Exosome परिसर के एकल कण इलेक्ट्रॉन माइक्रोस्कोपी पुनर्निर्माण रैंडम शंक्वाकार घुमाएँ विधि का प्रयोग
Summary
यह लेख जैविक अणुओं की एक तीन आयामी (3 डी) पुनर्निर्माण नकारात्मक धुंधला इलेक्ट्रॉन माइक्रोस्कोपी (EM) का उपयोग करने के लिए एक मानक विधि का वर्णन करता है. इस प्रोटोकॉल में, हम की व्याख्या कैसे मध्यम यादृच्छिक शंक्वाकार झुकाव पुनर्निर्माण विधि (RCT) का उपयोग कर संकल्प पर Saccharomyces cerevisiae exosome परिसर के 3D संरचना प्राप्त करने के लिए.
Abstract
Single particle electron microscopy (EM) reconstruction has recently become a popular tool to get the three-dimensional (3D) structure of large macromolecular complexes. Compared to X-ray crystallography, it has some unique advantages. First, single particle EM reconstruction does not need to crystallize the protein sample, which is the bottleneck in X-ray crystallography, especially for large macromolecular complexes. Secondly, it does not need large amounts of protein samples. Compared with milligrams of proteins necessary for crystallization, single particle EM reconstruction only needs several micro-liters of protein solution at nano-molar concentrations, using the negative staining EM method. However, despite a few macromolecular assemblies with high symmetry, single particle EM is limited at relatively low resolution (lower than 1 nm resolution) for many specimens especially those without symmetry. This technique is also limited by the size of the molecules under study, i.e. 100 kDa for negatively stained specimens and 300 kDa for frozen-hydrated specimens in general.
For a new sample of unknown structure, we generally use a heavy metal solution to embed the molecules by negative staining. The specimen is then examined in a transmission electron microscope to take two-dimensional (2D) micrographs of the molecules. Ideally, the protein molecules have a homogeneous 3D structure but exhibit different orientations in the micrographs. These micrographs are digitized and processed in computers as “single particles”. Using two-dimensional alignment and classification techniques, homogenous molecules in the same views are clustered into classes. Their averages enhance the signal of the molecule’s 2D shapes. After we assign the particles with the proper relative orientation (Euler angles), we will be able to reconstruct the 2D particle images into a 3D virtual volume.
In single particle 3D reconstruction, an essential step is to correctly assign the proper orientation of each single particle. There are several methods to assign the view for each particle, including the angular reconstitution1 and random conical tilt (RCT) method2. In this protocol, we describe our practice in getting the 3D reconstruction of yeast exosome complex using negative staining EM and RCT. It should be noted that our protocol of electron microscopy and image processing follows the basic principle of RCT but is not the only way to perform the method. We first describe how to embed the protein sample into a layer of Uranyl-Formate with a thickness comparable to the protein size, using a holey carbon grid covered with a layer of continuous thin carbon film. Then the specimen is inserted into a transmission electron microscope to collect untilted (0-degree) and tilted (55-degree) pairs of micrographs that will be used later for processing and obtaining an initial 3D model of the yeast exosome. To this end, we perform RCT and then refine the initial 3D model by using the projection matching refinement method3.
Protocol
Discussion
इस अनुच्छेद में हम नमूना तैयार करने की एक विस्तृत प्रोटोकॉल और exosome नकारात्मक धुंधला इलेक्ट्रॉन माइक्रोस्कोपी का उपयोग परिसर के तीन आयामी पुनर्निर्माण उपस्थित थे. इस पद्धति का उपयोग करके, हम 3 डी पुनर्?…
Declarações
The authors have nothing to disclose.
Acknowledgements
लेखकों के लिए UC बर्कले में प्रारंभिक और प्रोटोकॉल वांग प्रयोगशाला के सदस्यों को उनकी मदद में येल विश्वविद्यालय में स्थापित करने में मदद करने के लिए पूर्ण प्रोटोकॉल स्थापित करने में Nogales प्रयोगशाला के सदस्यों को धन्यवाद देना चाहूंगा. हम भी क्रायो – EM सुविधा और उनके समर्थन के लिए चिकित्सा के येल स्कूल में उच्च प्रदर्शन संगणना केंद्र में कर्मचारी को स्वीकार करते हैं. HW से एक स्मिथ परिवार पुरस्कार विजेता है.
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Polyvinyl Formal Resin | Electron Microscopy Science | 63450-15-7 | ||
| Uranyl Formate | Electron Microscopy Science | 22451 | ||
| Superfrost Microscope Slides | Thermo Scientist | 4951F-001 | ||
| 400 mesh grid regular | SPI Supplies | 3040C | ||
| Carbon coater Auto 306 | Edwards | |||
| Tecnai-12 Electron Microscope | FEI | |||
| Glow Discharger | BAL-TEC | Sputter Coater SCD 005 |
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