Cryo-electron microscopy (cryo-EM), the subject of the 2017 Nobel Prize in Chemistry, is a technology for determining the 3-D structure of macromolecules from many noisy 2-D projections of instances of these macromolecules, whose orientations and positions are unknown. The molecular structures are not rigid objects, but flexible objects involved in dynamical processes. The different conformations are exhibited by different instances of the macromolecule observed in a cryo-EM experiment, each of which is recorded as a particle image. The range of conformations and the conformation of each particle are not known a priori; one of the great promises of cryo-EM is to map this conformation space. Remarkable progress has been made in determining rigid structures from homogeneous samples of molecules in spite of the unknown orientation of each particle image and significant progress has been made in recovering a few distinct states from mixtures of rather distinct conformations, but more complex heterogeneous samples remain a major challenge. We introduce the hyper-molecule'' framework for modeling structures across different states of heterogeneous molecules, including continuums of states. The key idea behind this framework is representing heterogeneous macromolecules as high-dimensional objects, with the additional dimensions representing the conformation space. This idea is then refined to model properties such as localized heterogeneity. In addition, we introduce an algorithmic framework for recovering such maps of heterogeneous objects from experimental data using a Bayesian formulation of the problem and Markov chain Monte Carlo (MCMC) algorithms to address the computational challenges in recovering these high dimensional hyper-molecules. We demonstrate these ideas in a prototype applied to synthetic data.
More recently, the RELION framework has been extended to include multi-body refinement @cite_24 (also see @cite_0 @cite_6 @cite_34 @cite_34 @cite_1 @cite_64 ). In this approach, the user selects different rigid 3-D models that are to be refined separately from the main, or consensus, model. Each separate sub-model is then refined separately, with its own viewing direction and translation, allowing it to move with respect to the consensus model in a rigid-body fashion. This method is limited to rigid-body variability in a few sub-volumes, and cannot handle non-rigid deformations or other types of variability. In particular, the structure found at the interface between the sub-models is likely to vary as their relative positions vary, and it is therefore lost in this method.
@cite_64 @cite_1 @cite_6 @cite_24 @cite_0 @cite_34
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Electron cryomicroscopy can yield near-atomic resolution structures of highly ordered macromolecular complexes. Often however some subunits bind in a flexible manner, have different symmetry from the rest of the complex, or are present in sub-stoichiometric amounts, limiting the attainable resolution. Here we report a general method for the localized three-dimensional reconstruction of such subunits. After determining the particle orientations, local areas corresponding to the subunits can be extracted and treated as single particles. We demonstrate the method using three examples including a flexible assembly and complexes harbouring subunits with either partial occupancy or mismatched symmetry. Most notably, the method allows accurate fitting of the monomeric RNA-dependent RNA polymerase bound at the threefold axis of symmetry inside a viral capsid, revealing for the first time its exact orientation and interactions with the capsid proteins. Localized reconstruction is expected to provide novel biological insights in a range of challenging biological systems. An enzyme called gamma-secretase cuts other proteins in cells into smaller pieces. Like most enzymes, gamma-secretase is expected to move through several different three-dimensional shapes to perform its role, and identifying these structures could help us to understand how the enzyme works. One of the proteins that is targeted by gamma-secretase is called amyloid precursor protein, and cutting this protein results in the formation of so-called amyloid-beta peptides. When gamma-secretase doesn't work properly, these amyloid-beta peptides can accumulate in the brain and large accumulations of these peptides have been observed in the brains of patients with Alzheimer's disease. Earlier in 2015, a group of researchers used a technique called cryo-electron microscopy (cryo-EM) to produce a three-dimensional model of gamma-secretase. This revealed that the active site of the enzyme, that is, the region that is used to cut the other proteins, is particularly flexible. Now, – including many of the researchers from the earlier work – studied this flexibility in more detail. For the