Transmission Electron Microscopy Tilt-Series Data from In-Situ Chondrocyte Primary Cilia

The primary cilium has recently become the focus of intensive investigations into understanding the physical structure and processes of eukaryotic cells. This paper describes two tilt-series image datasets, acquired by transmission electron microscopy, of in situ chick-embryo sternal-cartilage primary cilia. These data have been released under an open-access licence, and are well suited to tomographic reconstruction and modelling of the cilium.

FastTomo: A SerialEM Script for Collecting Electron Tomography Data

FastTomo is a SerialEM script for collecting tilted specimen images in transmission electron microscopes to be further used in tomographic reconstruction. It achieves a speedup over conventional tracking methods by minimizing the usage of off-target tracking shots, and instead applies proportional control to the specimen images. Movement in the Z coordinate is estimated prior to each tilt series in a separate calibration routine. Overall, this method is fast and reliable when the field of view is at least 1 um, and can tolerate minor errors in setting eucentric height. The implemented tilt series schemes include the unidirectional, bidirectional, and dose-symmetric schemes.

In situ structure determination using single particle cryo-electron microscopy images

Cryo-electron microscopy (cryo-EM) tomography is a powerful tool for in situ structure determination. However, this method requires the acquisition of tilt series, and its time consuming throughput of acquiring tilt series severely slows determination of in situ structures. By treating the electron densities of non-target protein as non-Gaussian distributed noise, we developed a new target function that greatly improves the efficiency of the recognition of the target protein in a single cryo-EM image without acquiring tilt series. Moreover, we developed a sorting function that effectively eliminates the false positive detection, which not only improves the resolution during the subsequent structure refinement procedure but also allows using homolog proteins as models to recognize the target protein. Together, we developed an in situ single particle analysis (isSPA) method. Our isSPA method was successfully applied to solve structures of glycoproteins on the surface of a non-icosahedral virus and Rubisco inside the carboxysome. The cryo-EM data from both samples were collected within 24 hours, thus allowing fast and simple structural determination in situ.

In situ structure determination using single particle cryo-electron microscopy images

Cryo-electron microscopy (cryo-EM) tomography is a powerful tool for in situ structure determination. However, this method requires the acquisition of tilt series, and its time consuming throughput of acquiring tilt series severely slows determination of in situ structures. By treating the electron densities of non-target protein as non-Gaussian distributed noise, we developed a new target function that greatly improves the efficiency of the recognition of the target protein in a single cryo-EM image without acquiring tilt series. Moreover, we developed a sorting function that effectively eliminates the false positive detection, which not only improves the resolution during the subsequent structure refinement procedure but also allows using homolog proteins as models to recognize the target protein. Together, we developed an in situ single particle analysis (isSPA) method. Our isSPA method was successfully applied to solve structures of glycoproteins on the surface of a non-icosahedral virus and Rubisco inside the carboxysome. The cryo-EM data from both samples were collected within 24 hours, thus allowing fast and simple structural determination in situ.