Getting a Structure from Multiple Tomograms of HIV Capsids
The aim of this exercise is to get familiar with managing multiple tomograms through the Dynamo catalogue and to develop good strategies for alignment projects to reach a reasonable structure from a set of tomograms. This is an exercise and not a tutorial or walkthrough. Therefore only a few hints on how to proceed are given. The idea is that you come up with your own solutions by applying the knowledge that you acquired during the workshop to a more realistic case. It is recommended to get familiar with at the advanced starters guide and the walkthrough for lattices on vesicles before starting this exercise.
We prepared a set of 6 tomograms, each containing one Immature HIV-1 virus like particle(VLP) with a layer formed by a lattice of capsid proteins. The capsid proteins have a C6 symmetry and have a diameter of roughly 15nm and a molecular weight of about 150kDa. The pixelsize of the tomograms is 2.7 angstrom. The tomograms are part of the data that was used in the publication An atomic model of HIV-1 capsid-SP1 reveals structures regulating assembly and maturation by Schur FK, Obr M, Hagen WJ, Wan W, Jakobi AJ, Kirkpatrick JM, Sachse C, Kräusslich HG and Briggs JA. (2016). The full dataset can be found on the Electron Microscopy Public Image Archive (EMPIAR) under EMPIAR-10164. The 6 tomograms were extracted from the tilt series TS_03 and TS_43.
From the data given you should be able to get a final average in which you start seeing hints of alpha helices, i.e., it should be possible to reach a resolution close to roughly 10 angstrom.
Some Useful Hints
- Make sure you are familiar with the tutorial walkthrough for lattices on vesicles. Many of the described steps in the tutorial are needed in this exercise. You can also use similar parameters as used in the tutorial (adapted to the different pixelsize) for your first alignment projects.
- In the catalogue, you only need to prebin the tomograms with factor one (1x).
- Do not crop particles with a sidelength larger than 128px.
- Before using any C6 symmetries in the alignment projects, make sure to properly center the particles and template.
- To use the GPUs on sciCORE follow these steps.
- Without any postprocessing or elaborate refinement your final average may look similar to the one shown below: