On the explicit use of experimental images in high resolution cryo-EM refinement.

Single particle cryogenic electron microscopy (cryo-EM) is transforming structural biology by enabling the analysis of difficult macromolecular specimens, such as membrane proteins or large complexes with flexible elements, at near atomic resolution with an accuracy close to that of X-ray crystallography. As the technique continues to improve, it is important to assess and exploit its full potential to produce the most possible reliable atomic models. Here we propose to use the experimental images as the data for refinement and validation, instead of the reconstructed maps as currently used.

MglA functions as a three-state GTPase to control movement reversals of Myxococcus xanthus.

In Myxococcus xanthus, directed movement is controlled by pole-to-pole oscillations of the small GTPase MglA and its GAP MglB. Direction reversals require that MglA is inactivated by MglB, yet paradoxically MglA and MglB are located at opposite poles at reversal initiation. Here we report the complete MglA/MglB structural cycle combined to GAP kinetics and in vivo motility assays, which uncovers that MglA is a three-state GTPase and suggests a molecular mechanism for concerted MglA/MglB relocalizations.

Integrated conformational and lipid-sensing regulation of endosomal ArfGEF BRAG2.

The mechanisms whereby guanine nucleotide exchange factors (GEFs) coordinate their subcellular targeting to their activation of small GTPases remain poorly understood. Here we analyzed how membranes control the efficiency of human BRAG2, an ArfGEF involved in receptor endocytosis, Wnt signaling, and tumor invasion. The crystal structure of an Arf1-BRAG2 complex that mimics a membrane-bound intermediate revealed an atypical PH domain that is constitutively anchored to the catalytic Sec7 domain and interacts with Arf.

Structure of the Triatoma virus capsid.

The members of the Dicistroviridae family are non-enveloped positive-sense single-stranded RNA (+ssRNA) viruses pathogenic to beneficial arthropods as well as insect pests of medical importance. Triatoma virus (TrV), a member of this family, infects several species of triatomine insects (popularly named kissing bugs), which are vectors for human trypanosomiasis, more commonly known as Chagas disease. The potential use of dicistroviruses as biological control agents has drawn considerable attention in the past decade, and several viruses of this family have been identified, with their targets covering honey bees, aphids and field crickets, among others.

Fourier-space TEM reconstructions with symmetry adapted functions for all rotational point groups.

A general-purpose and simple expression for the coefficients of symmetry adapted functions referred to conveniently oriented symmetry axes is given for all rotational point groups. The expression involves the computation of reduced Wigner-matrix elements corresponding to an angle specific to each group and has the computational advantage of leading to Fourier-space TEM (transmission electron microscopy) reconstruction procedures involving only real valued unknowns. Using this expression, a protocol for ab initio view and center assignment and reconstruction so far used for icosahedral particles has been tested with experimental data in other point groups.

Cryo-electron microscopy reconstructions of triatoma virus particles: a clue to unravel genome delivery and capsid disassembly.

Triatoma virus (TrV) is a member of the insect virus family Dicistroviridae and consists of a small, non-enveloped capsid that encloses its positive-sense ssRNA genome. Using cryo-transmission electron microscopy and three-dimensional reconstruction techniques combined with fitting of the available crystallographic models, this study analysed the capsids corresponding to mature and several RNA-empty TrV particles. After genome release, the resulting reconstruction of the empty capsids displayed no prominent conformational changes with respect to the full virion capsid.

The crystal structure of human α2-macroglobulin reveals a unique molecular cage.

I'm your Venus: the crystal structure of the human methylamine-induced form of α(2)-macroglobulin (α(2)M) shows its large central cavity can accommodate two medium-sized proteinases. Twelve major entrances provide access for small substrates to the cavity and the still-active trapped "prey". The structure unveils the molecular basis of the unique "venus flytrap" mechanism of α(2)M.

Structure of a longitudinal actin dimer assembled by tandem w domains: implications for actin filament nucleation.

Actin filament nucleators initiate polymerization in cells in a regulated manner. A common architecture among these molecules consists of tandem WASP homology 2 domains (W domains) that recruit three to four actin subunits to form a polymerization nucleus. We describe a low-resolution crystal structure of an actin dimer assembled by tandem W domains, where the first W domain is cross-linked to Cys374 of the actin subunit bound to it, whereas the last W domain is followed by the C-terminal pointed end-capping helix of thymosin β4.

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.

Macromolecular crystal data phased by negative-stained electron-microscopy reconstructions.

The combination of transmission electron microscopy with X-ray diffraction data is usually limited to relatively large particles. Here, the approach is continued one step further by utilizing negative staining, a technique that is of wider applicability than cryo-electron microscopy, to produce models of medium-size proteins suitable for molecular replacement. The technique was used to solve the crystal structure of the dodecameric type II dehydroquinase enzyme from Candida albicans (approximately 190 kDa) and that of the orthologous Streptomyces coelicolor protein.

Structure of the outer membrane complex of a type IV secretion system.

