Structure Determination Of Macromolecular Assemblies Research Articles

AgarFix: Simple and accessible stabilization of challenging single-particle cryo-EM specimens through crosslinking in a matrix of agar

Cryogenic electron microscopy (cryo-EM) allows structure determination of macromolecular assemblies that have resisted other structural biology approaches because of their size and heterogeneity. These challenging multi-protein targets are typically susceptible to dissociation and/or denaturation upon cryo-EM grid preparation, and often require crosslinking prior to freezing. Several approaches for gentle on-column or in-tube crosslinking have been developed. On-column crosslinking is not widely applicable because of the poor separation properties of gel filtration techniques. In-tube crosslinking frequently causes sample aggregation and/or precipitation. Gradient-based crosslinking through the GraFix method is more robust, but very time-consuming and necessitates specialised expensive equipment. Furthermore, removal of the glycerol typically involves significant sample loss and may cause destabilization detrimental to the sample quality. Here, we introduce an alternative procedure: AgarFix (Agarose Fixation). The sample is embedded in an agarose matrix that keeps the molecules separated, thus preventing formation of aggregates upon cross-inking. Gentle crosslinking is accomplished by diffusion of the cross-linker into the agarose drop. The sample is recovered by diffusion or electroelution and can readily be used for cryo-EM specimen preparation. AgarFix requires minimal equipment and basic lab experience, making it widely accessible to the cryo-EM community.

Optimizing model representation for integrative structure determination of macromolecular assemblies

Integrative structure determination of macromolecular assemblies requires specifying the representation of the modeled structure, a scoring function for ranking alternative models based on diverse types of data, and a sampling method for generating these models. Structures are often represented at atomic resolution, although ad hoc simplified representations based on generic guidelines and/or trial and error are also used. In contrast, we introduce here the concept of optimizing representation. To illustrate this concept, the optimal representation is selected from a set of candidate representations based on an objective criterion that depends on varying amounts of information available for different parts of the structure. Specifically, an optimal representation is defined as the highest-resolution representation for which sampling is exhaustive at a precision commensurate with the precision of the representation. Thus, the method does not require an input structure and is applicable to any input information. We consider a space of representations in which a representation is a set of nonoverlapping, variable-length segments (i.e., coarse-grained beads) for each component protein sequence. We also implement a method for efficiently finding an optimal representation in our open-source Integrative Modeling Platform (IMP) software (https://integrativemodeling.org/). The approach is illustrated by application to three complexes of two subunits and a large assembly of 10 subunits. The optimized representation facilitates exhaustive sampling and thus can produce a more accurate model and a more accurate estimate of its uncertainty for larger structures than were possible previously.

Structural characterization of supramolecular assemblies by 13C spin dilution and 3D solid-state NMR

(13)C spin diluted protein samples can be produced using [1-(13)C] and [2-(13)C]-glucose (Glc) carbon sources in the bacterial growth medium. The (13)C spin dilution results in favorable (13)C spectral resolution and polarization transfer behavior. We recently reported the combined use of [1-(13)C]- and [2-(13)C]-Glc labeling to facilitate the structural analysis of insoluble and non-crystalline biological systems by solid-state NMR (ssNMR), including sequential assignment, detection of long-range contacts and structure determination of macromolecular assemblies. In solution NMR the beneficial properties of sparsely labeled samples using [2-(13)C]-glycerol ((13)C labeled Cα sites on a (12)C diluted background) have recently been exploited to provide a bi-directional assignment method (Takeuchi et al. in J Biomol NMR 49(1):17-26, 2011 ). Inspired by this approach and our own recent results using [2-(13)C]-Glc as carbon sources for the simplification of ssNMR spectra, we present a strategy for a bi-directional sequential assignment of solid-state NMR resonances and additionally the detection of long-range contacts using the combination of (13)C spin dilution and 3D NMR spectroscopy. We illustrate our results with the sequential assignment and the collection of distance restraints on an insoluble and non-crystalline supramolecular assembly, the Salmonella typhimurium type III secretion system needle.

