fo-Fc） 图。Fo-Fc 省略图表示实验图（Fo）和从模型派生的图（Fc）之间的差异。在模型中没有配体的情况下，Fo-Fc图谱中的正密度与实验EM图谱重叠通常表明配体的存在。这里的假设是蛋白质链在图谱中拟合良好，剩余的正密度表明配体的位置。然而，重要的是要仔细检查正密度是否源于建模的不准确性，例如蛋白质侧链的错误转子。
第二步涉及从可用的化学信息中获取或创建配体的笛卡尔坐标文件，该配体具有明确定义的几何形状。CCP4 单体库中已有的标准配体（例如，ATP 和 NADP+）可以通过其单体登录代码检索坐标和几何文件来用于精细化。但是，对于未知或非标准配体，可以使用各种工具来创建几何文件。其中一些示例包括 Phenix28 中的 eLBOW27 - （电子配体构建器和优化工作台）、Lidia - Coot29 中的内置工具、JLigand/ACEDRG30,31、CCP-EM23,24、Ligprep32-薛定谔套件中的 Glide 模块。然后，在实验冷冻电镜图和Coot中的差异图的指导下，将配体坐标文件拟合到密度中。接下来是 Phenix28 中的实时空间细化或 Refmac33 中的倒数细化。需要 Linux 工作站或配备优质显卡和上述软件的笔记本电脑。这些程序中的大多数都包含在各种套件中。CCP-EM24 和 Phenix28 可供学术用户免费使用，并包含本文中使用的各种工具，包括 Coot、Refmac533、34、35、36、Servalcat、phenix.real_space_refine 等。同样，Chimera37 和 ChimeraX38 为学术用户提供免费许可证。

<table><tbody><tr><td>CCP4-8.0</td><td>Consortium of several institutes</td><td>https://www.ccp4.ac.uk</td><td>Free for academic users and includes Coot and list of tools developed for X-ray crystallography</td></tr><tr><td>CCP-EM</td><td>Consortium of several institutes</td><td>https://www.ccpem.ac.uk/download.php</td><td>Free for academic users and includes Coot, Relion and many others</td></tr><tr><td>Coot&amp;nbsp;</td><td>Paul Emsley, LMB, Cambridge</td><td>https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/</td><td>General software for model building but also available with other suites described above</td></tr><tr><td>DockinMap (Phenix)</td><td>Consortium of several institutes</td><td>https://phenix-online.org/documentation/reference/dock_in_map.html</td><td>Software inside the Phenix suite for docking model into cryoEM maps</td></tr><tr><td>Electron Microscopy Data Bank&amp;nbsp;</td><td>Consortium of several institutes</td><td>https://www.ebi.ac.uk/emdb/</td><td>Public Repository for Electron Microscopy maps</td></tr><tr><td>Falcon</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>https://assets.thermofisher.com/TFS-Assets/MSD/Technical-Notes/Falcon-3EC-Datasheet.pdf</td><td>Commercial, camera from Thermo Fisher&amp;nbsp;</td></tr><tr><td>Phenix&amp;nbsp;</td><td>Consortium of several institutes</td><td>https://phenix-online.org/download</td><td>Free for academic users and includes Coot</td></tr><tr><td>Protein Data Bank</td><td>Consortium of several institutes</td><td>https://rcsb.org</td><td>Public database of macromolecular structures</td></tr><tr><td>Pymol</td><td>Schrodinger</td><td>https://pymol.org/2/</td><td>Molecular viusalization tool. Educational version is free but comes with limitation. The full version can be obtained with a small fee.</td></tr><tr><td>Relion</td><td>MRC-LMB, Cambridge</td><td>https://relion.readthedocs.io/en/release-4.0/Installation.html</td><td>Software for cryoEM image processing, also available with CCP-EM</td></tr><tr><td>Titan Krios</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>https://www.thermofisher.com/in/en/home/electron-microscopy/products/transmission-electron-microscopes/krios-g4-cryo-tem.html?cid=msd_ls_xbu_xmkt_tem-krios_285811_gl_pso_gaw_tpne1c&amp;amp;&lt;br/&gt; gad_source=1&amp;amp;gclid=CjwKCAiA-P-rBhBEEiwAQEXhHyw5c8MKThmdA&lt;br/&gt; AkZesWC4FYQSwIQRk&lt;br/&gt; ZApkj08MfYG040DtiiuL8&lt;br/&gt; RihoCebEQAvD_BwE</td><td>Commercial, cryoTEM from Thermo Fisher</td></tr><tr><td>UCSF Chimera</td><td>UCSF, USA</td><td>https://www.cgl.ucsf.edu/chimera/download.html</td><td>General purpose software for display, analysis and more</td></tr><tr><td>UCSF Chimera X</td><td>UCSF, USA</td><td>https://www.cgl.ucsf.edu/chimerax/</td><td>General purpose software for display, analysis and more</td></tr></tbody></table>

