Ab Initio Reconstruction Research Articles

Automated particle correspondence and accurate tilt-axis detection in tilted-image pairs

Tilted electron microscope images are routinely collected for an ab initio structure reconstruction as a part of the Random Conical Tilt (RCT) or Orthogonal Tilt Reconstruction (OTR) methods, as well as for various applications using the "free-hand" procedure. These procedures all require identification of particle pairs in two corresponding images as well as accurate estimation of the tilt-axis used to rotate the electron microscope (EM) grid. Here we present a computational approach, PCT (particle correspondence from tilted pairs), based on tilt-invariant context and projection matching that addresses both problems. The method benefits from treating the two problems as a single optimization task. It automatically finds corresponding particle pairs and accurately computes tilt-axis direction even in the cases when EM grid is not perfectly planar.

Ab-initio reconstruction of genetic code, prediction of T m , C-value paradox and correlation with ANISOU data based on cellular automaton theory with further applications to protein secondary structure prediction, codon-bias and degeneracy

Ab-initio reconstruction of genetic code, prediction of T m , C-value paradox and correlation with ANISOU data based on cellular automaton theory with further applications to protein secondary structure prediction, codon-bias and degeneracy

Quinine Binding by the Cocaine-Binding Aptamer. Thermodynamic and Hydrodynamic Analysis of High-Affinity Binding of an Off-Target Ligand

The cocaine-binding aptamer is unusual in that it tightly binds molecules other than the ligand it was selected for. Here, we study the interaction of the cocaine-binding aptamer with one of these off-target ligands, quinine. Isothermal titration calorimetry was used to quantify the quinine-binding affinity and thermodynamics of a set of sequence variants of the cocaine-binding aptamer. We find that the affinity of the cocaine-binding aptamer for quinine is 30-40 times stronger than it is for cocaine. Competitive-binding studies demonstrate that both quinine and cocaine bind at the same site on the aptamer. The ligand-induced structural-switching binding mechanism of an aptamer variant that contains three base pairs in stem 1 is retained with quinine as a ligand. The short stem 1 aptamer is unfolded or loosely folded in the free form and becomes folded when bound to quinine. This folding is confirmed by NMR spectroscopy and by the short stem 1 construct having a more negative change in heat capacity of quinine binding than is seen when stem 1 has six base pairs. Small-angle X-ray scattering (SAXS) studies of the free aptamer and both the quinine- and the cocaine-bound forms show that, for the long stem 1 aptamers, the three forms display similar hydrodynamic properties, and the ab initio shape reconstruction structures are very similar. For the short stem 1 aptamer there is a greater variation among the SAXS-derived ab initio shape reconstruction structures, consistent with the changes expected with its structural-switching binding mechanism.

Optimod – An automated approach for constructing and optimizing initial models for single-particle electron microscopy

Single-particle cryo-electron microscopy is now well established as a technique for the structural characterization of large macromolecules and macromolecular complexes. The raw data is very noisy and consists of two-dimensional projections, from which the 3D biological object must be reconstructed. The 3D object depends upon knowledge of proper angular orientations assigned to the 2D projection images. Numerous algorithms have been developed for determining relative angular orientations between 2D images, but the transition from 2D to 3D remains challenging and can result in erroneous and conflicting results. Here we describe a general, automated procedure, called OptiMod, for reconstructing and optimizing 3D models using common-lines methodologies. OptiMod approximates orientation angles and reconstructs independent maps from 2D class averages. It then iterates the procedure, while considering each map as a raw solution that needs to be compared with other possible outcomes. We incorporate procedures for 3D alignment, clustering, and refinement to optimize each map, as well as standard scoring metrics to facilitate the selection of the optimal model. We also show that small angle tilt-pair data can be included as one of the scoring metrics to improve the selection of the optimal initial model, and also to provide a validation check. The overall approach is demonstrated using two experimental cryo-EM data sets--the 80S ribosome that represents a relatively straightforward case for ab initio reconstruction, and the Tf-TfR complex that represents a challenging case in that it has previously been shown to provide multiple equally plausible solutions to the initial model problem.

