The authors thank B. Gruchmann for the mechanical drawings of the flow chamber. Further, we want to express our gratitude to Max Beckers and Florian Drechsler for insightful comments and discussions regarding NPS and the underlying sampling engine.

<ol>
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The authors have nothing to disclose.

我们目前的设置和实验程序准确地确定通过挠性接头到生物大分子， 例如 ，核酸和/或蛋白质附着染料之间的FRET效率。 为了确保精确的smFRET测量（第3节），关键的是在测量过程中的任何时间，以排除从流动室的空气。此外，确保不超载荧光流室。荧光团必须明确区分开来，以确保正确的分析。作为smFRET对，其不显示供体的漂白必须从分析中排除，确保在视场中的分子的&gt; 80％的在电影结束漂白。考虑到样品的β因子和γ-因子中的不均匀性，纠正供体和受体通道的串扰和相对的检测效率，分别计算每个FRET对个别。 有关荧光光谱仪（第7至9）的测量的信号强度与已记录的数据的光谱分辨率之间的良好折衷有被发现。为此在荧光光谱仪的激发和发射通路狭缝必须适应依赖于所使用的仪器和样品的浓度。 此外，我们目前的快速NPS分析方法来获得短暂的或动态MACROM的结构信息olecular复合物。 NPS已应用于以显示非模板DNA链的路径和转录起始因子在古细菌RNA聚合酶开放的复杂的位置。使用超过60种不同的距离测量网络，我们发现，快速NPS，配备了新实施的采样引擎（Eilert，T.，贝克尔，M.，德雷克斯勒，F.，与米氏，J.筹建中），减少了由幅度≈2订单所需此复杂smFRET网络的分析，由于相对于原来的全球NPS方法27的时间。该算法的鲁棒性是植根于都市中之吉布斯采样与并行回火方案相结合。快速NPS显示确切的网络结果的可重复性并与早期30日公布的结果一致。 几种不同的方法已经发表，旨在从smFRET测量11推断的结构信息</s向上&gt;，12，13，14，15，16，17，18。所有这些方法只提供一个特定染料模型。由此，染料，即不符合由各个模型所做的假设，不能使用，或者导致错误的结构信息。快速NPS中，与此相反，可以选择每个染料分子的不同的模型。这有助于解释两者的不同构象的行为时，染料分子本身，以及用于它的连接的连接体。染料分子的局部分子的环境中，以及它的物理性质将决定哪种模式是最合适的。 为古细菌起始复合物的分析smFRET网络，对于所有染料分子的各向同性假定导致急剧下降我n作为相比于经典模型的可信卷的大小。在具有动态位置平均为所有染料组合分子所有可信体积尺寸的中位数（95％）降低到小于0.5nm 3。然而，这些染料分子后验不再符合其smFRET测量，表明假设而导致虚假的结构信息。与此相反，在经典模型所确定的后验是与确定smFRET效率一致。 由于各向同性和/或动态位置求得所有染料的假设导致不一致，快速NPS使其中每个染料可分配的五款车型之一染料分子前科。每个型号都使用相同的访问卷。用于染料的AV计算的算法做了几个假设。首先，荧光团的空间形状由球形近似。因此，一个直径考虑到荧光团的WID次，高度和厚度应当使用（第12节）。此外，连接体的形状是由一柔性杆近似。第12节给出的数值计算了染料Alexa的647通过12-C接头附着。迄今为止，这是不可能精确地确定先验哪种模式是最适合给定的实验的几何形状，并且因此所有的模型进行测试。在一般情况下，人会选择这使可能的最小后尺寸的模型，同时仍然与数据一致。要测试的车型可以选择是否与smFRET数据是一致的，我们都计算后和可能性。一致性意味着90％以上的从后回收的样品中是可能性的95％置信区间内。 虽然这是事实，该染料分子的各向异性低，较小的距离的不确定性，在一个smFRET网络几何安排也必须加以考虑。因而人，而representing染料分子与一个iso模型低荧光各向异性是典型的首选，一致性检验提供了选择正确的染料模型更直接的手段。染料模型的最佳选择可以导致定位精度急剧增加，并在同一时间保持与它的FRET数据网络的一致性。 概括地说，快速NPS允许获得大的大分子复合物的结构和动态信息。在对比共同的结构的方法，如X射线晶体学或低温电子显微镜这允许监测高度灵活的或瞬态络合物，从而大大扩大了复杂的生物过程的机理的理解。

