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Pawel A Penczek

Publications and source records attributed to Pawel A Penczek.

At least 19 recordsLinked to original sources
PubMed2006

Structure of the ribosome-bound cricket paralysis virus IRES RNA.

Internal ribosome entry sites (IRESs) facilitate an alternative, end-independent pathway of translation initiation. A particular family of dicistroviral IRESs can assemble elongation-competent 80S ribosomal complexes in the absence of canonical initiation factors and initiator transfer RNA. We present here a cryo-EM reconstruction of a dicistroviral IRES bound to the 80S ribosome. The resolution of the cryo-EM reconstruction, in the subnanometer range, allowed the molecular structure of the complete IRES in its active, ribosome-bound state to be solved. The structure, harboring three pseudoknot-containing domains, each with a specific functional role, shows how defined elements of the IRES emerge from a compactly folded core and interact with the key ribosomal components that form the A, P and E sites, where tRNAs normally bind. Our results exemplify the molecular strategy for recruitment of an IRES and reveal the dynamic features necessary for internal initiation.

Animals↗
PubMed2006

SPARX, a new environment for Cryo-EM image processing.

SPARX (single particle analysis for resolution extension) is a new image processing environment with a particular emphasis on transmission electron microscopy (TEM) structure determination. It includes a graphical user interface that provides a complete graphical programming environment with a novel data/process-flow infrastructure, an extensive library of Python scripts that perform specific TEM-related computational tasks, and a core library of fundamental C++ image processing functions. In addition, SPARX relies on the EMAN2 library and cctbx, the open-source computational crystallography library from PHENIX. The design of the system is such that future inclusion of other image processing libraries is a straightforward task. The SPARX infrastructure intelligently handles retention of intermediate values, even those inside programming structures such as loops and function calls. SPARX and all dependencies are free for academic use and available with complete source.

Computational Biology↗
PubMed2006

The Transform Class in SPARX and EMAN2.

We describe the Transform Class in SPARX/EMAN2 that is designed to handle rigid body motions of two- and three-dimensional data. We describe relationships between Eulerian angles conventions used in different electron microscopy software packages, as well as give examples of the simple scripts that execute conversions of Eulerian angles between these packages and handle other related tasks. The Transform Class is also responsible for generating point-group symmetry operations as well as generating quasi-evenly distributed points on the sphere (which is used in creating reference projections for structure refinement procedures in single particle reconstruction). We discuss how this is carried out internally in the code, and how symmetry operations are accessed through the SPARX interactive interface. We present a comprehensive description of symmetry operations for all point-group symmetries, as implemented in the class. Finally, we provide solutions to a number of typical problems associated with rotation operations-alignment of markers for dual-axis tomography, delineations of asymmetric subunits, quasi-uniform distribution of projection directions and such, and provide examples how these problems are solved using operations in the Transform Class.

Computational Biology↗
PubMed2006

The parallelization of SPIDER on distributed-memory computers using MPI.

We describe the strategies and implementation details we employed to parallelize the SPIDER software package on distributed-memory parallel computers using the message passing interface (MPI). The MPI-enabled SPIDER preserves the interactive command line and batch interface used in the sequential version of SPIDER, thus does not require users to modify their existing batch programs. We show the excellent performance of the MPI-enabled SPIDER when it is used to perform multi-reference alignment and 3-D reconstruction operations on a number of different computing platforms. We point out some performance issues when the MPI-enabled SPIDER is used for a complete 3-D projection matching refinement run, and propose several ways to further improve the parallel performance of SPIDER on distributed-memory machines.

Algorithms↗
PubMed2006

A method of focused classification, based on the bootstrap 3D variance analysis, and its application to EF-G-dependent translocation.

