VJ和HS承认NIH R01 GM094246支持VJ。 HS承认克莱姆森大学创意咨询计划和克莱姆森大学光学材料科学和工程技术中心的创业资金。该项目还得到了墨西哥湾沿岸联盟跨学科生物科学培训凯克中心（NIGMS Grant No.1 T32GM089657-05）的训练奖学金和Schissler基金会DD普通人类疾病转化研究奖学金的支持。内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方观点。

所有作者都宣称与本文的内容没有竞争的经济利益。

在这项工作中，提出了使用PIE-MFD单分子FRET实验以高精度对准，校准和测量干涉距离的方案。通过仔细校准所有仪器参数，可以提高测量距离的精度并达到Angstrom精度。为了做到这一点，使用各种多维直方图来分析和识别人群以进一步表征。使用平均宏观时间来验证测量样品的稳定性，可以基于化学计量参数来校正供体和受体光漂白并选择FRET群体。然而，受体的光物理性质可以根据标签的位置而?...

结构生物学研究的一个基本目标是揭示生物分子机器的结构与功能之间的关系。生物分子（ 例如，蛋白质和核酸）的第一个视觉印象发生在20世纪50年代，通过开发X射线晶体学1,2 。 X射线晶体学提供了由晶体包装约束的高分辨率静态结构信息。因此，X射线结构模型的固有不动性可以避免生物分子的动态性质，这是影响大多数生物学功能的因素3,4,5 。核磁共振（NMR） 6,7,8提供了解决水溶液中结构模型的问题的替代方案。一个很大的优势的NMR是其恢复生物分子和构象集合的内在动力学特性的能力，有助于阐明结构，动力学和功能之间的内在关系3,4,5。然而，由样品量和大量样品限制的NMR对较大系统需要复杂的标记策略。因此，迫切需要开发结构生物学中的替代方法。 历史上，Förster共振能量转移（FRET） 9在结构生物学中没有发挥重要作用，因为FRET提供了低精度距离测量的误解。该协议的目的是重新审视FRET确定纳米尺度的距离，使得这些距离可用于构建生物分子的结构模型。第一个实验验证通过测量各种长度的聚合物作为"光谱尺"，1967年10号由Stryer在FRET效率方面的依赖性。在2005年的单分子水平上完成了类似的实验11 。聚脯氨酸分子被证明是非理想的，因此后来使用双链DNA分子12 。这打开了精确距离测量的窗口和使用FRET识别生物分子的结构特性的想法。 当干涉距离范围为〜0.6-1.3 R 0时，FRET最佳，其中R 0是Förster距离。对于在单分子FRET实验中使用的典型荧光团， R 0为〜50。通常，与其他方法相比，FRET在解决和区分结构和动态的能力方面提供了许多优势异质系统:（i）由于荧光的最终灵敏度，单分子FRET实验13,14,15,16可以通过直接计数和同时表征其各个成员的结构来解析异质集合。 （ii）复合反应途径可以在单分子FRET研究中直接解密，因为不需要整体的同步。 （iii）FRET可以及时访问跨越十多年的各种时间领域，涵盖各种各样的生物相关动态。 （iv）FRET实验可以在体外和体内的任何溶液条件下进行 。 FRET与荧光显微镜的组合允许直接在活细胞中研究分子结构和相互作用15,16 ， </<html> sup&gt; 17，18，19 ，甚至高精度20 。 （v）FRET可以应用于几乎任何大小的系统（ 例如，聚脯氨酸低聚物21,22,23,24，Hsp90 25 ，HIV逆转录酶26和核糖体27 ）。 （vi）最后，包含生物分子的所有维度的距离网络可用于导出静态或动态分子的结构模型18,28,29,30,31,32,33,34 ，参考"&gt; 35,36,37。 因此，单分子FRET光谱可用于导出足够精确的距离以用于距离限制结构建模26 。这可以通过利用八维尺寸的荧光信息（ 即激发光谱，荧光光谱，各向异性，荧光寿命，荧光量子产率等）的多参数荧光检测（MFD）28,38,39,40,41,42的优点，宏观时间，荧光强度和荧光团之间的距离）精确而准确地提供距离限制。另外，脉冲交错激励（PIE）与MFD组合（PIE-MFD） 42以监测直接激发受体荧光并选择由含有1:1供体 - 受体化学计量的样品产生的单分子事件。典型的PIE-MFD设置使用连接到共聚焦显微镜主体的双脉冲交错激光激光器，其中光子检测在不同光谱窗口和偏振特性中分为四个不同的通道。更多细节见图1 。 重要的是要注意，FRET必须结合计算方法来实现与FRET结果26,30相符合的类原子结构模型。目前的协议的目的不在于使用FRET衍生距离来构建结构模型的相关方法。然而，这些方法已经与其他技术（ 例如，小角度X射线散射）结合使用或电子顺磁共振），产生了整合结构生物学领域43,44,45,46。目前的目标是为FRET作为结构生物学的定量工具铺平道路。作为示例，该方法用于鉴定N-甲基-D-天冬氨酸（NMDA）受体的配体结合结构域（LBD）中的三种构象状态。最终的目标是克服上述限制，并通过提供高精度的测量距离将FRET引入用于结构测定生物分子的综合方法。