确定的生物分子的结构对于理解其功能的重要前提。两个用于结构测定行之有效的方法是低温电子显微镜和X-射线晶体学1,2。如今，这两种方法提供一个分辨率下到埃级高分辨率的结构信息。这两种方法已被广泛用来阐明大生物分子的结构，例如蛋白质复合物。虽然现有的方法都不断被整个过去的几十年改善，生物结构的复杂性，仍对结构生物学的一大挑战，尤其是当大，动态和短暂络合物进行了研究3。 为了研究大分子复合物的动力学，特别是结构与功能的关系，单分子的方法有省被提供的有用信息4。一些新的战略被开发提供有关收购的结构和动态信息的正交方式。实例是高速AFM 5，机械操作6，荧光定位显微镜7，以及单分子荧光共振能量转移（smFRET）8,9。由于相当早就FRET已称为分子尺 ，由于对生物大分子10的长度尺度的距离依赖性。 smFRET的一个特别有趣的应用是使用从smFRET测量获得的距离信息来推断结构信息11，12，13，14，15 </sup&gt;，16，17，18，19，20，21，22，23。由于smFRET的高时间分辨率，蛋白质结构的移动部分的位置可以本地化。然而，为了从约染料分子smFRET数据重要校正参数提取量化信息所需要的测量24中被确定。与这些校正因子，FRET的效率E FRET可以使用公式来计算<img alt="式（1）" src="/files/ftp_upload/54782/54782eq1.jpg" /> ， 在那里我 A和I D </sUB&gt;分别是供体和受体分子，的荧光强度（参见图2）。的β因子占串扰，供体发射的漏入受体信道和由下式计算<img alt="公式（2）" src="/files/ftp_upload/54782/54782eq2.jpg" />在那里我是A和I&#39;D是供体的荧光强度和受体分子的光漂白后的受体分子。 的γ因子校正两个通道中的相对探测效率的差异，以及在该供体的荧光量子产率和受体染料的差异。它是由每个人的时间跟踪的计算方法请注意，本说明书中忽略了受体分子，其有时变得很重要，并需要为待校正以及直接激励。用于确定这些修正因子是有用的，以便光的物理变化和结构动力学区分以激励这两个供体以及受体以交替的方案25。 为了不仅获得定量smFRET效率而且定量的结构信息，所述纳米定位系统（NPS）于2008年26被引入。名称是根据其相似性基于卫星的全球定位系统（GPS）的选择。该NPS是结合smFRET和X射线晶体学数据在生物大分子复合物不明染料位置的定位的混合技术。的Crystal结构用作基准帧和smFRET结果用于得到未知的荧光团的位置（ 天线 ），并从晶体结构（ 卫星 ）已知的位置之间的距离信息。在连续实验中，天线和多个卫星之间的距离被测量和天线的位置是由基于贝叶斯参数估计统计学上严格的分析方案的方法来确定。其结果，不仅天线的最可能的位置被计算，但其完整的3D不确定性分布，即所谓的后，由可信卷可视化。此外，NPS扩大到允许完整smFRET网络27的分析。 所述NPS已被用来解决一些在真核转录的重要问题，上游的DNA，非模板DNA和RNA聚合酶II延伸共内新生mRNA的即过程mplex 12，28，也显示出转录起始的影响因素26和一个开放式启动子的动态建筑群29。此外，NPS是用来阐明古细菌的RNA聚合酶开放的复杂30转录起始因子TFE的位置，这竞争结合相同位点的转录延伸因子Spt4 / 5 31的，特别是结构。 从那时起，许多基于smFRET结构方法已经发表15，18，21，23。