modeling ligands into maps derived from electron cryomicroscopy

破译大分子复合物中的蛋白质-配体相互作用对于理解分子机制、潜在的生物学过程和药物开发至关重要。近年来，低温样品电子显微镜（cryoEM）已成为确定大分子结构和研究近原子分辨率配体结合模式的有力技术。由于目标分子的各向异性分辨率和数据中固有的噪声，在冷冻电磁图谱中识别和建模非蛋白质分子通常具有挑战性。在本文中，向读者介绍了目前用于配体鉴定、模型构建和使用选定大分子细化原子坐标的各种软件和方法。如烯醇化酶所示，识别配体存在的最简单方法之一是减去有和没有配体获得的两张图谱。即使在更高的阈值下，配体的额外密度也可能在差异图中脱颖而出。在某些情况下，如代谢型谷氨酸受体 mGlu5 的情况所示，当无法生成这种简单的差异图时。最近引入的推导Fo-Fc省略图的方法可以作为验证和证明配体存在的工具。最后，以经过充分研究的β-半乳糖苷酶为例，分析了分辨率对冷冻电子显微镜图谱中配体和溶剂分子建模的影响，并对冷冻电子镜在药物发现中的应用进行了展望。

示例 1 来自结核分枝杆菌的烯醇化酶催化糖酵解的倒数第二步，并将 2-磷酸甘油酸转化为磷酸烯醇丙酮酸 （PEP），这是几种代谢途径的重要中间体44,45。在相同像素大小 1.07 Å 下收集 apo-烯醇化酶和 PEP 结合烯醇化酶样品的 CryoEM 数据，并使用 Relion 3.146,47 进行图像处理。载脂蒲-烯醇化酶?...

作者聚焦：通过在冷冻电镜图中对配体进行建模来探索细胞过程

该协议介绍了可用于在大分子的冷冻EM图谱中建模小分子配体的工具。

将配体建模为源自电子冷冻显微镜的图谱

Deciphering the protein-ligand interactions in a macromolecular complex is crucial for understanding the molecular mechanism, underlying biological ...

我们感兴趣的是了解整个细胞中发生的生理过程，特别是在膜界面处。我们使用冷冻电镜作为主要技术，并研究各种有趣和具有挑战性的生物学问题，研究多种大分子复合物。几十年前，确定膜蛋白和其他不稳定复合物的结构具有挑战性。然而，冷冻电镜（一种新型洗涤剂和脂质模拟物）的技术进步为这些复合物的快速结构测定铺平了道路。这些结构现在可以以高分辨率获得，并有可能用于药物发现。在单粒子冷冻电子显微镜中，挑战包括获取稳定和均匀的样本、在薄冰中以随机方向嵌入样本、处理大量低信噪比的图像，以及在 3D 重建过程中准确确定角度取向和分类。由于数据中固有的噪声以及各向同性且图谱分辨率低、样品异质性和配体灵活性，因此在配体蛋白复合物的冷冻电镜图谱中识别和建模小分子具有挑战性。该协议是在低至中分辨率冷冻电镜中识别和建模配体和溶剂分子的分步指南。

modeling-ligands-into-maps-derived-from-electron-cryomicroscopy

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

Modeling Ligands into Maps Derived from Electron Cryomicroscopy

1.1K 次观看.  Tata Institute of Fundamental Research. 我们感兴趣的是了解整个细胞中发生的生理过程，特别是在膜界面处。我们使用冷冻电镜作为主要技术，并研究各种有趣和具有挑战性的生物学问题，研究多种大分子复合物。几十年前，确定膜蛋白和其他不稳定复合物的结构具有挑战性。然而，冷冻电镜（一种新型洗涤剂和脂质模拟物）的技术进步为这些复合物的快速结构测定铺平了道路。这些结构现在可以以高分辨?...

