JoVE Journal > Biochemistry
Summary
Abstract
Introduction
Protocol
Risultati
Discussion
Divulgazioni
Acknowledgements
Materials
Riferimenti
Traduzione automatica
Please note that all translations are automatically generated. Click here for the English version.
Biochimica

用于膜蛋白结构分析和从头建模的小角中子散射实验中的对比匹配洗涤剂

Published: October 21, 2018
doi:
10.3791/57901
, , , , ,
1Neutron Scattering Division,Oak Ridge National Laboratory, 2School of Chemistry and Biochemistry,Georgia Institute of Technology

Summary

该协议演示了如何获得一个低分辨率的从头模型和洗涤剂-溶解膜蛋白在溶液中使用小角中子散射与洗涤剂的对比匹配的结构细节。

Abstract

橡树岭国家实验室高通量同位素反应器生物小角中子散射仪致力于生物材料、生物燃料加工和生物启发材料的研究, 涵盖纳米到微米长度刻度。本文介绍了在胶束成型洗涤剂溶液中研究膜蛋白 (这里, MmIAP, intramembrane aspartyl 蛋白酶, Methanoculleus marisnigri) 的物理性质 (即尺寸和形状) 的方法。非常适合这种小角度中子散射仪器, 等等。其他生物物理表征技术由于无法在蛋白质洗涤剂复合结构中处理洗涤剂的贡献而受到阻碍。此外, 对生物氘化实验室的访问提供了独特的能力, 以制备大规模培养和表达氘标记蛋白, 增强散射信号从蛋白质。虽然这种技术不提供高分辨率的结构细节, 但膜蛋白结构知识缺口包含许多可寻址的研究领域, 而无需近原子分辨率。例如, 这些区域包括寡聚状态的测定、复杂的形成、摄动过程中的构象变化和折叠/展开事件。通过这种方法的应用, 可以很容易地完成这些调查。

Introduction

膜蛋白是由估计的30% 的所有基因1编码, 并代表了大多数现代药物的目标。2这些蛋白质执行一系列重要的细胞功能,3但尽管它们的丰度和重要性-仅代表在结构生物信息学 (蛋白质) 蛋白研究磁场中沉积的总结构的约1%数据银行。4由于其部分疏水性, 对膜结合蛋白的结构测定一直是极其具有挑战性的。5,6,7

由于许多生物物理技术需要在溶液中单分散颗粒进行测量, 因此在本机膜中隔离膜蛋白和稳定这些蛋白质是近年来研究的一个活跃领域。几十 年。8,9,10这些调查导致许多新的两亲性组件的发展溶解膜蛋白, 如 nanodiscs,11,12,13 bicelles,14,15和 amphipols。16,17然而, 洗涤剂胶束的使用仍然是满足给定蛋白质溶解度要求的最常见和最直接的方法之一。18,19,20,21,22,23,24,25不幸的是, 目前尚无单一洗涤剂或混合洗涤剂, 满足所有膜蛋白的存在;因此, 这些条件必须经过经验筛选, 以满足每种蛋白质的独特要求。26,27

洗涤剂在其临界胶束浓度以上的溶液中自组装, 形成称为胶束的聚合结构。胶束由许多洗涤剂单体组成 (通常范围从 20-200), 疏水性烷基链形成胶束芯和亲水性头组排列在胶束壳层, 面对水性溶剂。洗涤剂和胶束形成的行为是典型的查尔斯 Tanford 在疏水性作用,28和大小和形状的胶束从常用洗涤剂在膜蛋白研究已采用小角度散射特征。29,30对膜蛋白的洗涤剂组织也进行了研究, 并预期蛋白质洗涤剂复合物 (PDCs) 的形成与洗涤剂分子周围的蛋白质在一个安排, 类似于整洁的洗涤剂胶 束。31