Structural elucidation of full-length nidogen and the laminin–nidogen complex in solution

Nidogen-1 is a key basement membrane protein that is required for many biological activities. It is one of the central elements in organizing basal laminae including those in the skin, muscle, and the nervous system. The self-assembling extracellular matrix that also incorporates fibulins, fibronectin and integrins is clamped together by networks formed between nidogen, perlecan, laminin and collagen IV. To date, the full-length version of nidogen-1 has not been studied in detail in terms of its solution conformation and shape because of its susceptibility to proteolysis. In the current study, we have expressed and purified full-length nidogen-1 and have investigated its solution behavior using size-exclusion chromatography (SEC), dynamic light scattering (DLS) and small angle X-ray scattering (SAXS). The ab initio shape reconstruction of the complex between nidogen-1 and the laminin γ-1 short arm confirms that the interaction is mediated solely by the C-terminal domains: the rest of the domains of both proteins do not participate in complex formation.

PRIME: Probabilistic Initial 3D Model Generation for Single-Particle Cryo-Electron Microscopy

Low-dose electron microscopy of cryo-preserved individual biomolecules (single-particle cryo-EM) is a powerful tool for obtaining information about the structure and dynamics of large macromolecular assemblies. Acquiring images with low dose reduces radiation damage, preserves atomic structural details, but results in low signal-to-noise ratio of the individual images. The projection directions of the two-dimensional images are random and unknown. The grand challenge is to achieve the precise three-dimensional (3D) alignment of many (tens of thousands to millions) noisy projection images, which may then be combined to obtain a faithful 3D map. An accurate initial 3D model is critical for obtaining the precise 3D alignment required for high-resolution (<10Å) map reconstruction. We report a method (PRIME) that, in a single step and without prior structural knowledge, can generate an accurate initial 3D map directly from the noisy images.

All-Atom Ensemble Modeling to Analyze Small-Angle X-Ray Scattering of Glycosylated Proteins

The flexible and heterogeneous nature of carbohydrate chains often renders glycoproteins refractory to traditional structure determination methods. Small-angle X-ray scattering (SAXS) can be a useful tool for obtaining structural information of these systems. All-atom modeling of glycoproteins with flexible glycan chains was applied to interpret the solution SAXS data for a set of glycoproteins. For simpler systems (single glycan, with a well-defined protein structure), all-atom modeling generates models in excellent agreement with the scattering pattern and reveals the approximate spatial occupancy of the glycan chain in solution. For more complex systems (several glycan chains, or unknown protein substructure), the approach can still provideinsightful models, though the orientations of glycans become poorly determined. Ab initio shape reconstructions appear to capture the global morphology of glycoproteins but in most cases offer littleinformation about glycan spatial occupancy. The all-atom modeling methodology is available as a web server at http://salilab.org/allosmod-foxs.

SIMPLE: Software for ab initio reconstruction of heterogeneous single-particles

The open source software suite SIMPLE: Single-particle IMage Processing Linux Engine provides data analysis methods for single-particle cryo-electron microscopy (cryo-EM). SIMPLE addresses the problem of obtaining 3D reconstructions from 2D projections only, without using an input reference volume for approximating orientations. The SIMPLE reconstruction algorithm is tailored to asymmetrical and structurally heterogeneous single-particles. Its basis is global optimization with the use of Fourier common lines. The advance that enables ab initio reconstruction and heterogeneity analysis is the separation of the tasks of in-plane alignment and projection direction determination via bijective orientation search - a new concept in common lines-based strategies. Bijective orientation search divides the configuration space into two groups of paired parameters that are optimized separately. The first group consists of the rotations and shifts in the plane of the projection; the second group consists of the projection directions and state assignments. In SIMPLE, ab initio reconstruction is feasible because the 3D in-plane alignment is approximated using reference-free 2D rotational alignment. The subsequent common lines-based search hence searches projection directions and states only. Thousands of class averages are analyzed simultaneously in a matter of hours. Novice SIMPLE users get a head start via the well documented front-end. The structured, object-oriented back-end invites advanced users to develop new alignment and reconstruction algorithms. An overview of the package is presented together with benchmarks on simulated data. Executable binaries, source code, and documentation are available at http://simple.stanford.edu.

