Ab initio resolution measurement for single particle structures

A computational method is described that allows the measurement of the signal-to-noise ratio and resolution of a three-dimensional structure obtained by single particle electron microscopy and reconstruction. The method does not rely on the availability of the original image data or the calculation of several structures from different parts of the data that are needed for the commonly used Fourier Shell Correlation criterion. Instead, the correlation between neighboring Fourier pixels is calculated and used to distinguish signal from noise. The new method has been conveniently implemented in a computer program called RMEASURE and is available to the microscopy community.     

Single particle refinement in electron crystallography: a pilot study

Electron crystallography can be used to determine the structures of membrane proteins at near-atomic resolution in some cases. However, most electron crystallography projects remain at a resolution around 10 Å. This might be partly due to lack of flatness of many two-dimensional crystals. We have investigated this problem and suggest single particle processing of locally averaged unit cells to improve the quality and possibly the resolution of three-dimensional maps. Applying this method to the secondary transporter melibiose permease we have calculated a three-dimensional map that is clearer and easier to interpret than the map derived using purely electron-crystallographic methods.     

Ensemble refinement of protein crystal structures: validation and application

X-ray crystallography typically uses a single set of coordinates and B factors to describe macromolecular conformations. Refinement of multiple copies of the entire structure has been previously used in specific cases as an alternative means of representing structural flexibility. Here, we systematically validate this method by using simulated diffraction data, and we find that ensemble refinement produces better representations of the distributions of atomic positions in the simulated structures than single-conformer refinements. Comparison of principal components calculated from the refined ensembles and simulations shows that concerted motions are captured locally, but that correlations dissipate over long distances. Ensemble refinement is also used on 50 experimental structures of varying resolution and leads to decreases in R(free) values, implying that improvements in the representation of flexibility observed for the simulated structures may apply to real structures. These gains are essentially independent of resolution or data-to-parameter ratio, suggesting that even structures at moderate resolution can benefit from ensemble refinement.     

Highly constrained multiple-copy refinement of protein crystal structures

In the course of refining atomic protein structures, one often encounters difficulty with molecules that are unusually flexible or otherwise disordered. We approach the problem by combining two relatively recent developments: simultaneous refinement of multiple protein conformations and highly constrained refinement. A constrained Langevin dynamics refinement is tested on two proteins: neurotrophin-3 and glutamine synthetase. The method produces closer agreement between the calculated and observed scattering amplitudes than standard, single-copy, Gaussian atomic displacement parameter refinement. This is accomplished without significantly increasing the number of fitting parameters in the model. These results suggest that loop motion in proteins within a crystal lattice can be extensive and that it is poorly modeled by isotropic Gaussian distributions for each atom. Proteins 29:426–432, 1997. © 1997 Wiley-Liss, Inc.     

Correlating ribosome function with high-resolution structures

Ribosome research has undergone astonishing progress in recent years. Crystal structures have shed light on the functional properties of the translation machinery and revealed how the striking architecture of the ribosome is ingeniously designed as the framework for its unique capabilities: precise decoding, substrate-mediated peptide-bond formation and efficient polymerase activity. New findings include the two concerted elements of tRNA translocation: sideways shift and a ribosomal-navigated rotatory motion; the dynamics of the nascent-chain exit tunnel and the shelter formed by the ribosome-bound trigger-factor, which acts as a chaperone to prevent nascent-chain aggregation and misfolding. The availability of these structures has also illuminated the action, selectivity, resistance and synergism of antibiotics that target ribosomes.     

Practical considerations for using K3 cameras in CDS mode for high-resolution and high-throughput single particle cryo-EM

Detector technology plays a pivotal role in high-resolution and high-throughput cryo-EM structure determination. Compared with the first-generation, single-electron counting direct detection camera (Gatan K2), the latest K3 camera is faster, larger, and now offers a correlated-double sampling mode (CDS). Importantly this results in a higher DQE and improved throughput compared to its predecessor. In this study, we focused on optimizing camera data collection parameters for daily use within a cryo-EM facility and explored the balance between throughput and resolution. In total, eight data sets of murine heavy-chain apoferritin were collected at different dose rates and magnifications, using 9-hole image shift data collection strategies. The performance of the camera was characterized by the quality of the resultant 3D reconstructions. Our results demonstrated that the Gatan K3 operating in CDS mode outperformed standard (nonCDS) mode in terms of reconstruction resolution in all tested conditions with 8 electrons per pixel per second being the optimal dose rate. At low magnification (64kx) we were able to achieve reconstruction resolutions of 149% of the physical Nyquist limit (1.8 Å with a 1.346 Å physical pixel size). Low magnification allows more particles to be collected per image, aiding analysis of heterogeneous samples requiring large data sets. At moderate magnification (105kx, 0.834 Å physical pixel size) we achieved a resolution of 1.65 Å within 8-h of data collection, a condition optimal for achieving high-resolution on well behaved samples. Our results also show that for an optimal sample like apoferritin, one can achieve better than 2.5 Å resolution with 5 min of data collection. Together, our studies validate the most efficient ways of imaging protein complexes using the K3 direct detector and will greatly benefit the cryo-EM community.     