当比较不同的基于smFRET结构的方法，它变得清晰，该方法的明显的精度在很大程度上取决于染料模型的特定选择。应该注意的是染料分子可能根据当地环境表现出不同的空间和取向行为。 为此，快NPS引入32。快速NPS采用了先进的采样算法大大减少计算时间。此外，快速NPS允许一个执行结构分析，并为每个染料分子，用户可以从一组五个不同染料的模型将在下面描述选择。最保守的模式，堪称经典之作，假设染料只占用一个，但不明，地位。在该位置，所述荧光团可以一个锥体，其尺寸从其相应（时间依赖性）荧光各向异性测定内自由转动。圆锥体的取向是未知的，转换测量smFRET效率成距离时导致大的不确定性。在这方面，该模型是保守的，因为这将导致最小精度相比，其他染料模式LS。只有很短的距离应该由经典机型率先作出一个明显不正确的位置确定的假设。对于典型smFRET价值观，正确的立场是始终封闭在较大音量可信。 然而，由于较高的精度是可取的，它开发和测试的替代染料模型，这可能有助于改善精度是重要的。如果染料旋转比其固有的荧光寿命快得多，所谓异模型可以应用。这里，取向因子κ2（所需要的计算特性各向同性福斯特半径<img alt="式（1）" src="/files/ftp_upload/54782/54782eq4.jpg" /> ）设定为2/3。其结果是，相比那些在经典机型的32可信计算量几乎是两个数量级较小。在于不仅能够快速reori该荧光团是在环境中发现的情况下entation，但另外的快速运动都在其访问的卷，应使用meanpos异模型。在此模型中，将染料有效地只占用一个平均位置，其中所述空间平均是由一个多项式距离转换15占。如果例如（通常疏水的）染料附着到亲水性区域， 例如，该DNA该模型适用。所述meanpos异模型的应用导致通过大约两个因素中的可信卷的尺寸进一步减小。然而，联系到蛋白质的染料可能会可逆地结合在其空间上访问的卷（AV）数的疏水补丁。这些区域之间的瞬时开关，但在一个区域发生自由转动和快速的局部运动，最好由无功meanpos异模型中描述的荧光团。对于类似的情况，其中染料是不能自由旋转VAR-meanpos模式适用。多个D-<html>有关这些车型etails可以在我们最近发表的32被发现。 这些模型提供了专门占了染料可能遇到的各种环境和应用它们明智地优化其定位精度丰富的剧目。在快速NPS附加到特定位置每染料分子可以被分配到一个单独的模式，使得FRET的伙伴被允许有不同的型号。这使得无限的贴近自然造型。然而，重要的是，一个执行严格的统计测试，以确保由最终模型组合获得的结果仍然与实验数据一致。这些测试包括在快速NPS软件。 为了快速NPS适用于实验数据是必需的（只）三个输入参数的测量。首先，该染料对特定各向同性福斯特半径（/54782/54782eq5.jpg"/&gt;）已被确定，因此，供体染料的量子产率（QY），供体荧光发射光谱和受体的吸收光谱需要测量，这些测量可以在进行散装，使用标准的光谱仪和一个标准的荧光光谱。对于每一对，则R 0然后使用免费软件PhotochemCAD计算，并且可以在NPS分析中使用。此外，（时间分辨）染料分子的荧光各向异性需要使用一个极化（和时间）敏感荧光光谱仪来获得。然而，对于快速NPS的最重要的输入参数是在单分子荧光显微镜的设置测量的smFRET效率，如一个全内反射荧光显微镜（TIRFM） 。 在这里，我们提出了一个一步一步的协议获得smFRET数据和应用快速NPS（ 图1）。