<table><tbody><tr><td>charcoal</td><td>Merck KGaA</td><td>K42964486 320</td><td></td></tr><tr><td>syringe filter</td><td>Fisherbrand</td><td>09-719C</td><td>size: 0.20 &amp;micro;m</td></tr><tr><td>chambered coverglass</td><td>Fisher Scientific</td><td>155409</td><td>1.5 borosilicate glass, 8 wells</td></tr><tr><td>microscope cover glass</td><td>Fisher Scientific</td><td>063014-9</td><td>size: 24 x 60-1.5</td></tr><tr><td>Nuclease free water</td><td>Fisher Scientific</td><td>148859</td><td>nuclease free</td></tr><tr><td>tween-20</td><td>Thermo Scientific</td><td>28320</td><td>10% solution of Polysorbate 20</td></tr><tr><td>acceptor DNA strand (High FRET)</td><td>Integrated DNA Technologies</td><td>178124895</td><td>5&amp;acute;-d(CGG CCT ATT TCG GAG TTG TAA ACA GAG AT(Cy5)C GCC TTA AAC GTT CGC CTA GAC TAG TCC AAG TAT TGC)</td></tr><tr><td>acceptor DNA strand (Low FRET)</td><td>Integrated DNA Technologies</td><td>177956424</td><td>5&amp;acute;-d(CGG CCT ATT TCG GAG TTG TAA ACA GAG ATC GCC TT(Cy5)A AAC GTT CGC CTA GAC TAG TCC AAG TAT TGC)</td></tr><tr><td>donor DNA strand</td><td>Integrated DNA Technologies</td><td>177951437</td><td>5&amp;acute; -d(GCA ATA CTT GGA CTA GTC TAG GCG AAC GTT TAA GGC GAT CTC TGT TT(Alexa488)A CAA CTC CGA AAT AGG CCG)</td></tr><tr><td>DNA strand (No FRET)</td><td>Integrated DNA Technologies</td><td></td><td>5&amp;acute; -d(CGG CCT ATT TCG GAG TTG TAA ACA GAG ATC GCC TTA AAC GTT CGC CTA GAC TAG TCC AAG TAT TGC)</td></tr><tr><td>thermal cycler</td><td>Eppendorf</td><td>E6331000025</td><td>nexus gradient</td></tr><tr><td>Alexa Fluor 488 C5&amp;nbsp;Maleimide</td><td>Thermo Scientific</td><td>A10254</td><td>termed cyan-green fluorophore in the manuscript</td></tr><tr><td>Alexa Fluor 647 C2&amp;nbsp;Maleimide</td><td>Thermo Scientific</td><td>A20347</td><td>termed far-red fluorophore in the manuscript</td></tr><tr><td>Rhodamine 110</td><td>Sigma-Aldrich</td><td>83695-250MG</td><td></td></tr><tr><td>Rhodamine 101</td><td>Sigma-Aldrich</td><td>83694-500MG</td><td></td></tr><tr><td>LB Broth, Miller</td><td>Fisher Scientific</td><td>BP1426</td><td>For culture of &lt;em&gt;E. coli&lt;/em&gt;</td></tr><tr><td>Ampicillin</td><td>Sigma-Aldrich</td><td>A0166</td><td>Used at 100 &amp;micro;g/mL final concentration in selective LB medium to maintain plasmid selection</td></tr><tr><td>Tetracyline&amp;nbsp;</td><td>Calbiochem</td><td>58346</td><td>Used at 12.5 &amp;micro;g/mL final concentration in selective LB medium to maintain gor (flutathione reductase) mutation in Origami B(DE3) strains to facilitate disulfide bond oxidation</td></tr><tr><td>Kanamycin</td><td>Fisher Scientific</td><td>BP906-5</td><td>Used at 15 &amp;micro;g/mL final concentration in selective LB medium to maintain trxB (rhioredoxin reductase) mutation in B(DE3) stains to facilitate disulfide bond oxidation</td></tr><tr><td>Origami B(DE3) Competent Cells</td><td>Millipore</td><td>70837-3</td><td>Competent &lt;em&gt;E. coli&lt;/em&gt; cells for expression of protein with disulfide bridges</td></tr><tr><td>Isopropyl-&amp;beta;-D-thiogalactopyranoside (IPTG)</td><td>Fisher Scientific</td><td>BP1755</td><td>For induction of &lt;em&gt;E. coli&lt;/em&gt; protein expression</td></tr><tr><td>HiTrap Chelating HP</td><td>GE Life Sciences</td><td>17-0409-01</td><td>For Large-scale FPLC Purification of His-tagged protein</td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich</td><td>56749</td><td></td></tr><tr><td>Ni-NTA Agarose&amp;nbsp;</td><td>Qiagen</td><td>30210</td><td></td></tr><tr><td>PD-10 Desalting Column</td><td>GE Life Sciences</td><td>17-0851-01</td><td></td></tr><tr><td>AktaPurifier</td><td>GE Life Sciences</td><td>28406264</td><td>FPLC Instrument</td></tr><tr><td>Dialysis tubing</td><td>Spectrum labs</td><td>132562</td><td>15 kD MWCO 24 mm Flath width, 10 meters/roll</td></tr><tr><td>Dichroics</td><td>Semrock</td><td>FF500/646-Di01-25x36</td><td>500/646 BrightLight</td></tr><tr><td>50/50 Beam splitter polarizer</td><td>Qioptiq Linos&amp;nbsp;</td><td>G33 5743 000</td><td>10 x 10 film polarizer</td></tr><tr><td>Green pass filer</td><td>Chroma</td><td>ET525/50m</td><td>ET525/50m 25 mm diameter mount</td></tr><tr><td>Red pass filter</td><td>Chroma</td><td>ET720/150m</td><td>ET720/150m 25 mm diameter mount</td></tr><tr><td>Power Meter</td><td>ThorLabd</td><td>PM200</td><td></td></tr><tr><td>UV-Vis spectrophotometer</td><td>Varian</td><td>Cary300Bio</td><td></td></tr><tr><td>Fluorolog 3 fluorometer</td><td>Horiba</td><td>FL3-22-R3</td><td></td></tr><tr><td>Fluorohub TCSPC controller</td><td>Horiba</td><td>Fluorohub-B</td><td>TCSPC electronics for ensemble measurements</td></tr><tr><td>NanoLed 485L</td><td>Horiba</td><td>485L</td><td>Blue diode laser</td></tr><tr><td>NanoLed 635L</td><td>Horiba</td><td>635L</td><td>Red diode laser</td></tr><tr><td>Olympus IX73 Microscope</td><td>Olympus</td><td>IX73P2F</td><td>Microscope frame</td></tr><tr><td>PMA 40 Hybrid Detector</td><td>PicoQuant GmbH</td><td>932200, PMA 40</td><td>Optimized for green detection</td></tr><tr><td>PMA 50 Hybrid Detector</td><td>PicoQuant GmbH</td><td>932201, PMA 50</td><td>Optimized for ed shifter sensitivity</td></tr><tr><td>485 nm laser</td><td>PicoQuant GmbH</td><td>LDH-D-C-485</td><td></td></tr><tr><td>640 nm laser</td><td>PicoQuant GmbH</td><td>LDH-D-C-640</td><td></td></tr><tr><td>Hydraharp 400 and TTTR acqusition software</td><td>PicoQuant</td><td>930021</td><td>Picosecond event timer and Time Correlated Single Photon Coutning Unit, includes TTTR acqusition software</td></tr><tr><td>SEPIA II SLM 828 and SEPIA software</td><td>PicoQuant</td><td>910028</td><td>Laser driver for picosecond pulses , includes SEPIA software controller.</td></tr><tr><td>computer</td><td>Dell</td><td>optiplex 7010</td><td>cpu: i7-3770 ram:16GB</td></tr><tr><td>FRET Positioning and Screening (FPS) software</td><td>Heinrich Heine Unviersity</td><td></td><td>It include the Accesibel Volume clacualtor available at http://www.mpc.hhu.de/software/fps.html</td></tr><tr><td>MFD suite</td><td>Heinrich Heine Unviersity</td><td></td><td>It includes the BIFL software package Paris; Margarita for visualization of the multiparameter hisotrams, and Probability Distribution Analysis software availabel at http://www.mpc.hhu.de/software/software-package.html</td></tr></tbody></table>

