Cyanotriazoles are selective topoisomerase II poisons that rapidly cure trypanosome infections.

Millions who live in Latin America and sub-Saharan Africa are at risk of trypanosomatid infections, which cause Chagas disease and human African trypanosomiasis (HAT). Improved HAT treatments are available, but Chagas disease therapies rely on two nitroheterocycles, which suffer from lengthy drug regimens and safety concerns that cause frequent treatment discontinuation. We performed phenotypic screening against trypanosomes and identified a class of cyanotriazoles (CTs) with potent trypanocidal activity both in vitro and in mouse models of Chagas disease and HAT.

The CRL4 cullin-RING ubiquitin ligase is activated following a switch in oligomerization state.

The cullin-4-based RING-type (CRL4) family of E3 ubiquitin ligases functions together with dedicated substrate receptors. Out of the ˜29 CRL4 substrate receptors reported, the DDB1- and CUL4-associated factor 1 (DCAF1) is essential for cellular survival and growth, and its deregulation has been implicated in tumorigenesis. We carried out biochemical and structural studies to examine the structure and mechanism of the CRL4 ligase.

Structure of the human C9orf72-SMCR8 complex reveals a multivalent protein interaction architecture.

A major cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) spectrum disorder is the hexanucleotide G4C2 repeat expansion in the first intron of the C9orf72 gene. Many underlying mechanisms lead to manifestation of disease that include toxic gain-of-function by repeat G4C2 RNAs, dipeptide repeat proteins, and a reduction of the C9orf72 gene product. The C9orf72 protein interacts with SMCR8 and WDR41 to form a trimeric complex and regulates multiple cellular pathways including autophagy.

Dynamic association of human Ebp1 with the ribosome.

Ribosomes are the macromolecular machines at the heart of protein synthesis; however, their function can be modulated by a variety of additional protein factors that directly interact with them. Here, we report the cryo-EM structure of human Ebp1 (p48 isoform) bound to the human 80S ribosome at 3.3 Å resolution.

Mechanisms of OCT4-SOX2 motif readout on nucleosomes.

Transcription factors (TFs) regulate gene expression through chromatin where nucleosomes restrict DNA access. To study how TFs bind nucleosome-occupied motifs, we focused on the reprogramming factors OCT4 and SOX2 in mouse embryonic stem cells. We determined TF engagement throughout a nucleosome at base-pair resolution in vitro, enabling structure determination by cryo-electron microscopy at two preferred positions.

Dynamics of uS19 C-Terminal Tail during the Translation Elongation Cycle in Human Ribosomes.

Ribosomes undergo multiple conformational transitions during translation elongation. Here, we report the high-resolution cryoelectron microscopy (cryo-EM) structure of the human 80S ribosome in the post-decoding pre-translocation state (classical-PRE) at 3.3-Å resolution along with the rotated (hybrid-PRE) and the post-translocation states (POST).

Live Analysis and Reconstruction of Single-Particle Cryo-Electron Microscopy Data with CryoFLARE.

Efficient, reproducible and accountable single-particle cryo-electron microscopy structure determination is tedious and often impeded by the lack of a standardized procedure for data analysis and processing. To address this issue, we have developed the FMI Live Analysis and Reconstruction Engine (CryoFLARE). CryoFLARE is a modular open-source platform offering easy integration of new processing algorithms developed by the cryo-EM community.

Structural Basis of BRCC36 Function in DNA Repair and Immune Regulation.

In mammals, ∼100 deubiquitinases act on ∼20,000 intracellular ubiquitination sites. Deubiquitinases are commonly regarded as constitutively active, with limited regulatory and targeting capacity. The BRCA1-A and BRISC complexes serve in DNA double-strand break repair and immune signaling and contain the lysine-63 linkage-specific BRCC36 subunit that is functionalized by scaffold subunits ABRAXAS and ABRO1, respectively.

Publisher Correction: DNA damage detection in nucleosomes involves DNA register shifting.