<table border="0" cellpadding="0" cellspacing="0" width="789">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Flowchamber preparation</td>
			<td style="width:167px;"></td>
			<td style="width:155px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Customized metall sample holder</td>
			<td style="width:167px;">self-built</td>
			<td style="width:155px;">n/a</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">quartz-glass slides, 76 x 26 mm</td>
			<td style="width:167px;">Technical Glass Products</td>
			<td align="right">26007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">coverslips, 60 x 24 mm</td>
			<td style="width:167px;">Marienfeld</td>
			<td align="right">101242</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">detergent, Hellmanex II</td>
			<td style="width:167px;">Hellma</td>
			<td align="right" style="width:155px;">320.001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">ultra-pure water from Synergy UV</td>
			<td style="width:167px;">Millipore</td>
			<td align="right">2512600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zepto plasma cleaner</td>
			<td style="width:167px;">Diener</td>
			<td style="width:155px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">(3-aminopropyl)-triethoxysilane, p.a.</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:155px;">A3648</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">methoxy PEG-succinimidyl valerate, 5 kDa</td>
			<td style="width:167px;">Laysan Bio Inc.</td>
			<td>MPEG-SVA-5000-1g</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">biotinylated PEG-succinimidyl valerate, 5 kDa</td>
			<td style="width:167px;">Laysan Bio Inc.</td>
			<td>BIOTIN-PEG-SVA-5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Sodium biocarbonate</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td>S5761 Sigma&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Sodium carbonate</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td>S2127 Sigma-Aldrich&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">sealing film (Nescofilm)</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td align="right">12981805</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">Tygon Flexible Silicone Tubing, 0.8 mm ID, 2.4 mm OD</td>
			<td style="width:167px;">Saint-Gobain Performance Plastics</td>
			<td align="right">720958</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">Fine-Bore Polyethylene Tubing, 0.58 mm ID, 0.96 mm OD (Smiths Medical)</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td align="right">12665497</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Neutravidin</td>
			<td style="width:167px;">Life Technologies</td>
			<td style="width:155px;">A2666</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Name</td>
			<td style="width:167px;">Company</td>
			<td style="width:155px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Total internal reflection fluorescence microscope</td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">Nd:YAG Laser, 532 nm</td>
			<td style="width:167px;">Newport Spectra-Physics</td>
			<td style="width:155px;">EXLSR-532-100-CDRH</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">diode-pumped solid-state laser, 491 nm, Calypso</td>
			<td style="width:167px;">Cobolt</td>
			<td align="right" style="width:155px;">904010050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">diode laser 643 nm, iBeam smart</td>
			<td style="width:167px;">Toptica</td>
			<td style="width:155px;">iBEAM-SMART-640-S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">dichroic mirror, 532 RDC</td>
			<td style="width:167px;">Chroma</td>
			<td style="width:155px;">F33-540</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">dichroic mirror, 476 RDC</td>
			<td style="width:167px;">Chroma</td>
			<td style="width:155px;">F33-476z</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">acousto-optic tunable filter</td>
			<td style="width:167px;">AA Opto-Electronic</td>
			<td style="width:155px;">AOTFnC-VIS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">plano-convex cylindrical lens, f = 75 mm</td>
			<td style="width:167px;">Thorlabs</td>
			<td>LJ1703L1-A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">plano-concave cylindrical, f = -300 mm</td>
			<td style="width:167px;">Thorlabs</td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">prism, PS 991</td>
			<td style="width:167px;">Thorlabs</td>
			<td style="width:155px;">PS991</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">focussing lens, f = 75 mm</td>
			<td style="width:167px;">Thorlabs</td>
			<td>LA1608-B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">syringe pump, PHD 2000</td>
			<td style="width:167px;">Harvard Apparatus</td>
			<td style="width:155px;">70-2002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">2 stepper motors, Z812B</td>
			<td style="width:167px;">Thorlabs</td>
			<td style="width:155px;">Z812B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">piezoelectric actuator, PE4</td>
			<td style="width:167px;">Thorlabs</td>
			<td style="width:155px;">PE4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">IR diode laser</td>
			<td style="width:167px;">Edmund Optics</td>
			<td style="width:155px;">CPS808</td>
			<td>part of the autofocus system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">dichroic mirror, 775 DCXR</td>
			<td style="width:167px;">Chroma</td>
			<td style="width:155px;">775 DCXR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">position-sensing detector (PSD), PDP90A</td>
			<td style="width:167px;">Thorlabs</td>
			<td style="width:155px;">PDP90A</td>
			<td>part of the autofocus system</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">water-immersion objective, Plan Apo 60X WI, NA 1.2</td>
			<td style="width:167px;">Nikon</td>
			<td style="width:155px;"></td>
			<td>MRD07601</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">dichroic mirror, 645 DCXR</td>
			<td style="width:167px;">Chroma</td>
			<td style="width:155px;">645 DCXR</td>
			<td>part of the emission pathway</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">emission filter, 3RD550-510</td>
			<td style="width:167px;">Omega Optical</td>
			<td style="width:155px;">3RD550-510</td>
			<td>green channel in the emission pathway</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">emission filter, 3RD660-760</td>
			<td style="width:167px;">Omega Optical</td>
			<td style="width:155px;">3RD660-760</td>
			<td>red channel in the emission pathway</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">EMCCD camera, iXon+ DU897EBV</td>
			<td style="width:167px;">Andor</td>
			<td style="width:155px;">AND-20-00032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">EMCCD camera, iXon3 DU897D-BV</td>
			<td style="width:167px;">Andor</td>
			<td style="width:155px;">AND-20-000141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Name</td>
			<td style="width:167px;">Company</td>
			<td style="width:155px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Miscellaneous</td>
			<td style="width:167px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Varian 50</td>
			<td style="width:167px;">Cary</td>
			<td style="width:155px;"></td>
			<td>UV-VIS spectrometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Fluorolog2</td>
			<td style="width:167px;">SPEX</td>
			<td style="width:155px;"></td>
			<td>fluorescence spectrometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Solis (V4.15)</td>
			<td style="width:167px;">Andor</td>
			<td style="width:155px;"></td>
			<td>control software for the EM-CCD camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Apt user utility&#160; (V1.022)</td>
			<td style="width:167px;">Thorlabs</td>
			<td style="width:155px;"></td>
			<td>control software for the piezo-motors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Norland Optical Adhesive 68</td>
			<td style="width:167px;">Thorlabs</td>
			<td style="width:155px;"></td>
			<td>adhesive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PC-AFN-0.8 Nile red</td>
			<td>Kisker Biotech</td>
			<td style="width:155px;"></td>
			<td>avidin-coated fluorescent multispec beads&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Matlab</td>
			<td style="width:167px;">Mathworks</td>
			<td style="width:155px;"></td>
			<td>technical computing language for custon written software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Origin (V9.0)</td>
			<td style="width:167px;">Originlab</td>
			<td style="width:155px;"></td>
			<td>scientific graphing and data analysis software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hellma 105-202-15-40</td>
			<td style="width:167px;">Hellma</td>
			<td>105-202-15-40</td>
			<td>absorption cuvette of 1 cm path length</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hellma 105-251-15-40</td>
			<td style="width:167px;">Hellma</td>
			<td>105-251-15-40</td>
			<td>fluorescence cuvette with 3 mm path length</td>
		</tr>
	</tbody>
</table>