high precision fret at single-molecule level for biomolecule structure determination

这里介绍了如何在多参数荧光检测（MFD）模式下以单分子水平进行Förster共振能量转移（FRET）进行高精度间距距离测量的协议。 MFD最大化荧光的所有"尺寸"的使用，以减少光物理和实验伪像，并允许在刚性生物分子中以高达±1的精度测量间距距离。该方法用于鉴定N-甲基-D-天冬氨酸（NMDA）受体的配体结合结构域的三个构象状态，以解释配体结合时受体的活化。当将已知的结晶结构与实验测量结果进行比较时，对于更多的动态生物分子，他们认为不到3Å。收集覆盖生物分子的整体维度的一套距离约束将使得有可能提供动态生物分子的结构模型ES。

在使用MFD设置（激光线:485nm在60μW和640nm，23μW，5.1节）的典型smFRET实验中，将荧光样品稀释至低皮摩尔浓度（10 -12M = 1pM）并放置在共焦显微镜中，亚纳秒激光脉冲激发标记分子通过激发体自由扩散。典型的共焦体积&lt;4毫升（fL）。在这样低的浓度下，一次只能检测到一个分子。通过目标收集来自标记分子的发射荧光，并使用针孔进行空间过滤。此步骤定义了有效的共聚?...

Watch this Scientific Journal Video about High Precision FRET at Single-molecule Level for Biomolecule Structure Determination at JoVE.com

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

本文介绍了单分子水平高精度FRET实验方案。此外，该方法可用于鉴定N-甲基-D-天冬氨酸（NMDA）受体的配体结合结构域中的三种构象状态。确定精确的距离是建立基于FRET实验的结构模型的第一步。

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 ...

high-precision-fret-at-single-molecule-level-for-biomolecule

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

10.6K 次观看.  Clemson University. 本文介绍了单分子水平高精度FRET实验方案。此外，该方法可用于鉴定N-甲基-D-天冬氨酸（NMDA）受体的配体结合结构域中的三种构象状态。确定精确的距离是建立基于FRET实验的结构模型的第一步。 

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

Structural Information from Single-molecule FRET Experiments Using the Fast Nano-positioning System