Determining pair distance distribution function from SAXS data using parametric functionals

Small angle X-ray scattering (SAXS) experiments are widely applied in structural biology. The SAXS experiments yield one-dimensional profile that needs further analysis to reveal structural information. The pair distance distribution function (PDDF), P(r), can provide molecular structures more intuitively, and it can be used to guide ab initio model reconstructions, making it a critical step to derive P(r) from experimental SAXS profiles. To calculate the P(r) curves, a new method based on a specially designed parametric functional form is developed, and implemented in pregxs. This method is tested against both synthetic and experimental data, the estimated P(r) functions are in good agreement with correct or known P(r). The method can also predict the molecular size. In summary, the pregxs method is robust and accurate in P(r) determination from SAXS profiles. The pregxs source code and an online server are available at http://www.sastbx.als.lbl.gov.

Abstract 929: Transcriptome sequencing identifies novel non-coding RNAs associated with prostate cancer progression

Abstract Long intervening non-coding RNAs (lincRNAs) have been implicated in diverse biological processes including p53 signaling and chromatin remodeling, but have not been thoroughly profiled in human cancers. Here, we have developed an approach for ab initio reconstruction of poly-A+ transcriptome sequencing (RNA-seq) data for the unbiased discovery of novel transcripts. To accomplish this, we developed AssemblyLine, a method that clusters and filters large collections of transcripts to produce a consensus transcriptome. To demonstrate AssemblyLine, we sequenced a cohort comprised of 81 prostatic tissues (20 benign, 47 localized tumors, and 14 metastases) and 21 prostatic cell lines using the Illumina Genome Analyzer II and generated 1.723 billion sequence fragments. We successfully aligned 1.42 billion reads with Tophat – a program capable of ab initio splice junction discovery – and then used Cufflinks to model sample-specific transcriptomes totaling 8.25 million transcripts. AssemblyLine condensed the 8.25 million original transcripts into 35,415 distinct transcriptional loci, of which 1,859 (5.2%) represented candidate lincRNAs that lacked genomic overlap with known gene annotations. These putative RNAs lacked robust open reading frames suggesting that the vast majority were non-coding. Further, they exhibited evolutionary conservation and were enriched with histone modifications supporting independent transcriptional start sites and active transcription. Together, these results add confidence to AssemblyLine's nomination process and suggest that these novel lincRNAs may be transcriptionally active in prostate cancer. We then selected 106 transcripts that were differentially expressed in localized prostate cancer when compared to benign adjacent tissue (False Discovery Rate &amp;lt; 0.05), and 15 transcripts with profound cancer outlier expression profiles for further study. These 121 Prostate Cancer Associated Transcripts (PCATs) accurately classified benign, localized, and metastatic prostate cancer tissues by unsupervised hierarchical clustering. Consistent with AssemblyLine's nominations, PCR-based experiments on selected transcripts in an independent tissue cohort showed high validation rates for the transcript structure and expression level predictions. Furthermore, in vitro studies of PCAT-1, a novel lincRNA observed in our dataset as highly upregulated in prostate cancer, revealed direct regulation by the histone methyltransferase EZH2. siRNA knockdown of PCAT-1 in LNCaP, a prostate cancer cell line, caused a 25-50% decrease in cell proliferation. Thus, this study establishes a paradigm for ab initio transcriptome annotation and discovery of novel lincRNAs in cancer tissues. Further, these results provide intriguing evidence that lincRNAs are aberrantly expressed and may play a role in prostate cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 929. doi:10.1158/1538-7445.AM2011-929

New Light on Disordered Ensembles:Ab InitioStructure Determination of One Particle from Scattering Fluctuations of Many Copies

We report on the first experimental abinitio reconstruction of an image of a single particle from fluctuations in the scattering from an ensemble of copies, randomly oriented about an axis. The method is applicable to identical particles frozen in space or time (as by snapshot diffraction from an x-ray free electron laser). These fluctuations enhance information obtainable from an experiment such as conventional small angle x-ray scattering.

