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igbmc - auto-picking
This auto-picking protocol is based on a highly parallel correlation-based particle picking tool, gEMpicker, developed at INRIA and IGBMC, France (). The program uses template-matching approach, when reference images are correlated with a set of micrographs in order to locate single particles similar to the references. To achieve high processing speeds, the calculations can be performed on multiple GPUs and CPU cores.
To launch auto-picking you will need to select first the Input micrographs, that can be any
SetOfMicrographs produced earlier (Fig. 1). Though the program can accept images up to 4096x4096 pixels size, it is recommended to coarse the micrographs to speed up the calculations. Then you have to select Input references, that can be any
SetOfAverages: either 2D class averages, calculated before, or reprojections of the known 3D structure (in this case, you have to import your reprojections as Averages). Note that the pixel size of the micrographs and the references should be the same. Also, we have noticed that low-pass filtering both micrographs and references gives better results. If the input micrographs and references do no have the same contrast (e.g., white particles on a dark background), select the option References have inverted contrast.
Among other options the most important one is the Threshold range: cross-correlation peaks only within specified range will be selected as particles. Usually, you should first run the protocol on a few micrographs with different defocus level to optimize threshold and other parameters before starting auto-picking on a whole dataset.
Additionally, you can provide a mask for the references, either a circular (you might use a wizard for this) or a custom one. The number of masks should be either one (a single mask for all references) or equal the number of references (one mask per reference). In the latter case, the order of the masks should correspond to the order of references. Of course, image size and pixel size of the references and masks should be the same.
To see all available options, choose the Advanced expert level and click on the Help button for any specific parameter (Fig. 2).
When the protocol is finished you may click on the Analyze Results button, that will launch Xmipp particle picker window and display picked particles for each micrograph (Fig. 3).
Sometimes the picking algorithm may fail to find the particles. If this is the case, you may help it by playing with threshold values as well as some advanced parameters, i.e. Max distance between particles.
[[]] Hoang T. V., Cavin X., Schultz P., Ritchie D. W. (2013). gEMpicker: a highly parallel GPU-accelerated particle picking tool for cryo-electron microscopy. BMC Structural Biology, 13(25): 1 - 10.