使用洗涤剂的一个优点是, 所产生的胶束特性可以通过结合其他洗涤剂来操作。许多洗涤剂表现出理想的混合, 而混合胶束的选择性能甚至可以从组分和混合比例预测。22然而, 洗涤剂的存在仍然会给整个信号带来生物物理表征的挑战。例如, 使用 X 射线和光散射技术, PDC 中洗涤剂的信号与蛋白质几乎没有区别。32使用单粒子低温电子显微镜 (低温 EM) 进行的调查通常依赖于被困 (冰冻) 粒子;蛋白质的结构细节仍然被某些洗涤剂或高浓度的洗涤剂掩盖, 增加了背景。33通过计算方法来解释整个 PDC 结构 (包括洗涤剂) 的替代方法, 它试图在给定的膜蛋白周围重建洗涤剂。34

在中子散射的情况下, 胶束中洗涤剂的芯壳排列会产生一种有助于观测散射的形状因子。幸运的是, 可以更改解决方案组件, 使其不会对观察到的网络散射造成影响。这种 “对比匹配” 过程是通过用氘代替氢气来实现与背景 (缓冲器) 相匹配的散射长度密度来实现的。明智的选择洗涤剂 (与可用的氘代对应) 和他们的混合比例必须考虑。对于洗涤剂胶束, 这种替代可以使用同一头组的洗涤剂进行, 但具有氘代烷基链 (d-尾部而不是 h 尾)。由于洗涤剂混合良好,35它们的骨料将具有散射长度密度, 这是两个分量 (h 尾和 d 尾) 的摩尔分数加权平均值。当这种平均对比度与头部组一致时, 可以完全匹配均匀骨料结构, 以消除所有对观测到散射的贡献。

我们在这里介绍了一种通过将化学相同的洗涤剂分子与氘标记的烷基链结合在一起来操纵洗涤剂胶束中子对比度的协议。19,36,37这使得胶束核和壳体的同时对比度匹配, 这是一种独特的中子散射能力。35,38有了这一显著细化的细节水平, 对比度匹配可以使膜蛋白结构的其他不可行的研究。此外, 这种对比匹配方法可以扩展到其他涉及洗涤剂的系统, 如聚合物交换反应39和油水分散剂,40甚至其他增溶制剂, 如 bicelles,41nanodiscs,42或嵌段共聚物。43本手稿中概述的类似方法, 但在烷基链和/或头组采用了单一的含氘替代剂, 最近发表。37虽然这可以预期改善氢和氘在整个洗涤剂中的随机分布与此处介绍的方法相比, 在替代品和两个步骤的洗涤剂的可用位置数量有限洗涤剂的合成需要考虑更多的挑战。

下面详细介绍的协议步骤1和2经常重叠, 因为必须进行初步的实验规划才能提交质量建议。然而, 提案提交在这里被认为是第一步, 强调这一过程应该在中子实验之前很好地开始。还应指出的是, 建议应说明的一个先决条件步骤是对需要进行中子研究的样品进行生化和物理表征 (包括纯度和稳定性)。关于小角中子散射 (san) 的一般讨论超出了本文的范围。考夫曼44材料的参考工作表征中提供了一个简短而全面的介绍, 最近出版了一本以生物小角度溶液散射为重点的综合教科书。45在讨论部分给出了进一步建议的阅读。小角度散射使用所谓的散射矢量 Q 作为描述散射过程的中心量。本文使用广泛接受的定义 Q = 4π sin (θ)/λ, 其中θ是入射和散射光束的一半角, λ是埃中子辐射的波长。还存在使用不同符号 (如 “散射矢量”) 的其他定义, 并且可能因2π或使用纳米代替埃而有所不同 (参见图 10的讨论)。

Protocol

1. 准备并提交中子设施光束时间和仪器方案 请查阅在线资源, 以确定提供一般用户中子束时间访问的中子散射设施, 如橡树岭国家实验室 (ORNL)。有关中子设施的地图和有关全球中子研究的信息可在线获得。46请注意, 这些设施通常有定期要求的建议;这将确定下一个光束时间何时可用。中子用于各种应用;搜索小角度中子散射 (san) 仪器, 特别是那些具有生物样品能力的设备。…

Representative Results

光束时间和仪器建议应清楚地将所需的所有资料传达给审查委员会, 以便对所提议的试验进行有效的评估。对于经验不足的用户, 强烈建议与 NSS 进行通信。NSS 可以评估初步可行性并指导提案提交, 以强调可行性、安全性和高影响科学的潜力。建议中提供的资料应包括背景资料和背景, 以说明研究的重要性;预计将获得的知识, 以及这对相关科学领域目前的理解有何影响;将使?…

Discussion

结构生物学研究人员利用互补的结构技术, 如溶液散射, 获得生物化学和结构细节 (如整体大小和形状) 从生物分子在溶液中。san 是一种特别吸引人的技术, 用于确定膜蛋白的低分辨率结构, 这是现代结构生物学和生物化学的核心重点。san 需要与晶体试验 (1 毫克/样品) 相媲美的纯化蛋白数量。最近扩大的商业可用性与膜蛋白研究相关的高纯度氘代洗涤剂使可获取的手段来操纵这种氢/氘含量的无膜蛋…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

生物和环境研究办公室支持 ORNL 的结构分子生物学 (CSMB) 和生物 san 中心的研究, 使用由美国能源部基础能源科学办公室科学用户设施部支持的设施。的能量。在利伯曼实验室的膜蛋白结构工作已得到 NIH (DK091357, GM095638) 和 NSF (0845445) 的支持。

Materials

Amicon Ultra MWCO 50KDa concentrator  EMD Millipore UFC905096 labware
Ammonium citrate dibasic Fisher Scientific A663 medium component
Ammonium sulfate EMD Millipore 2150 medium component
Bioflo 310 Bioreactor System Eppendorf M1287-2110 equipment
Calcium chloride dihydrate Acros 423525000 medium component
Carbenicillin IBI Scientific IB02025 antibiotic
Chloramphenicol EMD Millipore 3130 antibiotic
Cobalt (II) chloride Acros AC21413-0050 medium component
Copper (II) sulfate Acros AC19771-1000  medium component
Deuterium oxide Sigma-Aldrich 756822 medium component
Drierite Gas Purifier W.A. Hammond Drierite Co. Ltd. 27068
EDTA, disodium, dihydrate EMD Millipore 4010 medium component
Emulsiflex-C3 Avestin EF-C3 equipment
Äkta Purifier UPC100 GE Healthcare  equipment
Glycerol Sigma-Aldrich G5516 medium component
HEPES Sigma-Aldrich H4034
HiPrep 16/60 Sephacryl S-300 HR column GE Healthcare  17116701
Imidazole VWR 97064-622
IPTG Teknova I3325
Iron(III) chloride hexahydrate MP Biochemicals ICN19404590 medium component
LB Agar Miller Fisher Scientific BP1425-2
Magnesium sulfate heptahydrate VWR 97062-134 medium component
Manganese(II) sulfate monohydrate Acros AC20590-5000 medium component
MaxQ 6000 Incubated/Refrigerated Shaker Thermo Scientific SHKE6000-7  equipment
n-Dodecyl-d25-β-D-maltopyranoside Anatrace D310T
n-Dodecyl-β-D-maltopyranoside Anatrace D310A
Potassium phosphate monobasic VWR 97062-346 medium component
RC 6 Plus Centrifuge Thermo Scientific Sorvall 46910 equipment
SIGMAFAST protease inhibitor cocktail tablets, EDTA-free Sigma-Aldrich S8830
Sodium chloride Sigma-Aldrich S3014
Sodium hydroxide Sigma-Aldrich 795429
Sodium phosphate dibasic Sigma-Aldrich S7907 medium component
Sterile 25mm syringe filter with 0.2µm PES membrane VWR 28145-501 labware
Sterile disposable bottle top filter with 0.2µm PES membrane Thermo Scientific 596-4520  labware
Superdex 200 10/300 GL  GE Healthcare  17517501
Superose-12 10/300 GL column  GE Healthcare  17517301
Ultrospec 10 Cell Density Meter GE Healthcare  80211630 equipment
Zinc sulfate monohydrate Acros AC38980-2500  medium component
DOWNLOAD MATERIALS LIST

