cryo-EM Structure Determination Research Articles

Semi-automated selection of cryo-EM particles in RELION-1.3.

The selection of particles suitable for high-resolution cryo-EM structure determination from noisy micrographs may represent a tedious and time-consuming step. Here, a semi-automated particle selection procedure is presented that has been implemented within the open-source software RELION. At the heart of the procedure lies a fully CTF-corrected template-based picking algorithm, which is supplemented by a fast sorting algorithm and reference-free 2D class averaging to remove false positives. With only limited user-interaction, the proposed procedure yields results that are comparable to manual particle selection. Together with an improved graphical user interface, these developments further contribute to turning RELION from a stand-alone refinement program into a convenient image processing pipeline for the entire single-particle approach.

Ribosome structures to near-atomic resolution from thirty thousand cryo-EM particles

Although electron cryo-microscopy (cryo-EM) single-particle analysis has become an important tool for structural biology of large and flexible macro-molecular assemblies, the technique has not yet reached its full potential. Besides fundamental limits imposed by radiation damage, poor detectors and beam-induced sample movement have been shown to degrade attainable resolutions. A new generation of direct electron detectors may ameliorate both effects. Apart from exhibiting improved signal-to-noise performance, these cameras are also fast enough to follow particle movements during electron irradiation. Here, we assess the potentials of this technology for cryo-EM structure determination. Using a newly developed statistical movie processing approach to compensate for beam-induced movement, we show that ribosome reconstructions with unprecedented resolutions may be calculated from almost two orders of magnitude fewer particles than used previously. Therefore, this methodology may expand the scope of high-resolution cryo-EM to a broad range of biological specimens.DOI:http://dx.doi.org/10.7554/eLife.00461.001.

RELION: Implementation of a Bayesian approach to cryo-EM structure determination

RELION, for REgularized LIkelihood OptimizatioN, is an open-source computer program for the refinement of macromolecular structures by single-particle analysis of electron cryo-microscopy (cryo-EM) data. Whereas alternative approaches often rely on user expertise for the tuning of parameters, RELION uses a Bayesian approach to infer parameters of a statistical model from the data. This paper describes developments that reduce the computational costs of the underlying maximum a posteriori (MAP) algorithm, as well as statistical considerations that yield new insights into the accuracy with which the relative orientations of individual particles may be determined. A so-called gold-standard Fourier shell correlation (FSC) procedure to prevent overfitting is also described. The resulting implementation yields high-quality reconstructions and reliable resolution estimates with minimal user intervention and at acceptable computational costs.

Prevention of overfitting in cryo-EM structure determination

Prevention of overfitting in cryo-EM structure determination

A Bayesian View on Cryo-EM Structure Determination

Three-dimensional (3D) structure determination by single-particle analysis of cryo-electron microscopy (cryo-EM) images requires many parameters to be determined from extremely noisy data. This makes the method prone to overfitting, that is, when structures describe noise rather than signal, in particular near their resolution limit where noise levels are highest. Cryo-EM structures are typically filtered using ad hoc procedures to prevent overfitting, but the tuning of arbitrary parameters may lead to subjectivity in the results. I describe a Bayesian interpretation of cryo-EM structure determination, where smoothness in the reconstructed density is imposed through a Gaussian prior in the Fourier domain. The statistical framework dictates how data and prior knowledge should be combined, so that the optimal 3D linear filter is obtained without the need for arbitrariness and objective resolution estimates may be obtained. Application to experimental data indicates that the statistical approach yields more reliable structures than existing methods and is capable of detecting smaller classes in data sets that contain multiple different structures.

Functional Tests Of Purified Slack Channel Protein For Cryo-em Structure Determination

The Slack (slo2.1) gene product is a sodium-gated potassium channel. We seek to image it using single-particle electron cryo-microscopy (cryo-EM). A first step is to verify that the expressed and purified protein is functional. Slack is expressed in HEK cell lines, solubilized, and purified by antibody-affinity. We have employed three methods to check the integrity of the solubilized and reconstituted Slack protein. Western blots verify the reconstitution of protein into floated vesicles. Fusion of these vesicles with planar bilayers yields sodium-sensitive K+ channel activity, while flux assays using the voltage-sensitive dye JC-1 show a high specific activity.

From cryo-EM, multiple protein structures in one shot

A new maximum likelihood–based algorithm allows cryo-EM structure determination of macromolecular complexes even when conformational heterogeneity is present.