视频: 作者聚焦：通过在冷冻电镜图中对配体进行建模来探索细胞过程

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 &#197; structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

This paper presents a protocol for processing cryo-EM images using the software suite SPHIRE. The present protocol can be applied for nearly all single particle EM projects that target near-atomic resolution.

使用SPHIRE的电子冷冻显微镜图像的高分辨率单粒子分析

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

科研

生物化学

Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions

Differential scanning calorimetry (DSC) is a powerful technique for quantifying thermodynamic parameters governing biomolecular folding and binding interactions. This information is critical in the design of new pharmaceutical compounds. However, many pharmaceutically relevant ligands are chemically unstable at the high temperatures used in DSC analyses. Thus, measuring binding interactions is challenging because the concentrations of ligands and thermally-converted products are constantly changing within the calorimeter cell. Here, we present a protocol using thermolabile ligands and DSC for rapidly obtaining thermodynamic and kinetic information on the folding, binding, and ligand conversion processes. We have applied our method to the DNA aptamer MN4 that binds to the thermolabile ligand cocaine. Using a new global fitting analysis that accounts for thermolabile ligand conversion, the complete set of folding and binding parameters are obtained from a pair of DSC experiments. In addition, we show that the rate constant for thermolabile ligand conversion may be obtained with only one supplementary DSC dataset. The guidelines for identifying and analyzing data from several more complicated scenarios are presented, including irreversible aggregation of the biomolecule, slow folding, slow binding, and rapid depletion of the thermolabile ligand.

We present a protocol for rapid characterization of biomolecular folding and binding interactions with thermolabile ligands using differential scanning calorimetry.

用耐热配体测量生物分子 DSC 剖面快速表征折叠和结合的相互作用

Differential scanning calorimetry (DSC) is a powerful technique for quantifying thermodynamic parameters governing biomolecular folding and binding ...

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

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

Biopanning bacterial display libraries is a proven technique for discovery of peptide affinity reagents, a robust alternative to antibodies. The semi-automated sorting method herein has streamlined biopanning to decrease the occurrence of false positives. Here we illustrate the thought process and techniques applied in evaluating candidates and minimizing downstream analysis.

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. ...

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the pre-endosomal anthrax toxin is assembled and transitioned into the endosomal complex. First, the N-terminal domain of a cysteine mutant lethal factor (LFN) is attached to a biolayer interferometry (BLI) biosensor through disulfide coupling in an optimal orientation, allowing protective antigen (PA) prepore to bind (Kd 1 nM). The optimally oriented LFN-PAprepore complex then binds to soluble capillary morphogenic gene-2 (CMG2) cell surface receptor (Kd 170 pM), resulting in a representative anthrax pre-endosomal complex, stable at pH 7.5. This assembled complex is then subjected to acidification (pH 5.0) representative of the late endosome environment to transition the PAprepore into the membrane inserted pore state. This PApore state results in a weakened binding between the CMG2 receptor and the LFN-PApore and a substantial dissociation of CMG2 from the transition pore. The thio-attachment of LFN to the biosensor surface is easily reversed by dithiothreitol. Reduction on the BLI biosensor surface releases the LFN-PAprepore-CMG2 ternary complex or the acid transitioned LFN-PApore complexes into microliter volumes. Released complexes are then visualized and identified using electron microscopy and mass spectrometry. These experiments demonstrate how to monitor the kinetic assembly/disassembly of specific protein complexes using label-free BLI methodologies and evaluate the structure and identity of these BLI assembled complexes by electron microscopy and mass spectrometry, respectively, using easy-to-replicate sequential procedures.

Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively.

用质谱和电镜分析 Biolayer 干涉传感器上的动态蛋白复合物的组装和释放

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

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

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). A&#946;, which forms neurotoxic amyloid aggregates in Alzheimer&#39;s disease (AD), has been shown to interact with fibrinogen. This A&#946;-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. A&#946;-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the A&#946;-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of A&#946; and A&#946;-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of A&#946;-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate A&#946;-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified A&#946;-fibrinogen interaction inhibitor.

Presented here are two methods that can be used individually or in combination to analyze the effect of beta-amyloid on fibrin clot structure. Included is a protocol for creating an in vitro fibrin clot, followed by clot turbidity and scanning electron microscopy methods.