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.

转录基因表达的第一步中的所有生物。在古菌，转录由单个RNA聚合酶（RNAP）进行。相比于真核生物，古细菌RNAP有着惊人的相似结构，同时具有更简单转录机器的真核同行。因此，古细菌可以用作模型系统通过RNA聚合酶II（RNA聚合酶II）来研究真核转录起始。近日，古细菌RNA聚合酶开放的复杂的完整的体系结构已经从单分子荧光共振能量转移和NPS确定。从NPS分析的数据被用来构建完整的古开放的启动子复合物，它提供了有益的见解转录起始机制的典范。 为了阐明这种结构，分别位于开放的启动子COM中未知的天线染料分子之间测量smFRET效率复杂和已在该RNAP，其位置由晶体结构（PDB-ID:2WAQ）是已知的五个参考网站包含了一些已知的卫星染料分子37。天线染料被装上的非模板DNA，TFB，TBP或TFE的不同位置中的任一个。在这项研究中所用的完整网络包括超过60测量的距离。 图7描绘了从NPS分析建完整的古细菌开放启动子复合物的模型。它包括双链启动子的DNA（亮和暗蓝色），该RNA聚合酶（灰色）和转录起始因子TBP（紫色），TFB（绿色）和TFE（黄色）。该模型叠加从NPS分析，可信册，其中采用了经典模型（A），ISO模型（B）计算的，meanpos-ISO模式（C）的VAR-meanpos异模型结果（D）和VAR-meanpos<html>模型（E）。 <img alt="图1" src="/files/ftp_upload/54782/54782fig1.jpg" /> 图1: 所需快速NPS计算参数的采集和处理的工作流程。 <a href="http://ecsource.jove.com/files/ftp_upload/54782/54782fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54782/54782fig2.jpg" /> 图 2: 事件smFRET的示范性荧光强度实时跟踪。 smFRET，II:供体（绿色）和示出了三个特征阶段，即我的受体分子（红色）的荧光强度施主荧光受体漂白后，三:施主漂白后的背景荧光。g2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/54782/54782fig3.jpg" /> 图3: 流动室供smFRET实验的示意图。流室安装到与亚克力玻璃持有人定制的金属支架。流室的夹层设计包括一个石英玻璃（熔融石英）有两个孔用于安装入口和出口管道，密封膜和关闭所述流动室盖玻片滑动。为TIRF照明棱镜被安装到所述流室的下半部。空心螺丝卡提供流室入口和出口。 <a href="http://ecsource.jove.com/files/ftp_upload/54782/54782fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img<html> ALT ="图4"SRC ="/文件/ ftp_upload / 54782 / 54782fig4.jpg"/&gt; 图4: 在石英玻璃载玻片和密封膜的制备。石英玻璃载玻片的机械制图表示的孔的位置（以毫米为单位给出）。 <a href="http://ecsource.jove.com/files/ftp_upload/54782/54782fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54782/54782fig5.jpg" /> 图5: 流动室机械制图。对于铝棱镜支架的措施，有机玻璃支架和铝安装帧毫米给出。 <a href="http://ecsource.jove.com/files/ftp_upload/54782/54782fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> igure 6"SRC ="/文件/ ftp_upload / 54782 / 54782fig6.jpg"/&gt; 图6: 用于smFRET实验棱柱型的TIRF设置的示意图。缩写光学元件:A，孔; DM，分色镜;楼发射滤波器; L，镜头;男，镜; 0，客观的; P，棱镜; PSD中，位置敏感光电二极管; S，样品; PS，定位阶段; T，望远镜。 <a href="http://ecsource.jove.com/files/ftp_upload/54782/54782fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/54782/54782fig7.jpg" /> 图7: 不同的模型假设的仿真结果。所有的图片显示的古细菌RNA聚合酶（PDB ID:2WAQ，顶视图）与模型为启动子DNA（分别TDNA和ntDNA在蓝色和青色，）一起，TBP（紫色），TFB（绿色）和TFE（黄色）在古开复30。可信卷叠加的（A）的经典模型，（B）ISO模型，（ 三 ）meanpos异型号，（ 四 ）VAR-meanpos异模型和（E）变种的NPS模拟结果-meanpos模式。所有卷都在68％可信度所示。经典和VAR-meanpos网络与smFRET数据一致。相反，在所有染料选择了ISO，meanpos异或VAR-meanpos异模型网络与实测数据不一致。 <a href="http://ecsource.jove.com/files/ftp_upload/54782/54782fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Structural Information from Single-molecule FRET Experiments Using the Fast Nano-positioning System at JoVE.com

