Project description:Using a new Titan Krios stage equipped with a single-axis holder, we developed two methods to accelerate the collection of tilt-series. We demonstrate a continuous-tilting method that can record a tilt-series in seconds, but with loss of details finer than ∼4 nm. We also demonstrate a fast-incremental method that can record a tilt-series several-fold faster than current methods and with similar resolution. We characterize the utility of both methods in real biological electron cryotomography workflows. We identify opportunities for further improvements in hardware and software and speculate on the impact such advances could have on structural biology.
Project description:A method is proposed to reconstruct the 3D molecular structure from micrographs collected at just one sample tilt angle in the random conical tilt scheme in cryo-electron microscopy. The method uses autocorrelation analysis on the micrographs to estimate features of the molecule which are invariant under certain nuisance parameters such as the positions of molecular projections in the micrographs. This enables the molecular structure to be reconstructed directly from micrographs, completely circumventing the need for particle picking. Reconstructions are demonstrated with simulated data and the effect of the missing-cone region is investigated. These results show promise to reduce the size limit for single-particle reconstruction in cryo-electron microscopy.
Project description:Cryogenic electron microscopy can be widely applied to biological specimens from the molecular to the cellular scale. In single-particle analysis, 3D structures may be obtained in high resolution by averaging 2D images of single particles in random orientations. For pleomorphic specimens, structures may be obtained by recording the tilt series of a single example of the specimen and calculating tomograms. Where many copies of a single structure such as a protein or nucleic acid assembly are present within the tomogram, averaging of the sub-volumes (subtomogram averaging) has been successfully applied. The choice of data collection method for any given specimen may depend on the structural question of interest and is determined by the radiation sensitivity of the specimen. Here, we survey some recent developments on the use of hybrid methods for recording and analysing data from radiation-sensitive biological specimens. These include single-particle reconstruction from 2D images where additional views are recorded at a single tilt angle of the specimen and methods where image tilt series, initially used for tomogram reconstruction, are processed as individual single-particle images. There is a continuum of approaches now available to maximize structural information obtained from the specimen.
Project description:Recent evidence suggests that the beam-induced motion of the sample during tilt-series acquisition is a major resolution-limiting factor in electron cryo-tomography (cryoET). It causes suboptimal tilt-series alignment and thus deterioration of the reconstruction quality. Here we present a novel approach to tilt-series alignment and tomographic reconstruction that considers the beam-induced sample motion through the tilt-series. It extends the standard fiducial-based alignment approach in cryoET by introducing quadratic polynomials to model the sample motion. The model can be used during reconstruction to yield a motion-compensated tomogram. We evaluated our method on various datasets with different sample sizes. The results demonstrate that our method could be a useful tool to improve the quality of tomograms and the resolution in cryoET.
Project description:Obtaining the heterogeneous conformation of small proteins is important for understanding their biological role, but it is still challenging. Here, we developed a multi-tilt nanoparticle-aided cryo-electron microscopy sampling (MT-NACS) technique that enables the observation of heterogeneous conformations of small proteins and applied it to calmodulin. By imaging the proteins labeled by two gold nanoparticles at multiple tilt angles and analyzing the projected positions of the nanoparticles, the distributions of 3D interparticle distances were obtained. From the measured distance distributions, the conformational changes associated with Ca2+ binding and salt concentration were determined. MT-NACS was also used to track the structural change accompanied by the interaction between amyloid-beta and calmodulin, which has never been observed experimentally. This work offers an alternative platform for studying the functional flexibility of small proteins.
Project description:A computational method was developed to recover the three-dimensional coordinates of gold nanoparticles specifically attached to a protein complex from tilt-pair images collected by electron microscopy. The program was tested on a simulated dataset and applied to a real dataset comprising tilt-pair images recorded by cryo electron microscopy of RNA polymerase II in a complex with four gold-labeled single-chain antibody fragments. The positions of the gold nanoparticles were determined, and comparison of the coordinates among the tetrameric particles revealed the range of motion within the protein complexes.