Molecular dynamics-based refinement and validation for sub-5 Å cryo-electron microscopy maps

Abstract

Two structure determination methods, based on the molecular dynamics flexible fitting (MDFF) paradigm, are presented that resolve sub-5 Å cryo-electron microscopy (EM) maps with either single structures or ensembles of such structures. The methods, denoted cascade MDFF and resolution exchange MDFF, sequentially re-refine a search model against a series of maps of progressively higher resolutions, which ends with the original experimental resolution. Application of sequential re-refinement enables MDFF to achieve a radius of convergence of ~25 Å demonstrated with the accurate modeling of β-galactosidase and TRPV1 proteins at 3.2 Å and 3.4 Å resolution, respectively. The MDFF refinements uniquely offer map-model validation and B-factor determination criteria based on the inherent dynamics of the macromolecules studied, captured by means of local root mean square fluctuations. The MDFF tools described are available to researchers through an easy-to-use and cost-effective cloud computing resource on Amazon Web Services.

DOI: http://dx.doi.org/10.7554/eLife.16105.001

eLife digest

To understand the roles that proteins and other large molecules play inside cells, it is important to determine their structures. One of the techniques that researchers can use to do this is called cryo-electron microscopy (cryo-EM), which rapidly freezes molecules to fix them in position before imaging them in fine detail.

The cryo-EM images are like maps that show the approximate position of atoms. These images must then be processed in order to build a three-dimensional model of the protein that shows how its atoms are arranged relative to each other. One computational approach called Molecular Dynamics Flexible Fitting (MDFF) works by flexibly fitting possible atomic structures into cryo-EM maps. Although this approach works well with relatively undetailed (or ‘low resolution’) cryo-EM images, it struggles to handle the high-resolution cryo-EM maps now being generated.

Singharoy, Teo, McGreevy et al. have now developed two MDFF methods – called cascade MDFF and resolution exchange MDFF – that help to resolve atomic models of biological molecules from cryo-EM images. Each method can refine poorly guessed models into ones that are consistent with the high-resolution experimental images. The refinement is achieved by interpreting a range of images that starts with a ‘fuzzy’ image. The contrast of the image is then progressively improved until an image is produced that has a resolution that is good enough to almost distinguish individual atoms.

The method works because each cryo-EM image shows not just one, but a collection of atomic structures that the molecule can take on, with the fuzzier parts of the image representing the more flexible parts of the molecule. By taking into account this flexibility, the large-scale features of the protein structure can be determined first from the fuzzier images, and increasing the contrast of the images allows smaller-scale refinements to be made to the structure.

The MDFF tools have been designed to be easy to use and are available to researchers at low cost through cloud computing platforms. They can now be used to unravel the structure of many different proteins and protein complexes including those involved in photosynthesis, respiration and protein synthesis.

DOI: http://dx.doi.org/10.7554/eLife.16105.002