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) can be used to obtain structural information on biomolecular complexes in real-time. Thereby, multiple smFRET measurements are used to localize an unknown dye position inside a protein complex by means of trilateration. In order to obtain quantitative information, the Nano-Positioning System (NPS) uses probabilistic data analysis to combine structural information from X-ray crystallography with single-molecule fluorescence data to calculate not only the most probable position but the complete three-dimensional probability distribution, termed posterior, which indicates the experimental uncertainty. The concept was generalized for the analysis of smFRET networks containing numerous dye molecules. The latest version of NPS, Fast-NPS, features a new algorithm using Bayesian parameter estimation based on Markov Chain Monte Carlo sampling and parallel tempering that allows for the analysis of large smFRET networks in a comparably short time. Moreover, Fast-NPS allows the calculation of the posterior by choosing one of five different models for each dye, that account for the different spatial and orientational behavior exhibited by the dye molecules due to their local environment.
Here we present a detailed protocol for obtaining smFRET data and applying the Fast-NPS. We provide detailed instructions for the acquisition of the three input parameters of Fast-NPS: the smFRET values, as well as the quantum yield and anisotropy of the dye molecules. Recently, the NPS has been used to elucidate the architecture of an archaeal open promotor complex. This data is used to demonstrate the influence of the five different dye models on the posterior distribution.

We present the setup and experimental procedure to obtain smFRET data from large donor-acceptor networks with a TIRF microscope. The step-by-step analysis of these measurements with the Bayesian inference software Fast-NPS yields high-resolved structural information via the application of adapted dye models.

从单分子结构信息FRET实验使用快速纳米定位系统

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) can be used to obtain structural information on biomolecular complexes in real-time. ...

科研

生物化学

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. Atomic force microscopy (AFM) can address this problem by enabling stretching and relaxation of single protein molecules, which gives direct evidence of specific unfolding and refolding characteristics. AFM-based single-molecule force-spectroscopy (AFM-SMFS) provides a means to consistently measure high-energy conformations in proteins that are not possible in traditional bulk (biochemical) measurements. Although numerous papers were published to show principles of AFM-SMFS, it is not easy to conduct SMFS experiments due to a lack of an exhaustively complete protocol. In this study, we briefly illustrate the principles of AFM and extensively detail the protocols, procedures, and data analysis as a guideline to achieve good results from SMFS experiments. We demonstrate representative SMFS results of single protein mechanical unfolding measurements and we provide troubleshooting strategies for some commonly encountered problems.

We describe the detailed procedures and strategies to measure the mechanical properties and mechanical unfolding pathways of single protein molecules using an atomic force microscope. We also show representative results as a reference for selection and justification of good single protein molecule recordings.

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. ...

Investigating Single Molecule Adhesion by Atomic Force Spectroscopy

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. At the same time the AFM tip is passivated to prevent unspecific interactions between the tip and the substrate, which is a prerequisite to study single molecules attached to the AFM tip. Analyses to determine the adhesion force, the adhesion length, and the free energy of these molecules on solid surfaces and bio-interfaces are shortly presented and external references for further reading are provided. Example molecules are the poly(amino acid) polytyrosine, the graft polymer PI-g-PS and the phospholipid POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine). These molecules are desorbed from different surfaces like CH3-SAMs, hydrogen terminated diamond and supported lipid bilayers under various solvent conditions. Finally, the advantages of force spectroscopic single molecule experiments are discussed including means to decide if truly a single molecule has been studied in the experiment.

A protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. Procedures and examples to determine the adhesion force and free energy of these molecules on solid supports and bio-interfaces are provided.

调查单分子粘附的原子力谱

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single ...

工程学

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in time-traces that report on biomolecular dynamics with a broad range of timescales from milliseconds to minutes. To facilitate the acquisition of large number of traces for statistical analyses, the process must be automated and the sample environment should be tightly controlled over the entire measurement time (~12 hours). This is accomplished using an automated scanning confocal microscope that allows the interrogation of thousands of single molecules overnight, and a microfluidic cell that permits the controlled exchange of buffer, with restricted oxygen content and maintains a constant temperature throughout the entire measuring period. Here we show how to assemble the microfluidic device and how to activate its surface for DNA immobilization. Then we explain how to prepare a buffer to maximize the photostability and lifetime of the fluorophores. Finally, we show the steps involved in preparing the setup for the automated acquisition of time-resolved single molecule FRET traces of DNA molecules.