Characterizing Intrinsically Disordered Proteins by Small-Angle Neutron Scattering

Intrinsically disordered proteins (IDPs) that undergo a coupled folding and binding event are found to be important in many recognition and signaling processes. However, exactly how this mechanism is linked to these functions is not completely understood. This is primarily because the structural flexibility of IDPs limits the number of suitable characterization techniques. We are using small-angle neutron scattering (SANS) to investigate the structure and binding interaction properties between NCBD, an IDP region of CREB binding protein (CBP), and its binding partner, ACTR, which also is an IDP. CBP is a transcription co-activator that is essential in embryonic development, growth control, and homeostasis, and its dysfunction is implicated in neurological disorders such as Huntington's disease and some cancers. SANS indicates the NCBD/ACTR complex is a globular, folded structure with a smaller radius of gyration compared to ACTR alone, which is mostly unfolded. Using ab initio shape reconstruction programs to gain further insight into structural flexibility, we find good agreement between the shape reconstruction and NMR structure for the NCBD/ACTR complex. In contrast, SANS reveals the nature of how ACTR is more expanded when in its unbound, unfolded state. This research should provide new possibilities for the study of disordered protein regions and yield unique perspectives into the mechanism of IDP function.

Structural Variations in the Aggregation Pathways of Normal and Pathological Huntingtin-Like Peptides

The current challenge in the amyloid field is to obtain structural information on the progression of associated aggregation pathways, relate them to the cause of disease, and specifically point to the species truly responsible for neurotoxicity. Huntington's disease (HD) is linked to the aggregation of mutant huntingtin (htt) peptide, which contains an abnormally long polyglutamine (polyGln) repeat that leads to the formation of fibrils with stable, β-sheet rich structures. Analogous to other neurodegenerative diseases like Alzheimer's, mature htt fibrils are thought to be overall protective while the earlier smaller intermediates are proposed to be toxic to neuronal cells. We performed time-resolved small-angle neutron scattering (SANS) experiments to monitor the structural kinetics for htt peptides having polyGln repeats in the normal (22 Glns) and pathological (42 Glns) range. In addition to observing the expected faster aggregation rate for longer polyGln repeats, we find distinct conformational differences between normal and pathological htt. We also present, for the first time, three-dimensional structures of intermediates formed at the earliest stages of htt aggregation as detected by SANS and obtained using ab initio shape reconstruction methods. Finally, we are able to investigate the internal structure of the mature fibrils, where the mass-per-length of pathological htt fibrils is considerably less than normal htt fibrils. These findings provide the first steps toward characterizing the oligomers formed by htt peptides and illustrate the utility of SANS for identifying different aggregate intermediates formed in the development of neurodegenerative diseases.

Biophysical analysis and small-angle X-ray scattering-derived structures of MeCP2-nucleosome complexes.

MeCP2 is a highly abundant chromatin architectural protein with key roles in post-natal brain development in humans. Mutations in MeCP2 are associated with Rett syndrome, the main cause of mental retardation in girls. Structural information on the intrinsically disordered MeCP2 protein is restricted to the methyl-CpG binding domain; however, at least four regions capable of DNA and chromatin binding are distributed over its entire length. Here we use small angle X-ray scattering (SAXS) and other solution-state approaches to investigate the interaction of MeCP2 and a truncated, disease-causing version of MeCP2 with nucleosomes. We demonstrate that MeCP2 forms defined complexes with nucleosomes, in which all four histones are present. MeCP2 retains an extended conformation when binding nucleosomes without extra-nucleosomal DNA. In contrast, nucleosomes with extra-nucleosomal DNA engage additional DNA binding sites in MeCP2, resulting in a rather compact higher-order complex. We present ab initio envelope reconstructions of nucleosomes and their complexes with MeCP2 from SAXS data. SAXS studies also revealed unexpected sequence-dependent conformational variability in the nucleosomes themselves.

Ab initioreconstruction of difference densities by charge flipping

The charge-flipping algorithm in its band-flipping variant is capable of ab initio reconstructions of scattering densities with positive and negative values. It is shown that the method can be applied to reconstructions of difference electron densities of superstructures, i.e. densities obtained as a difference between the true scattering density and the average density over two or more subcells of the true structure. The amplitudes of reflections lying on the reciprocal lattice of the subcell are not required for the procedure. A series of examples shows applications of the method to the solution of superstructures in periodic crystals or quasicrystals as well as the application to ab initio solution of modulation of an incommensurately modulated structure from satellite reflections only and solution of a structure from a crystal twinned by reticular pseudomerohedry. The method is especially suited for solving pseudosymmetry problems occurring frequently in superstructures.