Riferimenti

  1. Wallin, E., Heijne, G. V. Genome-wide analysis of integral membrane proteins from eubacterial, archaean, and eukaryotic organisms. Protein Science. 7 (4), 1029-1038 (1998).
  2. Tautermann, C. S. GPCR structures in drug design, emerging opportunities with new structures. Bioorganic & Medicinal Chemistry Letters. 24 (17), 4073-4079 (2014).
  3. Cournia, Z., et al. Membrane protein structure, function, and dynamics: A perspective from experiments and theory. The Journal of Membrane Biology. 248 (4), 611-640 (2015).
  4. Doerr, A. Membrane protein structures. Nature Methods. 6, 35 (2008).
  5. Bill, R. M., et al. Overcoming barriers to membrane protein structure determination. Nature Biotechnology. 29, 335 (2011).
  6. Carpenter, E. P., Beis, K., Cameron, A. D., Iwata, S. Overcoming the challenges of membrane protein crystallography. Current Opinion in Structural Biology. 18 (5), 581-586 (2008).
  7. Sanders, C. R., Sönnichsen, F. Solution NMR of membrane proteins: Practice and challenges. Magnetic Resonance in Chemistry. 44 (S1), S24-S40 (2006).
  8. Garavito, R. M., Ferguson-Miller, S. Detergents as tools in membrane biochemistry. Journal of Biological Chemistry. 276 (35), 32403-32406 (2001).
  9. Privé, G. G. Detergents for the stabilization and crystallization of membrane proteins. Methods. 41 (4), 388-397 (2007).
  10. Wessel, D., Flügge, U. I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Analytical Biochemistry. 138 (1), 141-143 (1984).
  11. Bayburt, T. H., Grinkova, Y. V., Sligar, S. G. Self-assembly of discoidal phospholipid bilayer nanoparticles with membrane scaffold proteins. Nano Letters. 2 (8), 853-856 (2002).
  12. Bayburt, T. H., Sligar, S. G. Membrane protein assembly into Nanodiscs. FEBS Letters. 584 (9), 1721-1727 (2010).
  13. Skar-Gislinge, N., et al. Elliptical structure of phospholipid bilayer nanodiscs encapsulated by scaffold proteins: Casting the roles of the lipids and the protein. Journal of the American Chemical Society. 132 (39), 13713-13722 (2010).
  14. Sanders, C. R., Schwonek, J. P. Characterization of magnetically orientable bilayers in mixtures of dihexanoylphosphatidylcholine and dimyristoylphosphatidylcholine by solid-state NMR. Biochimica. 31 (37), 8898-8905 (1992).
  15. Vestergaard, M., Kraft, J. F., Vosegaard, T., Thøgersen, L., Schiøtt, B. Bicelles and other membrane mimics: comparison of structure, properties, and dynamics from MD simulations. The Journal of Physical Chemistry B. 119 (52), 15831-15843 (2015).
  16. Popot, J. -. L., et al. Amphipols From A to Z. Annual Review of Biophysics. 40 (1), 379-408 (2011).
  17. Tribet, C., Audebert, R., Popot, J. -. L. Amphipols: Polymers that keep membrane proteins soluble in aqueous solutions. Proceedings of the National Academy of Sciences. 93 (26), 15047 (1996).
  18. Fernández, C., Wüthrich, K. NMR solution structure determination of membrane proteins reconstituted in detergent micelles. FEBS Letters. 555 (1), 144-150 (2003).
  19. Hiruma-Shimizu, K., Shimizu, H., Thompson, G. S., Kalverda, A. P., Patching, S. G. Deuterated detergents for structural and functional studies of membrane proteins: Properties, chemical synthesis and applications. Molecular Membrane Biology. 32 (5-8), 139-155 (2015).
  20. Krueger-Koplin, R. D., et al. An evaluation of detergents for NMR structural studies of membrane proteins. Journal of Biomolecular NMR. 28 (1), 43-57 (2004).
  21. Linke, D., Burgess, R. R., Deutscher, M. P. . Methods in Enzymology. 463, 603-617 (2009).
  22. Oliver, R. C., et al. Tuning micelle dimensions and properties with binary surfactant mixtures. Langmuir. 30 (44), 13353-13361 (2014).