In this Article, in Fig. 1a, the 5' and 3' labels were reversed in the DNA sequence, and Fig. 4 was missing panel labels a-e.

DNA damage detection in nucleosomes involves DNA register shifting.

Access to DNA packaged in nucleosomes is critical for gene regulation, DNA replication and DNA repair. In humans, the UV-damaged DNA-binding protein (UV-DDB) complex detects UV-light-induced pyrimidine dimers throughout the genome; however, it remains unknown how these lesions are recognized in chromatin, in which nucleosomes restrict access to DNA. Here we report cryo-electron microscopy structures of UV-DDB bound to nucleosomes bearing a 6-4 pyrimidine-pyrimidone dimer or a DNA-damage mimic in various positions.

A pipeline for comprehensive and automated processing of electron diffraction data in IPLT.

Electron crystallography of two-dimensional crystals allows the structural study of membrane proteins in their native environment, the lipid bilayer. Determining the structure of a membrane protein at near-atomic resolution by electron crystallography remains, however, a very labor-intense and time-consuming task. To simplify and accelerate the data processing aspect of electron crystallography, we implemented a pipeline for the processing of electron diffraction data using the Image Processing Library and Toolbox (IPLT), which provides a modular, flexible, integrated, and extendable cross-platform, open-source framework for image processing.

Critical phosphoprotein elements that regulate polymerase architecture and function in vesicular stomatitis virus.

The RNA-dependent RNA polymerase (RdRP) of nonsegmented negative-sense RNA viruses consists of a large catalytic protein (L) and a phosphoprotein cofactor (P). During infection, the RdRP replicates and transcribes the viral genome, which resides inside an oligomer of nucleocapsid protein (N-RNA). The classical view of P as a cofactor for L assigns a primary role of P as a bridge mediating the access of L to the RNA template, whereby its N-terminal domain (P(NTD)) binds L and its C-terminal domain (P(CTD)) binds N-RNA.

Molecular driving forces defining lipid positions around aquaporin-0.

Lipid-protein interactions play pivotal roles in biological membranes. Electron crystallographic studies of the lens-specific water channel aquaporin-0 (AQP0) revealed atomistic views of such interactions, by providing high-resolution structures of annular lipids surrounding AQP0. It remained unclear, however, whether these lipid structures are representative of the positions of unconstrained lipids surrounding an individual protein, and what molecular determinants define the lipid positions around AQP0.

Simulation and correction of electron images of tilted planar weak-phase samples.

The phase contrast theory describes the transfer of information from a weak-phase object to the image plane of a transmission electron microscope. For a tilted sample where the distance from the focal plane varies continuously across the field of view, the recently introduced Tilted Contrast Imaging Function (TCIF) model provides the mathematical description of this information transfer. Here we expand the TCIF model to account for astigmatism, and present several methods to generate simulated images of tilted samples and compare them to experimental results.

Molecular architecture of the vesicular stomatitis virus RNA polymerase.

Nonsegmented negative-strand (NNS) RNA viruses initiate infection by delivering into the host cell a highly specialized RNA synthesis machine comprising the genomic RNA completely encapsidated by the viral nucleocapsid protein and associated with the viral polymerase. The catalytic core of this protein-RNA complex is a 250-kDa multifunctional large (L) polymerase protein that contains enzymatic activities for nucleotide polymerization as well as for each step of mRNA cap formation. Working with vesicular stomatitis virus (VSV), a prototype of NNS RNA viruses, we used negative stain electron microscopy (EM) to obtain a molecular view of L, alone and in complex with the viral phosphoprotein (P) cofactor.

Assembly of a functional Machupo virus polymerase complex.

Segmented negative-sense viruses of the family Arenaviridae encode a large polymerase (L) protein that contains all of the enzymatic activities required for RNA synthesis. These activities include an RNA-dependent RNA polymerase (RdRP) and an RNA endonuclease that cleaves capped primers from cellular mRNAs to prime transcription. Using purified catalytically active Machupo virus L, we provide a view of the overall architecture of this multifunctional polymerase and reconstitute complex formation with an RNA template in vitro.