An Approach for De Novo Structure Determination of Dynamic Molecular Assemblies by Electron Cryomicroscopy

Single-particle electron cryomicroscopy is a powerful method for three-dimensional (3D) structure determination of macromolecular assemblies. Here we address the challenge of determining a 3D structure in the absence of reference models. The 3D structures are determined by alignment and weighted averaging of densities obtained by native cryo random conical tilt (RCT) reconstructions including consideration of missing data. Our weighted averaging scheme (wRCT) offers advantages for potentially heterogeneous 3D densities of low signal-to-noise ratios. Sets of aligned RCT structures can also be analyzed by multivariate statistical analysis (MSA) to provide insights into snapshots of the assemblies. The approach is used to compute 3D structures of the Escherichia coli 70S ribosome and the human U4/U6.U5 tri-snRNP under vitrified unstained cryo conditions, and to visualize by 3D MSA the L7/L12 stalk of the 70S ribosome and states of tri-snRNP. The approach thus combines de novo 3D structure determination with an analysis of compositional and conformational heterogeneity.

Integrating diverse data for structure determination of macromolecular assemblies.

To understand the cell, we need to determine the macromolecular assembly structures, which may consist of tens to hundreds of components. First, we review the varied experimental data that characterize the assemblies at several levels of resolution. We then describe computational methods for generating the structures using these data. To maximize completeness, resolution, accuracy, precision, and efficiency of the structure determination, a computational approach is required that uses spatial information from a variety of experimental methods. We propose such an approach, defined by its three main components: a hierarchical representation of the assembly, a scoring function consisting of spatial restraints derived from experimental data, and an optimization method that generates structures consistent with the data. This approach is illustrated by determining the configuration of the 456 proteins in the nuclear pore complex (NPC) from baker's yeast. With these tools, we are poised to integrate structural information gathered at multiple levels of the biological hierarchy--from atoms to cells--into a common framework.

Automated image acquisition and processing using a new generation of 4K × 4K CCD cameras for cryo electron microscopic studies of macromolecular assemblies

We have previously reported the development of AutoEM, a software package for semi-automated acquisition of data from a transmission electron microscope. In continuing efforts to improve the speed of structure determination of macromolecular assemblies by electron microscopy, we report here on the performance of a new generation of 4K CCD cameras for use in cryo electron microscopic applications. We demonstrate that at 120 kV, and at a nominal magnification of 67 000×, power spectra and signal-to-noise ratios for the new 4K CCD camera are comparable to values obtained for film images scanned using a Zeiss scanner to resolutions as high as ∼1/6.5 Å −1. The specimen area imaged for each exposure on the 4K CCD is about one-third of the area that can be recorded with a similar exposure on film. The CCD camera also serves the purpose of recording images at low magnification from the center of the hole to measure the thickness of vitrified ice in the hole. The performance of the camera is satisfactory under the low-dose conditions used in cryo electron microscopy, as demonstrated here by the determination of a three-dimensional map at 15 Å for the catalytic core of the 1.8 MDa Bacillus stearothermophilus icosahedral pyruvate dehydrogenase complex, and its comparison with the previously reported atomic model for this complex obtained by X-ray crystallography.

An Integrated System for Transmission Electron Microscopy.

Macromolecular microscopy is becoming an increasingly important tool for structural biology. The development of improved capabilities for three-dimensional electron microscopy is critical for optimal progress in emerging integrative research in molecular cell biology. These techniques currently suffer from several severe disadvantages related to the tremendous time and effort required to acquire and analyze the data.For several years we have been developing software for automated and intelligent acquisition of transmission electron micrographs [1,2,3]. Our overall goal is to develop a system for rapid and routine structure determination of macromolecular assemblies from specimens preserved in vitreous ice. Ultimately we plan to develop an integrated system that can produce an electron density map of a structure within a few hours of inserting a specimen into the electron microscope. With this goal in mind it is essential that the images be acquired using a digital camera rather than film.

X-Ray crystallography in structural biology.

X-ray crystallography is an important part in elucidating molecular mechanisms of biological reactions since begining of biophysics. Advance in x-ray crystallography techniques has made possible the structure determination of macromolecular assemblies and time-resolved structure analysis. X-ray crystallography will preserve a central role in understanding biological processes at atomic level.