  23. Orwick-Rydmark, M., Arnold, T., Linke, D. The use of detergents to purify membrane proteins. Current Protocols in Protein Science. 84 (1), (2016).
  24. Tanford, C., Reynolds, J. A. Characterization of membrane proteins in detergent solutions. Biochimica et Biophysica Acta (BBA) – Reviews on Biomembranes. 457 (2), 133-170 (1976).
  25. Tulumello, D. V., Deber, C. M. Efficiency of detergents at maintaining membrane protein structures in their biologically relevant forms. Biochimica et Biophysica Acta (BBA) – Biomembranes. 1818 (5), 1351-1358 (2012).
  26. Arachea, B. T., et al. Detergent selection for enhanced extraction of membrane proteins. Protein Expression and Purification. 86 (1), 12-20 (2012).
  27. Seddon, A. M., Curnow, P., Booth, P. J. Membrane proteins, lipids and detergents: Not just a soap opera. Biochimica et Biophysica Acta (BBA) – Biomembranes. 1666 (1), 105-117 (2004).
  28. Tanford, C. . The Hydrophobic Effect: Formation of Micelles and Biological Membranes. , (1980).
  29. Littrell, K., Urban, V., Tiede, D., Thiyagarajan, P. Solution structure of detergent micelles at conditions relevant to membrane protein crystallization. Journal of Applied Crystallography. 33 (3 Part 1), 577-581 (2000).
  30. Oliver, R. C., et al. Dependence of micelle size and shape on detergent alkyl chain length and head group. PLOS ONE. 8 (5), e62488 (2013).
  31. Maire, M., Champeil, P., Møller, J. V. Interaction of membrane proteins and lipids with solubilizing detergents. Biochimica et Biophysica Acta (BBA) – Biomembranes. 1508 (1), 86-111 (2000).
  32. Hong, X., Weng, Y. -. X., Li, M. Determination of the topological shape of integral membrane protein light-harvesting complex LH2 from photosynthetic bacteria in the detergent solution by small-angle X-ray scattering. Biophysical Journal. 86 (2), 1082-1088 (2004).
  33. Vinothkumar, K. R. Membrane protein structures without crystals, by single particle electron cryomicroscopy. Current Opinion in Structural Biology. 33, 103-114 (2015).
  34. Pérez, J., Koutsioubas, A. Memprot: A program to model the detergent corona around a membrane protein based on S-SAXS data. Acta Crystallographica Section D. 71 (1), 86-93 (2015).
  35. Oliver, R. C., Pingali, S. V., Urban, V. S. Designing mixed detergent micelles for uniform neutron contrast. The Journal of Physical Chemistry Letters. 8 (20), 5041-5046 (2017).
  36. Hiruma-Shimizu, K., Kalverda, A. P., Henderson, P. J. F., Homans, S. W., Patching, S. G. Synthesis of uniformly deuterated n-dodecyl-β-D-maltoside (d39-DDM) for solubilization of membrane proteins in TROSY NMR experiments. Journal of Labelled Compounds & Radiopharmaceuticals. 57 (14), 737-743 (2014).
  37. Midtgaard, S. R., et al. Invisible detergents for structure determination of membrane proteins by small-angle neutron scattering. The FEBS Journal. 285 (2), 357-371 (2018).
  38. Gabel, F., Chaudhuri, B., Muñoz, I. G., Qian, S., Urban, V. S. . Biological Small Angle Scattering: Techniques, Strategies and Tips. , 201-214 (2017).
  39. Schantz, A. B., et al. PEE-PEO Block copolymer exchange rate between mixed micelles is detergent and temperature activated. Macromolecules. 50 (6), 2484-2494 (2017).
  40. Liyana-Arachchi, T. P., et al. Bubble bursting as an aerosol generation mechanism during an oil spill in the deep-sea environment: Molecular dynamics simulations of oil alkanes and dispersants in atmospheric air/salt water interfaces. Environmental Science: Processes & Impacts. 16 (1), 53-64 (2014).