Ab initio high-resolution single-particle 3D reconstructions: The symmetry adapted functions way

A protocol to attain high-resolution single-particle reconstructions is presented. The protocol is the concatenation of two procedures: one to obtain an ab initio low-resolution reconstruction, the other to determine a fixed point of the consecutive applications of fast projection matching and 3D reconstruction. It is a reciprocal space formulation where the Fourier coefficients of the 3D scattering density are expressed in terms of symmetry adapted functions and the 2D particle images are represented by their Fourier-Bessel transforms. The new protocol shows advantages in terms of speed and accuracy when compared to other methods currently in use. We illustrate its performance as applied to high-resolution cryo-electron micrographs of rotavirus.

Erratum: Corrigendum: Ab initio reconstruction of cell type–specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincRNAs

Nat. Biotechnol. 28, 503–510 (2010); published online 02 May 2010; corrected after print 9 July 2010 In the version of this article initially published, the fourth sentence in the Online Methods section “RNA extraction and library preparation,” that read in part “procedure that combines a random priming step with a shearing step8,9, 28 and results in fragments of ∼700 bp in size,” should have read, “procedure that combines fragmentation of mRNA to a peak size of ∼750 nucleotides by heating6 followed by random-primed reverse transcription8.

Ab initio reconstruction of cell type–specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincRNAs

RNA-Seq provides an unbiased way to study a transcriptome, including both coding and non-coding genes. To date, most RNA-Seq studies have critically depended on existing annotations, and thus focused on expression levels and variation in known transcripts. Here, we present Scripture, a method to reconstruct the transcriptome of a mammalian cell using only RNA-Seq reads and the genome sequence. We apply it to mouse embryonic stem cells, neuronal precursor cells, and lung fibroblasts to accurately reconstruct the full-length gene structures for the vast majority of known expressed genes. We identify substantial variation in protein-coding genes, including thousands of novel 5′-start sites, 3′-ends, and internal coding exons. We then determine the gene structures of over a thousand lincRNA and antisense loci. Our results open the way to direct experimental manipulation of thousands of non-coding RNAs, and demonstrate the power of ab initio reconstruction to render a comprehensive picture of mammalian transcriptomes.

A Toolbox for ab initio 3-D reconstructions in single-particle electron microscopy

Structure determination of a novel macromolecular complex via single-particle electron microscopy depends upon overcoming the challenge of establishing a reliable 3-D reconstruction using only 2-D images. There are a variety of strategies that deal with this issue, but not all of them are readily accessible and straightforward to use. We have developed a "toolbox" of ab initio reconstruction techniques that provide several options for calculating 3-D volumes in an easily managed and tightly controlled work-flow that adheres to standard conventions and formats. This toolbox is designed to streamline the reconstruction process by removing the necessity for bookkeeping, while facilitating transparent data transfer between different software packages. It currently includes procedures for calculating ab initio reconstructions via random or orthogonal tilt geometry, tomograms, and common lines, all of which have been tested using the 50S ribosomal subunit. Our goal is that the accessibility of multiple independent reconstruction algorithms via this toolbox will improve the ease with which models can be generated, and provide a means of evaluating the confidence and reliability of the final reconstructed map.

Cryo-EM Reveals Promoter DNA Binding and Conformational Flexibility of the General Transcription Factor TFIID

The general transcription factor IID (TFIID) is required for initiation of RNA polymerase II-dependent transcription at many eukaryotic promoters. TFIID comprises the TATA-binding protein (TBP) and several conserved TBP-associated factors (TAFs). Recognition of the core promoter by TFIID assists assembly of the preinitiation complex. Using cryo-electron microscopy in combination with methods for ab initio single-particle reconstruction and heterogeneity analysis, we have produced density maps of two conformational states of Schizosaccharomyces pombe TFIID, containing and lacking TBP. We report that TBP-binding is coupled to a massive histone-fold domain rearrangement. Moreover, docking of the TBP-TAF1(N-terminus) atomic structure to the TFIID map and reconstruction of a TAF-promoter DNA complex helps to account for TAF-dependent regulation of promoter-TBP and promoter-TAF interactions.