Using Cryo-Electron Microscopy to Determine the 3-Dimensional Structure of Fibrinogen
Viverette, Elizabeth G
Fibrinogen is a large protein that is the soluble protein precursor to fibrin, which forms fibers that hold blood cells together to create blood clots. Research into the properties of fibrinogen is necessary because of its role in many thrombotic ailments, including strokes and heart attacks. The structure of fibrinogen has been studied through various methods, though certain regions of fibrinogen have not yet had their structures conclusively determined. These regions all have apparent flexibility and mobility of the region that is lost in sample preparation of other imaging methods such as the formation of crystal structures for x-ray crystallography. Cryogenic Electron Microscopy (cryo-EM) is a technique for imaging proteins and other macromolecules at resolutions of 1-3 Å while preserving native structures such as flexibility. Cryo-EM involves freezing samples in a layer of amorphous ice to remove interference from frozen crystal structures. Because of this sample preparation procedure, fibrinogen can be imaged in its natural state. CryoSPARC and other Cryo-EM analysis software works by identifying isolated molecules and grouping them into relative angle orientations that are then condensed and averaged into 2-D structures. The initial process of 2-D angle classification and data refinement can be automated to some degree with neural networks, such as Topaz, developed and trained specifically to assist with Cryo-EM analysis. Once 2-D structures have been optimized, a complete three-dimensional render of the protein can be reconstructed using Gaussian best-fit algorithms. A data set of over 500 GB has been collected using the Cryo-EM microscope at the National Institute of Environmental Health Sciences. This data set includes samples collected via negative staining. Multiple software packages for Cryo-EM analysis will be used to choose appropriate particles for use in the final structure and render initial and final structures, including EMAN2, CryoSPARC, and a neural network specifically trained to process fibrinogen data. The particles to be used to image 2-D and 3-D maps of fibrinogen will be both manually and automatically selected and sorted into angle templates. This is complicated by the numerous possible flexibility states of fibrinogen. These templates will be refined and averaged over multiple iterations to create an optimal data set for the construction of a high-resolution structure map of fibrinogen in its natural, flexible form.
Viverette, Elizabeth G. (May 2021). Using Cryo-Electron Microscopy to Determine the 3-Dimensional Structure of Fibrinogen (Honors Thesis, East Carolina University). Retrieved from the Scholarship. (http://hdl.handle.net/10342/9204.)
Viverette, Elizabeth G. Using Cryo-Electron Microscopy to Determine the 3-Dimensional Structure of Fibrinogen. Honors Thesis. East Carolina University, May 2021. The Scholarship. http://hdl.handle.net/10342/9204. October 03, 2023.
Viverette, Elizabeth G, “Using Cryo-Electron Microscopy to Determine the 3-Dimensional Structure of Fibrinogen” (Honors Thesis., East Carolina University, May 2021).
Viverette, Elizabeth G. Using Cryo-Electron Microscopy to Determine the 3-Dimensional Structure of Fibrinogen [Honors Thesis]. Greenville, NC: East Carolina University; May 2021.
East Carolina University