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

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. ...

structural-information-from-single-molecule-fret-experiments-using

Research

JoVE Journal

Biochemistry

12.1K 次观看.  Ulm University. 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.

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

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

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

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

单分子水平的高精度FRET用于生物分子结构测定

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

科研

生物化学

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together&#160;with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail.
Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates.
By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.

Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated interactions.

应用三色单分子焦虑研究蛋白质相互作用的相关性

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

单分子力谱中蛋白质共价固定化的研究

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy

The field of cryo-electron microscopy (cryo-EM) is rapidly developing with new hardware and processing algorithms, producing higher resolution structures and information on more challenging systems. Sample preparation for cryo-EM is undergoing a similar revolution with new approaches being developed to supersede the traditional blotting systems. These include the use of piezo-electric dispensers, pin printing and direct spraying. As a result of these developments, the speed of grid preparation is going from seconds to milliseconds, providing new opportunities, especially in the field of time-resolved cryo-EM where proteins and substrates can be rapidly mixed before plunge freezing, trapping short lived intermediate states. Here we describe, in detail, a standard protocol for making grids on our in-house time-resolved EM device both for standard fast grid preparation and also for time-resolved experiments. The protocol requires a minimum of about 50 &#181;L sample at concentrations of &#8805; 2 mg/mL for the preparation of 4 grids. The delay between sample application and freezing can be as low as 10 ms. One limitation is increased ice thickness at faster speeds and compared to the blotting method. We hope this protocol will aid others in designing their own grid making devices and those interested in designing time-resolved experiments.

Here, we provide a detailed protocol for the use of a rapid grid making device for both fast grid-making and for rapid mixing and freezing to conduct time-resolved experiments.

快速网格准备时间解决低温电子显微镜

The field of cryo-electron microscopy (cryo-EM) is rapidly developing with new hardware and processing algorithms, producing higher resolution structures ...

gP2S, an Information Management System for CryoEM Experiments

Cryogenic electron microscopy (cryoEM) has become an integral part of many drug-discovery projects because crystallography of the protein target is not always achievable and cryoEM provides an alternative means to support structure-based ligand design. When dealing with a large number of distinct projects, and within each project a potentially large number of ligand-protein co-structures, accurate record keeping rapidly becomes challenging. Many experimental parameters are tuned for each target, including at the sample preparation, grid preparation, and microscopy stages. Therefore, accurate record keeping can be crucially important to enable long-term reproducibility, and to facilitate efficient teamwork, especially when steps of the cryoEM workflow are performed by different operators. To help deal with this challenge, we developed a web-based information management system for cryoEM, called gP2S. 
The application keeps track of each experiment, from sample to final atomic model, in the context of projects, a list of which is maintained in the application, or externally in a separate system. User-defined controlled vocabularies of consumables, equipment, protocols and software help describe each step of the cryoEM workflow in a structured manner. gP2S is widely configurable and, depending on the team's needs, may exist as a standalone product or be a part of a broader ecosystem of scientific applications, integrating via REST APIs with project management tools, applications tracking the production of proteins or of small molecules ligands, or applications automating data collection and storage. Users can register details of each grid and microscopy session including key experimental metadata and parameter values, and the lineage of each experimental artifact (sample, grid, microscopy session, map, etc.) is recorded. gP2S serves as a cryoEM experimental workflow organizer that enables accurate record keeping for teams, and is available under an open-source license.

gP2S is a web application for the tracking of cryoEM experiments. Its main features are described, as are the steps required to install and configure the application. Once configured, the application allows one to accurately record metadata associated with negative stain and cryoEM experiments.

gP2S，冷冻实验信息管理系统

Cryogenic electron microscopy (cryoEM) has become an integral part of many drug-discovery projects because crystallography of the protein target is not ...

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 ...

Production of Recombinant PRMT Proteins using the Baculovirus Expression Vector System

Protein arginine methyltransferases (PRMTs) methylate arginine residues on a wide variety of proteins that play roles in numerous cellular processes. PRMTs can either mono- or dimethylate arginine guanidino groups symmetrically or asymmetrically. The enzymology of these proteins is a complex and intensely investigated area that requires milligram quantities of high-quality recombinant protein. The baculovirus expression vector system (BEVS) employing Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Spodoptera frugiperda 9 (Sf9) insect cells has been used for expression screening and production of many PRMTs, including PRMT 1, 2, and 4 through 9. To simultaneously screen for the expression of multiple constructs of these proteins, including domains and truncated fragments as well as the full-length proteins, we have applied scalable methods utilizing adjustable and programmable multichannel pipettes, combined with 24- and 96-well plates and blocks. Overall, these method adjustments enabled a large-scale generation of bacmid DNA, recombinant viruses, and protein expression screening. Using culture vessels with a high-fill volume of Sf9 cell suspension helped to overcome space limitations in the production pipeline for single batch large-scale protein production. Here, we describe detailed protocols for the efficient and cost-effective expression of functional PRMTs for biochemical, biophysical, and structural studies.