Type IV secretion systems are secretion nanomachines spanning the two membranes of Gram-negative bacteria. Three proteins, VirB7, VirB9 and VirB10, assemble into a 1.05 megadalton (MDa) core spanning the inner and outer membranes.

UROX 2.0: an interactive tool for fitting atomic models into electron-microscopy reconstructions.

Electron microscopy of a macromolecular structure can lead to three-dimensional reconstructions with resolutions that are typically in the 30-10 A range and sometimes even beyond 10 A. Fitting atomic models of the individual components of the macromolecular structure (e.g.

The picobirnavirus crystal structure provides functional insights into virion assembly and cell entry.

Double-stranded (ds) RNA virus particles are organized around a central icosahedral core capsid made of 120 identical subunits. This core capsid is unable to invade cells from outside, and animal dsRNA viruses have acquired surrounding capsid layers that are used to deliver a transcriptionally active core particle across the membrane during cell entry. In contrast, dsRNA viruses infecting primitive eukaryotes have only a simple core capsid, and as a consequence are transmitted only vertically.

Domain organization in Clostridium botulinum neurotoxin type E is unique: its implication in faster translocation.

Clostridium botulinum produces seven antigenically distinct neurotoxins [C. botulinum neurotoxins (BoNTs) A-G] sharing a significant sequence homology. Based on sequence and functional similarity, it was believed that their three-dimensional structures will also be similar.

An approach to control of bioreactors. Application of the gain-scheduling method.

A control system based on a combination of the gain-scheduling control method and an adaptive PID controller was designed for the production of xylose from hardwood hemicellulose using a stirring tank reactor. Different operating conditions have been considered for estimating the adjustable parameter (theta) to take into account the changes of energy of the system. The performance of the control system was studied first by numerical simulation, and after implementation in the stirred tank reactor where the controller actually works.

Stabilization by extra-helical thymines of a DNA duplex with Hoogsteen base pairs.

We present the crystal structure of the DNA duplex formed by d(ATATATCT). The crystals contain seven stacked antiparallel duplexes in the asymmetric unit with A.T Hoogsteen base pairs.

Fast projection matching for cryo-electron microscopy image reconstruction.

A new FFT-accelerated projection matching method is presented and tested. The electron microscopy images are represented by their Fourier-Bessel transforms and the 3D model by its expansion in spherical harmonics, or more specifically in terms of symmetry-adapted functions. The rotational and translational properties of these representations are used to quickly access all the possible 2D projections of the 3D model, which allow an exhaustive inspection of the whole five-dimensional domain of parameters associated to each particle.

Phasing of the Triatoma virus diffraction data using a cryo-electron microscopy reconstruction.

The blood-sucking reduviid bug Triatoma infestans, one of the most important vector of American human trypanosomiasis (Chagas disease) is infected by the Triatoma virus (TrV). TrV has been classified as a member of the Cripavirus genus (type cricket paralysis virus) in the Dicistroviridae family. This work presents the three-dimensional cryo-electron microscopy (cryo-EM) reconstruction of the TrV capsid at about 25 A resolution and its use as a template for phasing the available crystallographic data by the molecular replacement method.

Geometric mismatches within the concentric layers of rotavirus particles: a potential regulatory switch of viral particle transcription activity.

Rotaviruses are prototypical double-stranded RNA viruses whose triple-layered icosahedral capsid constitutes transcriptional machinery activated by the release of the external layer. To understand the molecular basis of this activation, we studied the structural interplay between the three capsid layers by electron cryo-microscopy and digital image processing. Two viral particles and four virus-like particles containing various combinations of inner (VP2)-, middle (VP6)-, and outer (VP7)-layer proteins were studied.

Combining X-ray and electron-microscopy data to solve crystal structures.

Low-resolution electron-microscopy reconstructions can be used as search models in molecular replacement or may be combined with existing monomeric structures in order to produce multimeric models suitable for molecular replacement. The technique is described in the case of viral and subviral particles as well as in the case of oligomeric proteins.

AMoRe: classical and modern.

An account is given of the latest developments of the AMoRe package: new rotational search algorithms, exploitation of noncrystallographic symmetry, generation and use of ensemble models and interactive graphical molecular replacement.

The concept of resolution in the domain of rotations.

The metric of the SO(3) group of rotations can be used to define the angular resolution of a function of rotations. The resolution is related to the degree of the highest representation present in the expansion of the function in terms of Wigner functions. The peculiar non-Euclidean metric of the rotation domain, however, implies that the terms which effectively contribute to the expansion vary through two-dimensional sections of the rotation domain and are within limiting resolution circles in two-dimensional reciprocal sections.

SCA: symmetry-based center assignment of 2D projections of symmetric 3D objects.

A method for finding the center of cryo-EM images which correspond to the projections of a symmetric 3D structure, based on mathematical properties of symmetry adapted functions and the Fourier-Bessel transform, is presented. It is a model independent one-step procedure with no parameters to be chosen by the user. The proposed method is tested in one synthetic tetrahedral case with different noise levels and in two real cases with D7 and icosahedral symmetries.