Isolation and characterization of a novel ribonucleoprotein particle: large structures contain a single species of small RNA

Rat liver coated vesicle preparations were frequently found to contain small ovoid bodies, which resembled coated vesicles in morphology. We have purified these bodies to homogeneity using sucrose density gradients and preparative agarose gel electrophoresis. When negatively stained and viewed by electron microscopy, the purified structures display a very distinct and complex morphology, resembling the multiple arches which form cathedral vaults. They measure 35 X 65 nm and are therefore considerably larger than ribosomes. When subjected to SDS PAGE, these structures, which we refer to as vaults, appear to contain several minor and five major species: Mr 210,000, 192,000, 104,000, 54,000, and 37,000. One of these (Mr 104,000) greatly predominates, accounting for greater than 70% of the total Coomassie Brilliant Blue-staining protein. Another major species of Mr 37,000 has been identified as a species of small RNA of unusual base composition (adenosine 12.0%, guanosine 29.7%, uridine 30.9%, and 27.4% cytidine), which migrates as a single species in urea PAGE between the 5S and 5.8S ribosomal standards, containing approximately 140 bases. Although the RNA constitutes only 4.6% of the entire structure, the large size of the particle requires that each one contains approximately 9 molecules of this RNA. Antibodies prepared against the entire particle are largely specific for the major (Mr 104,000) polypeptide species. Although they do not directly react with the RNA constituent on Western blots, these antibodies immunoprecipitate a 32P-labeled RNA of identical size from metabolically-labeled rat hepatoma cells. Vaults are observed in partially purified fractions from human fibroblasts, murine 3T3 cells, glial cells, and rabbit alveolar macrophages. It therefore appears that these novel ribonucleoprotein structures are broadly distributed among different cell types. The function of vaults is at present unknown.     

Ribosomal crystallography: from poorly diffracting microcrystals to high-resolution structures

The cellular organelles translating the genetic code into proteins, the ribosomes, are large, asymmetric, flexible, and unstable ribonucleoprotein assemblies, hence they are difficult to crystallize. Despite two decades of intensive effort and thorough searches for suitable sources, so far only three crystal types have yielded high-resolution structures: two large subunits (from an archaean and from a mesophilic eubacterium) and one thermophilic small subunit. These structures have added to our understanding of decoding, have revealed dynamic aspects of the biosynthetic process, and have indicated the strategies adopted by ribosomes for interacting between themselves as well as with inhibitors, factors and substrates.     

Real space refinement of acto-myosin structures from sectioned muscle

We have adapted a real space refinement protocol originally developed for high-resolution crystallographic analysis for use in fitting atomic models of actin filaments and myosin subfragment 1 (S1) to 3-D images of thin-sectioned, plastic-embedded whole muscle. The rationale for this effort is to obtain a refinement protocol that will optimize the fit of the model to the density obtained by electron microscopy and correct for poor geometry introduced during the manual fitting of a high-resolution atomic model into a lower resolution 3-D image. The starting atomic model consisted of a rigor acto-S1 model obtained by X-ray crystallography and helical reconstruction of electron micrographs. This model was rebuilt to fit 3-D images of rigor insect flight muscle at a resolution of 7 nm obtained by electron tomography and image averaging. Our highly constrained real space refinement resulted in modest improvements in the agreement of model and reconstruction but reduced the number of conflicting atomic contacts by 70% without loss of fit to the 3-D density. The methodology seems to be well suited to the derivation of stereochemically reasonable atomic models that are consistent with experimentally determined 3-D reconstructions computed from electron micrographs.     

Acoustically mounted microcrystals yield high-resolution X-ray structures

We demonstrate a general strategy for determining structures from showers of microcrystals. It uses acoustic droplet ejection to transfer 2.5 nL droplets from the surface of microcrystal slurries, through the air, onto mounting micromesh pins. Individual microcrystals are located by raster-scanning a several-micrometer X-ray beam across the cryocooled micromeshes. X-ray diffraction data sets merged from several micrometer-sized crystals are used to determine 1.8 ?? resolution crystal structures.     