experiments, gamma-secretase was exposed to an inhibitor molecule that stopped it from cutting other proteins. This meant that the structure of gamma-secretase became more rigid than normal, which made it possible to collect more detailed structural information using cryo-EM. also developed new methods for processing images to separate the images of individual enzyme molecules based on the different shapes they had adopted at the time. These methods make it possible to view a mixture of very similar enzyme structures that differ only in a small region of the protein (in this case the active site). In the future, it would be useful to repeat these imaging experiments using a range of different molecules that alter the activity of gamma-secretase. Furthermore, the new image processing methods developed by could be used to study flexibility in the shapes of other proteins. Each year, malaria kills more than 600,000 people, mostly children younger than 5 years old. Humans who have been bitten by mosquitoes infected with malaria-causing parasites become ill as the parasites rapidly multiply in blood cells. Although there are several drugs that are currently used to treat malaria, the parasites are rapidly developing resistance to them, setting off an urgent hunt for new malaria drugs. Developing new antimalarial medications from scratch is likely to take decades—too long to combat the current public health threat posed by emerging strains of drug-resistant parasites. To speed up the process, scientists are investigating whether drugs developed for other illnesses may also act as therapies for malaria, either when used alone or in combination with existing malaria drugs. Certain antibiotics—including one called emetine—have already shown promise as antimalarial drugs. These antibiotics prevent the parasites from multiplying by interfering with the ribosome—the part of a cell that builds new proteins. However, humans become ill after taking emetine for long periods because it also blocks the production of human proteins. Tweaking emetine so that it acts only against the production of parasite proteins would make it a safer malaria treatment. To do this, scientists must first map the precise interactions between the drug and the ribosomes in parasites. have now used a technique called cryo-electron microscopy to examine the ribosome of the most virulent form of malaria parasite. This technique uses very cold temperatures to rapidly freeze molecules, allowing scientists to look at molecular-level details without distorting the structure of the molecule—a problem sometimes encountered in other techniques. The images of the parasitic ribosome taken by Wong, Bai, show that emetine binds to the end of the ribosome where the instructions for how to assemble amino acids into a protein are copied from strands of RNA. In addition, the images revealed features of the parasitic ribosome that are not found in the human form. Drug makers could exploit these features to improve emetine so that it more specifically targets the production of proteins by the parasite and is less toxic to humans. Macromolecular complexes that exhibit continuous forms of structural flexibility pose a challenge for many existing tools in cryo-EM single-particle analysis. We describe a new tool, called multi-b ... Mitochondria have specialized ribosomes that have diverged from their bacterial and cytoplasmic counterparts. We have solved the structure of the yeast mitoribosomal large subunit using single-particle cryo–electron microscopy. The resolution of 3.2 angstroms enabled a nearly complete atomic model to be built de novo and refined, including 39 proteins, 13 of which are unique to mitochondria, as well as expansion segments of mitoribosomal RNA. The structure reveals a new exit tunnel path and architecture, unique elements of the E site, and a putative membrane docking site. N-ethylmaleimide-sensitive factor (NSF) and α soluble NSF attachment proteins (α-SNAPs) work together within a 20S particle to disassemble and recycle the SNAP receptor (SNARE) complex after intracellular membrane fusion. To understand the disassembly mechanism of the SNARE complex by NSF and α-SNAP, we performed single-particle cryo-electron microscopy analysis of 20S particles and determined the structure of the α-SNAP-SNARE assembly portion at a resolution of 7.35 A. The structure illustrates that four α-SNAPs wrap around the single left-handed SNARE helical bundle as a right-handed cylindrical assembly within a 20S particle. A conserved hydrophobic patch connecting helices 9 and 10 of each α-SNAP forms a chock protruding into the groove of the SNARE four-helix bundle. Biochemical studies proved that this structural element was critical for SNARE complex disassembly. Our study suggests how four α-SNAPs may coordinate with the NSF to tear the SNARE complex into individual proteins.