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 ...

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium homeostasis, particularly store-operated calcium entry, which is critical to maintaining proper function of synaptic vesicle release and intracellular signaling pathways. Accordingly, TRPC channels have been implicated in a variety of human diseases including cardiovascular disorders such as cardiac hypertrophy, neurodegenerative disorders such as Parkinson&#39;s disease, and neurologic disorders such as spinocerebellar ataxia. Therefore, TRPC channels represent a potential pharmacologic target in human diseases. However, the molecular mechanisms of gating in these channels are still unclear. The difficulty in obtaining large quantities of stable, homogeneous, and purified protein has been a limiting factor in structure determination studies, particularly for mammalian membrane proteins such as the TRPC ion channels. Here, we present a protocol for the large-scale expression of mammalian ion channel membrane proteins using a modified baculovirus gene transfer system and the purification of these proteins by affinity and size-exclusion chromatography. We further present a protocol to collect single-particle cryo-electron microscopy images from purified protein and to use these images to determine the protein structure. Structure determination is a powerful method for understanding the mechanisms of gating and function in ion channels.

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

单粒子冷冻电子显微镜在结构中测定脂质敏感分通道 trpc3 的表达与纯化

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium ...

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 ...

Modeling an Enzyme Active Site using Molecular Visualization Freeware

Biomolecular visualization skills are paramount to understanding key concepts in the biological sciences, such as structure-function relationships and molecular interactions. Various programs allow a learner to manipulate 3D structures, and biomolecular modeling promotes active learning, builds computational skills, and bridges the gap between two dimensional textbook images and the three dimensions of life. A critical skill in this area is to model a protein active site, displaying parts of the macromolecule that can interact with a small molecule, or ligand, in a way that shows binding interactions. In this protocol, we describe this process using four freely available macromolecular modeling programs: iCn3D, Jmol/JSmol, PyMOL, and UCSF ChimeraX. This guide is intended for students seeking to learn the basics of a specific program, as well as instructors incorporating biomolecular modeling into their curriculum. The protocol enables the user to model an active site using a specific visualization program, or to sample several of the free programs available. The model chosen for this protocol is human glucokinase, an isoform of the enzyme hexokinase, which catalyzes the first step of glycolysis. The enzyme is bound to one of its substrates, as well as a non-reactive substrate analog, which allows the user to analyze interactions in the catalytic complex.

A key skill in biomolecular modeling is displaying and annotating active sites in proteins. This technique is demonstrated using four popular free programs for macromolecular visualization: iCn3D, Jmol, PyMOL, and UCSF ChimeraX.

使用分子可视化免费软件对酶活性位点进行建模

Biomolecular visualization skills are paramount to understanding key concepts in the biological sciences, such as structure-function relationships and ...

Simplified Volumetric Models as an Effective Strategy for Segmenting Actin Networks in Cryo-Electron Tomograms

Efficient methods for the extraction of features of interest remain one of the biggest challenges for the interpretation of cryo-electron tomograms. Various automated approaches have been proposed, many of which work well for high-contrast datasets where the features of interest can be easily detected and are clearly separated from one another. Our inner ear stereocilia cryo-electron tomographic datasets are characterized by a dense array of hexagonally packed actin filaments that are frequently cross-connected. These features make automated segmentation very challenging, further aggravated by the high-noise environment of cryo-electron tomograms and the high complexity of the densely packed features. Using prior knowledge about the actin bundle organization, we have placed layers of a highly simplified ball-and-stick actin model to first obtain a global fit to the density map, followed by regional and local adjustments of the model. We show that volumetric model building not only allows us to deal with the high complexity, but also provides precise measurements and statistics about the actin bundle. Volumetric models also serve as anchoring points for local segmentation, such as in the case of the actin-actin cross connectors. Volumetric model building, particularly when further augmented by computer-based automated fitting approaches, can be a powerful alternative when conventional automated segmentation approaches are not successful.

Here, we present a protocol to place simplified volumetric models into noisy, complex, tomographic 3D volumes. This allows the fast segmentation of actin filament densities, detection of systematic filament bending and of gaps in hair bundle filaments, as well as convenient quantification of volumetric model properties, such as distances.

Efficient methods for the extraction of features of interest remain one of the biggest challenges for the interpretation of cryo-electron tomograms. ...