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

表面固定生物分子的单分子荧光检测的自动化系统

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

生物学

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes measurements on freely diffusing biomolecules more accessible. This overview includes a step-by-step protocol for using this instrument to make measurements of precise FRET efficiencies in duplex DNA samples, including details of the sample preparation, instrument setup and alignment, data acquisition, and complete analysis routines. The presented approach, which includes how to determine all the correction factors required for accurate FRET-derived distance measurements, builds on a large body of recent collaborative work across the FRET Community, which aims to establish standard protocols and analysis approaches. This protocol, which is easily adaptable to a range of biomolecular systems, adds to the growing efforts in democratising smFRET for the wider scientific community.

This article provides step-by-step instructions for making fully-corrected accurate FRET measurements on individual, freely diffusing biomolecules using the open-source, inexpensive smfBox, from switch on, through alignment and focusing, to data collection and analysis.

使用开源 smfBox 进行精确和准确的单分子 FRET 测量

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes ...

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One &#945;-Synuclein Monomer at a Time

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural dynamics and its contributions to biology. While the FRET-based ruler reports on inter-dye distances in the 3-10 nm range, other spectroscopic techniques, such as protein-induced fluorescence enhancement (PIFE), report on the proximity between a dye and a protein surface in the shorter 0-3 nm range. Regardless of the method of choice, its use in measuring freely-diffusing biomolecules one at a time retrieves histograms of the experimental parameter yielding separate centrally-distributed sub-populations of biomolecules, where each sub-population represents either a single conformation that stayed unchanged within milliseconds, or multiple conformations that interconvert much faster than milliseconds, and hence an averaged-out sub-population. In single-molecule FRET, where the reported parameter in histograms is the inter-dye FRET efficiency, an intrinsically disordered protein, such as the &#945;-Synuclein monomer in buffer, was previously reported as exhibiting a single averaged-out sub-population of multiple conformations interconverting rapidly. While these past findings depend on the 3-10 nm range of the FRET-based ruler, we sought to put this protein to the test using single-molecule PIFE, where we track the fluorescence lifetime of site-specific sCy3-labeled &#945;-Synuclein proteins one at a time. Interestingly, using this shorter range spectroscopic proximity sensor, sCy3-labeled &#945;-Synuclein exhibits several lifetime sub-populations with distinctly different mean lifetimes that interconvert in 10-100 ms. These results show that while &#945;-Synuclein might be disordered globally, it nonetheless attains stable local structures. In summary, in this work we highlight the advantage of using different spectroscopic proximity sensors that track local or global structural changes one biomolecule at a time.

Time-resolved single-molecule protein-induced fluorescence enhancement is a useful fluorescence spectroscopic proximity sensor sensitive to local structural changes in proteins. Here we show it can be used to uncover stable local conformations in &#945;-Synuclein, which is otherwise known as globularly unstructured and unstable when measured using the longer range FRET ruler.

利用时间分辨蛋白诱导的荧光增强来鉴定稳定的局部构象，一次一α-突触核蛋白单体

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural ...

Picometer-Precision Atomic Position Tracking through Electron Microscopy

The modern aberration-corrected scanning transmission electron microscopes (AC-STEM) have successfully achieved direct visualization of atomic columns with sub-angstrom resolution. With this significant progress, advanced image quantification and analysis are still at the early stages. In this work, we present the complete pathway for the metrology of atomic resolution scanning transmission electron microscopy (STEM) images. This includes (1) tips for acquiring high-quality STEM images; (2) denoising and drift-correction for enhancing measurement accuracy; (3) obtaining initial atomic positions; (4) indexing the atoms based on unit cell vectors; (5) quantifying the atom column positions with either 2D-Gaussian single peak fitting or (6) multi-peak fitting routines for slightly overlapping atomic columns; (7) quantification of lattice distortion/strain within the crystal structures or at the defects/interfaces where the lattice periodicity is disrupted; and (8) some common methods to visualize and present the analysis.
Furthermore, a simple self-developed free MATLAB app (EASY-STEM) with a graphical user interface (GUI) will be presented. The GUI can assist in the analysis of STEM images without the need for writing dedicated analysis code or software. The advanced data analysis methods presented here can be applied for the local quantification of defect relaxations, local structural distortions, local phase transformations, and non-centrosymmetry in a wide range of materials.