  41. Dos Santos Morais, R., et al. Contrast-matched isotropic bicelles: A versatile tool to specifically probe the solution structure of peripheral membrane proteins using SANS. Langmuir. 33 (26), 6572-6580 (2017).
  42. Maric, S., et al. Stealth carriers for low-resolution structure determination of membrane proteins in solution. Acta Crystallographica Section D. 70 (2), 317-328 (2014).
  43. Pedersen, J. S., Svaneborg, C., Almdal, K., Hamley, I. W., Young, R. N. A small-angle neutron and x-ray contrast variation scattering study of the structure of block copolymer micelles: Corona shape and excluded volume interactions. Macromolecules. 36 (2), 416-433 (2003).
  44. Urban, V. S., Kaufmann, E. N. . Characterization of Materials. , (2012).
  45. Chaudhuri, B., Muñoz, I. G., Qian, S., Urban, V. S., Cohen, I. R., et al. . Advances in Experimental Medicine and Biology. 1009, 1-268 (2017).
  46. . Submitting a Research Proposal Available from: https://neutrons.ornl.gov/users/proposals (2018)
  47. . Accompanying Bio-Deuteration Laboratory Proposal Available from: https://www.ornl.gov/sites/default/files/BDL_info_request.docx (2014)
  48. Whitten, A. E., Cai, S., Trewhella, J. MULCh: Modules for the analysis of small-angle neutron contrast variation data from biomolecular assemblies. Journal of Applied Crystallography. 41 (1), 222-226 (2008).
  49. . Scattering Length Density Calculator by National Institute of Standards and Technology (NIST) Center for Neutron Research Available from: https://www.ncnr.nist.gov/resources/activation/ (2018)
  50. Ibel, K., Stuhrmann, H. B. Comparison of neutron and X-ray scattering of dilute myoglobin solutions. Journal of Molecular Biology. 93 (2), 255-265 (1975).
  51. Holme, T., Arvidson, S., Lindholm, B., Pavlu, B. Enzymes: Laboratory-scale production. Process Biochemistry. 5 (9), 62-66 (1970).
  52. Larsson, G., Enfors, S. -. O. Protein release and foaming in Escherichia coli cultures grown in minimal medium. Bioprocess Engineering. 15 (5), 231-237 (1996).
  53. Artero, J. -. B., Hartlein, M., McSweeney, S., Timmins, P. A comparison of refined X-ray structures of hydrogenated and perdeuterated rat [gamma]E-crystallin in H2O and D2O. Acta Crystallographica Section D. 61 (11), 1541-1549 (2005).
  54. Paliy, O., Bloor, D., Brockwell, D., Gilbert, P., Barber, J. Improved methods of cultivation and production of deuteriated proteins from E. coli strains grown on fully deuteriated minimal medium. Journal of applied microbiology. 94 (4), 580-586 (2003).
  55. Sivashanmugam, A., et al. Practical protocols for production of very high yields of recombinant proteins using Escherichia coli. Protein Science. 18 (5), 936-948 (2009).
  56. Hoopes, J. T., Elberson, M. A., Preston, R. J., Reddy, P. T., Kelman, Z., Kelman, Z. . Methods in Enzymology. 565, 27-44 (2015).
  57. Leiting, B., Marsilio, F., O’Connell, J. F. Predictable deuteration of recombinant proteins expressed in Escherichia coli. Analytical Biochemistry. 265 (2), 351-355 (1998).
  58. Perkins, S. J. Estimation of deuteration levels in whole cells and cellular proteins by 1H n.m.r. spectroscopy and neutron scattering. Biochemical Journal. 199 (1), 163-170 (1981).
  59. Obom, K. M., Magno, A., Cummings, P. J. Operation of a benchtop bioreactor. Journal of Visualized Experiments. (79), e50582 (2013).
  60. Duff, A. P., Wilde, K. L., Rekas, A., Lake, V., Holden, P. J., Kelman, Z. . Methods in Enzymology. 565, 3-25 (2015).
  61. Haertlein, M., Kelman, Z., et al. . Methods in Enzymology. 566, 113-157 (2016).
  62. Meilleur, F., Weiss, K. L., Myles, D. A. A. T Micro and Nano Technologies in Bioanalysis. Methods in Molecular Biology. 544, 281-292 (2009).