The baculovirus expression vector system (BEVS) is a robust platform for expression screening and production of protein arginine methyltransferases (PRMTs) to be used for biochemical, biophysical, and structural studies. Milligram quantities of material can be produced for the majority of PRMTs and other proteins of interest requiring a eukaryotic expression platform.

使用巴库洛病毒表达载体系统生产重组PRMT蛋白

Protein arginine methyltransferases (PRMTs) methylate arginine residues on a wide variety of proteins that play roles in numerous cellular processes. ...

Visualizing the Conformational Dynamics of Membrane Receptors Using Single-Molecule FRET

The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the environment, a cell must be able to recognize and process it. This task mainly relies on the function of membrane receptors, whose role is to convert signals into the biochemical language of the cell. G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptor proteins in humans. Among GPCRs, metabotropic glutamate receptors (mGluRs) are a unique subclass that function as obligate dimers and possess a large extracellular domain that contains the ligand-binding site. Recent advances in structural studies of mGluRs have improved the understanding of their activation process. However, the propagation of large-scale conformational changes through mGluRs during activation and modulation is poorly understood. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to visualize and quantify the structural dynamics of biomolecules at the single-protein level. To visualize the dynamic process of mGluR2 activation, fluorescent conformational sensors based on unnatural amino acid (UAA) incorporation were developed that allowed site-specific protein labeling without perturbation of the native structure of receptors. The protocol described here explains how to perform these experiments, including the novel UAA labeling approach, sample preparation, and smFRET data acquisition and analysis. These strategies are generalizable and can be extended to investigate the conformational dynamics of a variety of membrane proteins.

This study presents a detailed procedure to perform single-molecule fluorescence resonance energy transfer (smFRET) experiments on G protein-coupled receptors (GPCRs) using site-specific labeling via unnatural amino acid (UAA) incorporation. The protocol provides a step-by-step guide for smFRET sample preparation, experiments, and data analysis.

使用单分子FRET可视化膜受体的构象动力学

The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the ...

IDR 结构敏感性的 FRET 分析以研究酵母中的高渗应激

Estimation of Structural Sensitivity of Intrinsically Disordered Regions in Response to Hyperosmotic Stress in Living Cells Using FRET

Intrinsically disordered regions (IDRs) are protein domains that participate in crucial cellular processes. During stress conditions, the physicochemical properties of the cellular environment change, directly impacting the conformational ensemble of IDRs. IDRs are inherently sensitive to environmental perturbations. Studying how the physicochemical properties of the cell regulate the conformational ensemble of IDRs is essential for understanding the environmental control of their function. Here, we describe a step-by-step method for measuring the structural sensitivity of IDRs in living Saccharomyces cerevisiae cells in response to hyperosmotic stress conditions. We present the use of ensemble fluorescence resonance energy transfer (FRET) to estimate how the global dimensions of IDRs change during a progressive increase of hyperosmotic stress imposed on cells with any osmolyte. In addition, we provide a script for processing fluorescence measurements and comparing structural sensitivity for different IDRs. By following this method, researchers can obtain valuable insights into the conformational changes that IDRs undergo in the complex intracellular milieu upon changing environments.

作者聚焦：通过细胞感应和响应解锁固有无序区域的世界

Intrinsically disordered regions (IDRs) are flexible protein domains that modify their conformation in response to environmental changes. Ensemble fluorescence resonance energy transfer (FRET) can estimate protein dimensions under different conditions. We present a FRET approach to assess IDR structural sensitivity in living Saccharomyces cerevisiae cells under hyperosmotic stress.

使用 FRET估计活细胞中响应高渗应激的固有无序区域的结构敏感性

Intrinsically disordered regions (IDRs) are protein domains that participate in crucial cellular processes. During stress conditions, the physicochemical ...