Quantitative high-resolution genomic analysis of single cancer cells

During cancer progression, specific genomic aberrations arise that can determine the scope of the disease and can be used as predictive or prognostic markers. The detection of specific gene amplifications or deletions in single blood-borne or disseminated tumour cells that may give rise to the development of metastases is of great clinical interest but technically challenging. In this study, we present a method for quantitative high-resolution genomic analysis of single cells. Cells were isolated under permanent microscopic control followed by high-fidelity whole genome amplification and subsequent analyses by fine tiling array-CGH and qPCR. The assay was applied to single breast cancer cells to analyze the chromosomal region centred by the therapeutical relevant EGFR gene. This method allows precise quantitative analysis of copy number variations in single cell diagnostics.     

SwarmPS: rapid, semi-automated single particle selection software

Single particle analysis (SPA) coupled with high-resolution electron cryo-microscopy is emerging as a powerful technique for the structure determination of membrane protein complexes and soluble macromolecular assemblies. Current estimates suggest that approximately 10(4)-10(5) particle projections are required to attain a 3A resolution 3D reconstruction (symmetry dependent). Selecting this number of molecular projections differing in size, shape and symmetry is a rate-limiting step for the automation of 3D image reconstruction. Here, we present Swarm(PS), a feature rich GUI based software package to manage large scale, semi-automated particle picking projects. The software provides cross-correlation and edge-detection algorithms. Algorithm-specific parameters are transparently and automatically determined through user interaction with the image, rather than by trial and error. Other features include multiple image handling (approximately 10(2)), local and global particle selection options, interactive image freezing, automatic particle centering, and full manual override to correct false positives and negatives. Swarm(PS) is user friendly, flexible, extensible, fast, and capable of exporting boxed out projection images, or particle coordinates, compatible with downstream image processing suites.     

Fitting of high-resolution structures into electron microscopy reconstruction images

Dynamic macromolecular assemblies, such as ribosomes, viruses, and muscle protein complexes, are often more amenable to visualization by electron microscopy than by high-resolution X-ray crystallography or NMR. When high-resolution structures of component structures are available, it is possible to build an atomic model that gives information about the molecular interactions at greater detail than the experimental resolution, due to constraints of modeling placed upon the interpretation. There are now several competing computational methods to search systematically for orientations and positions of components that match the experimental image density, and continuing developments will be reviewed. Attention is now also moving toward the related task of optimization, with flexible and/or multifragment models and sometimes with stereochemically restrained refinement methods. This paper will review the various approaches and describe advances in the authors' methods and applications of real-space refinement.     

Real space refinement of crystal structures with canonical distributions of electrons

Recurring groups of atoms in molecules are surrounded by specific canonical distributions of electrons. Deviations from these distributions reveal unrealistic molecular geometries. Here, we show how canonical electron densities can be combined with classical electron densities derived from X-ray diffraction experiments to drive the real space refinement of crystal structures. The refinement process generally yields superior molecular models with reduced excess electron densities and improved stereochemistry without compromising the agreement between molecular models and experimental data.     

Computational refinement of functional single nucleotide polymorphisms associated with ATM gene

Background

Understanding and predicting molecular basis of disease is one of the major challenges in modern biology and medicine. SNPs associated with complex disorders can create, destroy, or modify protein coding sites. Single amino acid substitutions in the ATM gene are the most common forms of genetic variations that account for various forms of cancer. However, the extent to which SNPs interferes with the gene regulation and affects cancer susceptibility remains largely unknown.

Principal findings

We analyzed the deleterious nsSNPs associated with ATM gene based on different computational methods. An integrative scoring system and sequence conservation of amino acid residues was adapted for a priori nsSNP analysis of variants associated with cancer. We further extended our approach on SNPs that could potentially influence protein Post Translational Modifications in ATM gene.

Significance

In the lack of adequate prior reports on the possible deleterious effects of nsSNPs, we have systematically analyzed and characterized the functional variants in both coding and non coding region that can alter the expression and function of ATM gene. In silico characterization of nsSNPs affecting ATM gene function can aid in better understanding of genetic differences in disease susceptibility.     

Estimation of uncertainties in X-ray refinement results by use of perturbed structures

The uncertainties in the refined parameters for a 1.5-A X-ray structure of carbon-monoxy (FeII) myoglobin are estimated by combining energy minimization with least-squares refinement against the X-ray data. The energy minimizations, done without reference to the X-ray data, provide perturbed structures which are used to restart conventional X-ray refinement. The resulting refined structures have the same, or better, R-factor and stereochemical parameters as the original X-ray structure, but deviate from it by 0.13 A rms for the backbone atoms and 0.31 A rms for the sidechain atoms. Atoms interacting with a disordered sidechain, Arg 45 CD3, are observed to have larger positional uncertainties. The uncertainty in the B-factors, within the isotropic harmonic motion approximation, is estimated to be 15%. The resulting X-ray structures are more consistent with the energy parameters used in simulations.