  63. Naing, S. -. H., Oliver, R. C., Weiss, K. L., Urban, V. S., Lieberman, R. L. Solution structure of an intramembrane aspartyl protease via small angle neutron scattering. Biophysical Journal. 114 (3), 602-608 (2018).
  64. . Training Requirements for First Time and Repeat Users Available from: https://neutrons.ornl.gov/users (2018)
  65. . Remote Analysis Cluster Available from: https://analysis.sns.gov (2018)
  66. Franke, D., et al. ATSAS 2.8: A comprehensive data analysis suite for small-angle scattering from macromolecular solutions. Journal of Applied Crystallography. 50 (4), 1212-1225 (2017).
  67. . Software Suite Available from: https://www.embl-hamburg.de/biosaxs/download.html (2018)
  68. . Software Individual Programs Available from: https://www.embl-hamburg.de/biosaxs/software.html (2018)
  69. Konarev, P. V., Volkov, V. V., Sokolova, A. V., Koch, M. H. J., Svergun, D. I. PRIMUS: A Windows PC-based system for small-angle scattering data analysis. Journal of Applied Crystallography. 36 (5), 1277-1282 (2003).
  70. Jacques, D. A., Trewhella, J. Small-angle scattering for structural biology-Expanding the frontier while avoiding the pitfalls. Protein Science. 19 (4), 642-657 (2010).
  71. Svergun, D. Determination of the regularization parameter in indirect-transform methods using perceptual criteria. Journal of Applied Crystallography. 25 (4), 495-503 (1992).
  72. Putnam, C. D., Hammel, M., Hura, G. L., Tainer, J. A. X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution. Quarterly Reviews of Biophysics. 40 (3), 191-285 (2007).
  73. Svergun, D. I., et al. Protein hydration in solution: Experimental observation by x-ray and neutron scattering. Proceedings of the National Academy of Sciences of the United States of America. 95 (5), 2267-2272 (1998).
  74. Franke, D., Svergun, D. I. DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering. Journal of Applied Crystallography. 42 (2), 342-346 (2009).
  75. Svergun, D. I. Restoring Low Resolution Structure of Biological Macromolecules from Solution Scattering Using Simulated Annealing. Biophysical Journal. 76 (6), 2879-2886 (1999).
  76. Jacques, D. A., Guss, J. M., Svergun, D. I., Trewhella, J. Publication guidelines for structural modelling of small-angle scattering data from biomolecules in solution. Acta Crystallographica Section D. 68 (6), 620-626 (2012).
  77. Heller, W. T., et al. The Bio-SANS instrument at the High Flux Isotope Reactor of Oak Ridge National Laboratory. Journal of Applied Crystallography. 47 (4), 1238-1246 (2014).
  78. Svergun, D. I., Koch, M. H. J. Small-angle scattering studies of biological macromolecules in solution. Reports on Progress in Physics. 66 (10), 1735 (2003).
  79. Johnson, J. L., Kalyoncu, S., Lieberman, R. L., Mus-Veteau, I. . Heterologous Expression of Membrane Proteins: Methods and Protocols. , 281-301 (2016).

Tags

Keywords: Membrane ProteinSmall-angle Neutron ScatteringContrast MatchingDeuterium LabelingAb Initio ModelingOligomeric StateSizeShapeLow Resolution StructureDetergentScattering Length DensityContrast Match PointE. ColiDeuterated ProteinMinimal MediumBioreactor
check_url/it/57901?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Citazione di questo articolo
Oliver, R. C., Naing, S., Weiss, K. L., Pingali, S. V., Lieberman, R. L., Urban, V. S. Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling. J. Vis. Exp. (140), e57901, doi:10.3791/57901 (2018).
Scarica citazione
Ristampe e autorizzazioni

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image