Electron cryo-tomography (cryo-ET) is a technique that is used to produce three-dimensional pictures (tomograms) of complex objects like asymmetric viruses, cellular organelles or whole cells from a series of tilted electron cryo-microscopy (cryo-EM) images. single-particle analysis, has recently undergone significant progress with the development of highly efficient direct-electron detectors and improved image processing software. Notably, this technique now allows near-atomic resolution structures to be calculated without the need for crystallisation and from as little as 10-100 g of purified material 1, 2. In electron tomography (ET) multiple images are taken of the same sample region at different tilt angles in the microscope. From such a series of tilted images, a 3D reconstruction, or tomogram, of a single 3D object such as an entire cell 3 may be obtained. Thereby, this technique provides the unique possibility to image complexes in their native environment. Moreover, if many copies of a complex of interest are present in tomograms, then the reconstructed 3D density corresponding to 84485-00-7 supplier each complex may be computationally extracted, and the resulting 3D sub-tomograms may be averaged together to increase the signal-to-noise ratio and thereby produce a higher resolution 3D structure 4. This technique is called sub-tomogram averaging, and it has been successfully applied in numerous cases to reveal biological structures or in environments that are otherwise not amenable to single-particle analysis 5C9. To date, the use of sub-tomogram averaging is not as widespread as that of single-particle analysis. An important limitation of sub-tomogram averaging is usually that the best resolved structures 84485-00-7 supplier by this technique are markedly lower in resolution than those from single-particle analysis 4. Tomographic data collection is usually slower, and sub-tomogram averaging requires more complicated image processing, since tomographic reconstruction needs to be followed by alignment and classification of the sub-tomograms. Furthermore, due to increased effective specimen thickness at high tilt angles the sample cannot be imaged at high tilt angles, which leads to a wedge-shaped region in the Fourier domain name where data is usually absent. This ‘missing-wedge’ leads to blurring artefacts in tomograms. Still, the advantage of being able to study macromolecules (e.g. inside an entire cell) remains extremely attractive. This is powerfully illustrated by the recent application of sub-nanometer resolution cryo-ET sub-tomogram averaging to the HIV-1 capsid 10 and to membrane-bound ribosomes 11. Further developments of both experimental data acquisition procedures 12 and image processing algorithms 13 will continue to drive this technique towards higher resolutions and wider applicability. Recently, we introduced a new image processing approach to sub-tomogram averaging 14 that is based on a regularized likelihood optimization algorithm in the RELION program 15, 16. This program was originally designed for single-particle analysis and has been used to calculate numerous near-atomic resolution structures 1. Because the sub-tomogram averaging approach in RELION was modelled around the single-particle analysis workflow, existing RELION users will find many similarities (Physique 1). The main deviation from the single-particle workflow lies in the generation of a 3D model Rabbit polyclonal to ZNF449.Zinc-finger proteins contain DNA-binding domains and have a wide variety of functions, most ofwhich encompass some form of transcriptional activation or repression. The majority of zinc-fingerproteins contain a Krppel-type DNA binding domain and a KRAB domain, which is thought tointeract with KAP1, thereby recruiting histone modifying proteins. As a member of the krueppelC2H2-type zinc-finger protein family, ZNF449 (Zinc finger protein 449), also known as ZSCAN19(Zinc finger and SCAN domain-containing protein 19), is a 518 amino acid protein that containsone SCAN box domain and seven C2H2-type zinc fingers. ZNF449 is ubiquitously expressed andlocalizes to the nucleus. There are three isoforms of ZNF449 that are produced as a result ofalternative splicing events for the information transfer in each sub-tomogram, which is used 84485-00-7 supplier to compensate for both the missing wedge as well as the effects of the contrast transfer function (CTF) in the tomogram 14. A significant effort was made to build on existing 84485-00-7 supplier tools inside RELION, rather than writing new tools specifically for sub-tomogram averaging. This facilitates transitioning between sub-tomogram averaging and single-particle analysis, and thus naturally supports a hybrid approach of combining cryo-EM and cryo-ET data 17C19. Physique 1 Workflow of the image processing protocol. In this protocol we describe the practical use of RELION for sub-tomogram averaging. Our approach complements various single-particle analysis software packages that also offer functionalities for sub-tomogram averaging 20, 21, as well as multiple specialized packages for sub-tomogram averaging 8, 9, 13, 22C24. As many structure determination projects in practice resort to a combination of different software packages, we will explicitly indicate those points in the workflow that are likely points of conversion between option approaches. Recommended procedures for single-particle.