This work presents a workflow for atomic position tracking in atomic resolution transmission electron microscopy imaging. This workflow is performed using an open-source Matlab app (EASY-STEM).

通过电子显微镜进行精确原子位置跟踪

The modern aberration-corrected scanning transmission electron microscopes (AC-STEM) have successfully achieved direct visualization of atomic columns ...

化学

Time-Resolved Fluorescence Anisotropy from Single Molecules for Characterizing Local Flexibility in Biomolecules

We describe a protocol for conducting time-resolved fluorescence anisotropy at the single-molecule level using confocal microscopy to investigate the local flexibility and dynamics of the deoxyribonucleic acid (DNA)-binding forkhead (FKH) domain of the FoxP1 transcription factor. FoxP1 dimerizes through a three-dimensional domain-swapping (3D-DS) mechanism, forming a disordered intermediate with or without DNA. Since 3D-DS involves an intrinsically disordered region, understanding its behavior is crucial for elucidating the structural and functional properties of FoxP1. Using a single-cysteine-labeled FoxP1, we conducted single-molecule fluorescence anisotropy (smFA) experiments, applying dynamic anisotropy Photon Distribution Analysis (daPDA) and time-resolved anisotropy Burst Variance Analysis (traBVA) approaches to probe local flexibility and dynamics. This protocol provides a detailed, step-by-step guide for smFA measurements, emphasizing time-resolved analyses, variance, and probability distribution techniques to capture structural dynamics across different timescales. This approach enabled us to relate dynamics and heterogeneity to FoxP1 dimerization and DNA binding, highlighting the complex action mechanism that characterizes this transcription factor.

Here, we present the protocol to study the local flexibility and dynamics of biomolecules using time-resolved fluorescence anisotropy at the single-molecule level in confocal microscopy mode.

We describe a protocol for conducting time-resolved fluorescence anisotropy at the single-molecule level using confocal microscopy to investigate the ...

Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes

Single-molecule F&#246;rster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. It has been used in a wide range of biophysical and molecular biological experiments, including the measurement of molecular forces, characterization of conformational dynamics of biomolecules, observation of intracellular colocalization of proteins, and determination of receptor-ligand interaction times. In a widefield microscopy configuration, experiments are typically performed using surface-immobilized probes. Here, a method combining single-molecule tracking with alternating excitation (ALEX) smFRET experiments is presented, permitting the acquisition of smFRET time traces of surface-bound, yet mobile probes in plasma membranes or glass-supported lipid bilayers. For the analysis of recorded data, an automated, open-source software collection was developed supporting (i) the localization of fluorescent signals, (ii) single-particle tracking, (iii) determination of FRET-related quantities including correction factors, (iv) stringent verification of smFRET traces, and (v) intuitive presentation of the results. The generated data can conveniently be used as input for further exploration via specialized software, e.g., for the assessment of the diffusional behavior of probes or the investigation of FRET transitions.

This article presents a method for spatiotemporal analysis of mobile, single-molecule F&#246;rster resonance energy transfer (smFRET)-based probes using widefield fluorescence microscopy. The newly developed software toolkit allows the determination of smFRET time traces of moving probes, including the correct FRET efficiency and the molecular positions, as functions of time.

移动式单分子FRET探头的自动化二维时空分析

Single-molecule F&#246;rster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. ...

生物工程

Super-resolution Imaging of the Bacterial Division Machinery

Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell1,2. Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan3,4. The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator5,6. This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (~200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus cells7, but is difficult to apply to larger cells such as E. coli or B. subtilis. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli Z-ring8.
	PALM imaging offers both high spatial resolution (~35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm9. During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with &lt;20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules.
	Using this method, we found that the Z-ring has a fixed width of ~100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring10 and can be applied to other proteins of interest.

We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.

超分辨率成像细菌科机械

Bacterial cell division requires the coordinated  assembly of more than ten essential proteins at midcell1,2. Central  to this process is the formation of ...