The bootstrap-based method for calculation of the 3D variance in cryo-EM maps reconstructed from sets of their projections was applied to a dataset of functional ribosomal complexes containing the Escherichia coli 70S ribosome, tRNAs, and elongation factor G (EF-G). The variance map revealed regions of high variability in the intersubunit space of the ribosome: in the locations of tRNAs, in the putative location of EF-G, and in the vicinity of the L1 protein. This result indicated heterogeneity of the dataset. A method of focused classification was put forward in order to sort out the projection data into approximately homogenous subsets. The method is based on the identification and localization of a region of high variance that a subsequent classification step can be focused on by the use of a 3D spherical mask. After initial classification, template volumes are created and are subsequently refined using a multireference 3D projection alignment procedure. In the application to the ribosome dataset, the two resulting structures were interpreted as resulting from ribosomal complexes with bound EF-G and an empty A site, or, alternatively, from complexes that had no EF-G bound but had both A and P sites occupied by tRNA. The proposed method of focused classification proved to be a successful tool in the analysis of the heterogeneous cryo-EM dataset. The associated calculation of the correlations within the density map confirmed the conformational variability of the complex, which could be interpreted in terms of the ribosomal elongation cycle.

Cryoelectron Microscopy↗
PubMed2006

Estimation of variance in single-particle reconstruction using the bootstrap technique.

Density maps of a molecule obtained by single-particle reconstruction from thousands of molecule projections exhibit strong changes in local definition and reproducibility, as a consequence of conformational variability of the molecule and non-stoichiometry of ligand binding. These changes complicate the interpretation of density maps in terms of molecular structure. A three-dimensional (3-D) variance map provides an effective tool to assess the structural definition in each volume element. In this work, the different contributions to the 3-D variance in a single-particle reconstruction are discussed, and an effective method for the estimation of the 3-D variance map is proposed, using a bootstrap technique of sampling. Computations with test data confirm the viability, computational efficiency, and accuracy of the method under conditions encountered in practical circumstances.

Algorithms↗
PubMed2006

Cryo-electron microscopy studies of human TFIID: conformational breathing in the integration of gene regulatory cues.

The multisubunit transcription factor TFIID is essential for directing eukaryotic promoter recognition and mediating interactions with activators/cofactors during assembly of the preinitiation complex. Despite its central role in transcription initiation and regulation, structural knowledge of the TFIID complex has so far been largely limited to electron microscopy studies of negatively stained samples. Here, we present a cryo-electron microscopy 3D reconstruction of the large endogenous human TFIID complex. The improved cryopreservation has allowed for a more detailed definition of the structural elements in the complex and for the detection, by an extensive statistical analysis of the data, of a conformational opening and closing of the cavity central to the TFIID architecture. We propose that these density rearrangements in the structure are a likely reflection of the plasticity of the interactions between TFIID and its many partner proteins.

Cryoelectron Microscopy↗
PubMed2006

The endosome-associated protein Hrs is hexameric and controls cargo sorting as a "master molecule".

The structure of the endosomal-associated protein, Hrs, has been determined with cryo-electron microscopy. Hrs interacts with a number of proteins, including SNAP-25 and STAM1, forming a complex that binds ubiquitin moieties. Analytical ultracentrifugation studies revealed that Hrs exists as a hexamer. The symmetry and the structure of the hexameric form of Hrs were determined with the single-particle reconstruction method. Hrs comprises three antiparallel dimers with a central core and distinct caps on either end. Crystal structures of VHS and FYVE domains fit into the Hrs end caps in the EM density map. Thus, the location of domains that interact with the endosomal membrane, the VHS, FYVE, and C-terminal domains, facilitates the anchorage of Hrs to the membrane, initiating the functional processes of Hrs on the endosome. Based on our model, the Hrs hexamer interacts with the membrane and acts as a "master molecule" that presents multiple sites for protein binding.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Unified 3-D structure and projection orientation refinement using quasi-Newton algorithm.

We describe an algorithm for simultaneous refinement of a three-dimensional (3-D) density map and of the orientation parameters of two-dimensional (2-D) projections that are used to reconstruct this map. The application is in electron microscopy, where the 3-D structure of a protein has to be determined from a set of 2-D projections collected at random but initially unknown angles. The design of the algorithm is based on the assumption that initial low resolution approximation of the density map and reasonable guesses for orientation parameters are available. Thus, the algorithm is applicable in final stages of the structure refinement, when the quality of the results is of main concern. We define the objective function to be minimized in real space and solve the resulting nonlinear optimization problem using a Quasi-Newton algorithm. We calculate analytical derivatives with respect to density distribution and the finite difference approximations of derivatives with respect to orientation parameters. We demonstrate that calculation of derivatives is robust with respect to noise in the data. This is due to the fact that noise is annihilated by the back-projection operations. Our algorithm is distinguished from other orientation refinement methods (i) by the simultaneous update of the density map and orientation parameters resulting in a highly efficient computational scheme and (ii) by the high quality of the results produced by a direct minimization of the discrepancy between the 2-D data and the projected views of the reconstructed 3-D structure. We demonstrate the speed and accuracy of our method by using simulated data.

