Abstract
The α-β tubulin heterodimer undergoes subtle conformational changes during microtubule assembly. These can be modulated by external factors, whose effects on microtubule structure can be characterized on 2D views obtained by cryo-electron microscopy. Analysis of microtubule images is facilitated if they are straight enough to interpret and filter their image Fourier transform, which provide useful information concerning the arrangement of tubulin molecules inside the microtubule lattice. Here, we describe the use of the TubuleJ software to straighten microtubules and determine their lattice parameters. Basic 3D reconstructions can be performed to evaluate the relevance of these parameters. This approach can be used to analyze the effects of nucleotide analogues, drugs or MAPs on microtubule structure, or to select microtubule images prior to high-resolution 3D reconstructions.
Highlights
[Background] Microtubules are polymers of the α-β tubulin heterodimer that form tubes of about 25 nm in diameter and several μm in length
Tubulin binds two molecules of guanosine triphosphate (GTP), one of which is hydrolyzed to GDP during assembly
Works with GTP analogues such as guanylyl-(α, β)-methylene-diphosphonate (GMPCPP) or guanosine 5'-(γ-thio)-triphosphate (GTPγS) gave rise to a model in which GTP-tubulin undergoes a compaction and rotation between its subunits during assembly and GTP-hydrolysis (Zhang et al, 2015). This model was recently challenged by a study that involved a range of nucleotide analogues and structural approaches, including X-ray crystallography, small angle X-ray diffraction and cryo-electron microscopy (Estévez-Gallego et al, 2020)
Summary
Click on ‘Select fiber’ and create a folder named ‘MTa’ Select this folder as your working directory. 5. Draw a line on the fiber of interest (Figure 2B) and press ‘OK’. Draw a line on the fiber of interest (Figure 2B) and press ‘OK’ This defines the rotation angle of the image. Select the whole image (Edit->Select All) and resize the image to, e.g., 189 in height, to minimize background (Image->Crop; Figure 2E) Save this image in the working directory. As the plugins starts with the current image opened in ImageJ, any file format that can be opened in ImageJ can be used with TubuleJ. It includes classical formats such as tiff. With the install of TomoJ, formats dedicated to electron microscopy such as dm[3], mrc, spider or mrc.bz[2] are accepted
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