Algorithms↗
PubMed2005

Estimating alignment errors in sets of 2-D images.

We describe a robust and accurate method for the estimation of alignment errors for a set of two-dimensional images, in the case where the true pattern is unknown. The intended application of the proposed method is cryo-electron microscopy, where two-dimensional views of individual proteins in random orientations are observed in the electron microscope at low signal-to-noise ratio. By representing images in the basis of Fourier-harmonic coordinates and constructing averages and average intensities, we demonstrate that the variances of translation and rotational errors as well as of the Gaussian noise can be recovered. This machinery therefore allows one to isolate the various categories of errors that impede the quality of results in single particle reconstructions into constituent parts: translational errors, rotational errors, and additive noise.

Algorithms↗
PubMed2005

ERj1p uses a universal ribosomal adaptor site to coordinate the 80S ribosome at the membrane.

Ribosomes translating secretory and membrane proteins are targeted to the endoplasmic reticulum membrane and attach to the protein-conducting channel and ribosome-associated membrane proteins (RAMPs). Recently, a new RAMP, ERj1p, has been identified that recruits BiP to ribosomes and regulates translational activity. Here we present the cryo-EM structure of a ribosome-ERj1p complex, revealing how ERj1p coordinates the ribosome at the membrane and how allosteric effects may mediate ERj1p's regulatory activity.

Animals↗
PubMed2004

Rearrangement of the 16S precursor subunits is essential for the formation of the active 20S proteasome.

Proteasome-dependent proteolysis is essential for a number of key cellular processes and requires a sophisticated biogenesis pathway to function. Here, we have arrested the assembly process in its dynamic progression at the short-lived 16S state. Structural analysis of the 16S proteasome precursor intermediates by electron microscopy, and single particle analysis reveals major conformational changes in the structure of the beta-ring in comparison with one-half of the 20S proteasome. The individual beta-subunits in the 16S precursor complex rotate with respect to their positions in the x-ray crystallographic structure of the fully assembled 20S. This rearrangement results in a movement of the catalytic residue threonine-1 from the protected location in 16S precursor complexes to a more exposed position in the 20S structure. Thereby, our findings provide a molecular explanation for the structural rearrangements necessary for the dimerization of two 16S precursor complexes and the subsequent final maturation to active 20S proteasomes.

Computer Simulation↗
PubMed2004

Domain movements of elongation factor eEF2 and the eukaryotic 80S ribosome facilitate tRNA translocation.

An 11.7-A-resolution cryo-EM map of the yeast 80S.eEF2 complex in the presence of the antibiotic sordarin was interpreted in molecular terms, revealing large conformational changes within eEF2 and the 80S ribosome, including a rearrangement of the functionally important ribosomal intersubunit bridges. Sordarin positions domain III of eEF2 so that it can interact with the sarcin-ricin loop of 25S rRNA and protein rpS23 (S12p). This particular conformation explains the inhibitory action of sordarin and suggests that eEF2 is stalled on the 80S ribosome in a conformation that has similarities with the GTPase activation state. A ratchet-like subunit rearrangement (RSR) occurs in the 80S.eEF2.sordarin complex that, in contrast to Escherichia coli 70S ribosomes, is also present in vacant 80S ribosomes. A model is suggested, according to which the RSR is part of a mechanism for moving the tRNAs during the translocation reaction.

Antifungal Agents↗
PubMed2004

Gridding-based direct Fourier inversion of the three-dimensional ray transform.

We describe a fast and accurate direct Fourier method for reconstructing a function f of three variables from a number of its parallel beam projections. The main application of our method is in single particle analysis, where the goal is to reconstruct the mass density of a biological macromolecule. Typically, the number of projections is extremely large, and each projection is extremely noisy. The projection directions are random and initially unknown. However, it is possible to determine both the directions and f by an iterative procedure; during each stage of the iteration, one has to solve a reconstruction problem of the type considered here. Our reconstruction algorithm is distinguished from other direct Fourier methods by the use of gridding techniques that provide an efficient means to compute a uniformly sampled version of a function g from a nonuniformly sampled version of Fg, the Fourier transform of g, or vice versa. We apply the two-dimensional reverse gridding method to each available projection of f, the function to be reconstructed, in order to obtain Ff on a special spherical grid. Then we use the three-dimensional gridding method to reconstruct f from this sampled version of Ff. This stage requires a proper weighting of the samples of Ff to compensate for their nonuniform distribution. We use a fast method for computing appropriate weights that exploits the special properties of the spherical sampling grid for Ff and involves the computation of a Voronoi diagram on the unit sphere. We demonstrate the excellent speed and accuracy of our method by using simulated data.

Algorithms↗
PubMed2002

Efficiency of 2D alignment methods.

In single particle analysis, the alignment of two-dimensional images is a fundamental step aimed at bringing into register various particle views of biological macromolecules observed with the electron microscope. The computational efficiency of this step is a deciding factor in design of alignment strategies for large sets of noisy data and in development of three-dimensional structure refinement methods. In addition, the accuracy of the alignment method varies depending on the numerical solutions adopted to efficiently perform exhaustive searches for three orientation parameters. The selected alignment methods are analyzed in terms of their computational complexity and the estimates of numbers of arithmetic operations for each method are given. The tests of alignment accuracy are performed using images simulated in accordance with the linear theory of image formation in the electron microscope. It is demonstrated that the efficiency of the alignment methods can be improved if approximate centers of gravity of particle views are known. The accuracy of the methods considered is largely affected, particularly for high noise levels, by the order in which interpolation steps are applied.

Algorithms↗
PubMedDate not supplied

Automatic particle selection: results of a comparative study.

Manual selection of single particles in images acquired using cryo-electron microscopy (cryoEM) will become a significant bottleneck when datasets of a hundred thousand or even a million particles are required for structure determination at near atomic resolution. Algorithm development of fully automated particle selection is thus an important research objective in the cryoEM field. A number of research groups are making promising new advances in this area. Evaluation of algorithms using a standard set of cryoEM images is an essential aspect of this algorithm development. With this goal in mind, a particle selection "bakeoff" was included in the program of the Multidisciplinary Workshop on Automatic Particle Selection for cryoEM. Twelve groups participated by submitting the results of testing their own algorithms on a common dataset. The dataset consisted of 82 defocus pairs of high-magnification micrographs, containing keyhole limpet hemocyanin particles, acquired using cryoEM. The results of the bakeoff are presented in this paper along with a summary of the discussion from the workshop. It was agreed that establishing benchmark particles and using bakeoffs to evaluate algorithms are useful in promoting algorithm development for fully automated particle selection, and that the infrastructure set up to support the bakeoff should be maintained and extended to include larger and more varied datasets, and more criteria for future evaluations.

Algorithms↗
PubMedDate not supplied

Automated determination of parameters describing power spectra of micrograph images in electron microscopy.

The current theory of image formation in electron microscopy has been semi-quantitatively successful in describing data. The theory involves parameters due to the transfer function of the microscope (defocus, spherical aberration constant, and amplitude constant ratio) as well as parameters used to describe the background and attenuation of the signal. We present empirical evidence that at least one of the features of this model has not been well characterized. Namely the spectrum of the noise background is not accurately described by a Gaussian and associated "B-factor;" this becomes apparent when one studies high-quality far-from focus data. In order to have both our analysis and conclusions free from any innate bias, we have approached the questions by developing an automated fitting algorithm. The most important features of this routine, not currently found in the literature, are (i). a process for determining the cutoff for those frequencies below which observations and the currently adopted model are not in accord, (ii). a method for determining the resolution at which no more signal is expected to exist, and (iii). a parameter-with units of spatial frequency-that characterizes which frequencies mainly contribute to the signal. Whereas no general relation is seen to exist between either of these two quantities and the defocus, a simple empirical relationship approximately relates all three.

Algorithms↗