3D reconstruction from 2D crystal image and diffraction data.

Electron crystallography of 2D protein crystals can determine the structure of membrane embedded proteins at high resolution. Images or electron diffraction patterns are recorded with the electron microscope of the frozen hydrated samples, and the 3D structure of the proteins is then determined by computer data processing. Here we introduce the image-processing algorithms for crystallographic Fourier space based methods using the Medical Research Council (MRC) programs, and illustrate the usage of the software packages 2dx, XDP, and IPLT.

Electron crystallography and aquaporins.

Electron crystallography of two-dimensional (2D) crystals can provide information on the structure of membrane proteins at near-atomic resolution. Originally developed and used to determine the structure of bacteriorhodopsin (bR), electron crystallography has recently been applied to elucidate the structure of aquaporins (AQPs), a family of membrane proteins that form pores mostly for water but also other solutes. While electron crystallography has made major contributions to our understanding of the structure and function of AQPs, structural studies on AQPs, in turn, have fostered a number of technical developments in electron crystallography.

Collecting electron crystallographic data of two-dimensional protein crystals.

Similar to X-ray crystallography, which requires three-dimensional (3D) crystals, electron crystallography is used to obtain structural information for proteins that form two-dimensional (2D) crystals. However, unlike data collection in X-ray crystallography, which is typically fast and straightforward, data collection in electron crystallography can take months to years and requires substantial expertise. In this contribution, we first discuss the proper preparation of 2D crystals for electron microscopy, which, besides the quality of the 2D crystals, may be the most defining parameter for successful data collection.

OpenStructure: a flexible software framework for computational structural biology.

Motivation: Developers of new methods in computational structural biology are often hampered in their research by incompatible software tools and non-standardized data formats. To address this problem, we have developed OpenStructure as a modular open source platform to provide a powerful, yet flexible general working environment for structural bioinformatics. OpenStructure consists primarily of a set of libraries written in C++ with a cleanly designed application programmer interface.

Anabolic and catabolic responses of human articular chondrocytes to varying oxygen percentages.

Introduction: Oxygen is a critical parameter proposed to modulate the functions of chondrocytes ex-vivo as well as in damaged joints. This article investigates the effect of low (more physiological) oxygen percentage on the biosynthetic and catabolic activity of human articular chondrocytes (HAC) at different phases of in vitro culture.

Methods: HAC expanded in monolayer were cultured in pellets for two weeks (Phase I) or up to an additional two weeks (Phase II).

Projection structure of a member of the amino acid/polyamine/organocation transporter superfamily.

The L-arginine/agmatine antiporter AdiC is a key component of the arginine-dependent extreme acid resistance system of Escherichia coli. Phylogenetic analysis indicated that AdiC belongs to the amino acid/polyamine/organocation (APC) transporter superfamily having sequence identities of 15-17% to eukaryotic and human APC transporters. For functional and structural characterization, we cloned, overexpressed, and purified wild-type AdiC and the point mutant AdiC-W293L, which is unable to bind and consequently transport L-arginine.

Projection maps of three members of the KdgM outer membrane protein family.

We present the projection structures of the three outer membrane porins KdgM and KdgN from Erwinia chrysanthemi and NanC from Escherichia coli, based on 2D electron crystallography. A wide screening of 2D crystallization conditions yielded tubular crystals of a suitable size and quality to perform high-resolution electron microscopy. Data processing of untilted samples allowed us to separate the information of the two crystalline layers and resulted in projection maps to a resolution of up to 7A.

Collaborative EM image processing with the IPLT image processing library and toolbox.

We present the Image Processing Library and Toolbox, IPLT, in the context of a collaborative electron microscopy processing effort, which has driven the evolution of our software architecture over the last years. The high-level interface design as well as the underlying implementations are described to demonstrate the flexibility of the IPLT framework. It aims to support the wide range of skills and interests of methodologically oriented scientists who wish to implement their ideas and algorithms as processing code.

The 4.5 A structure of human AQP2.

Located in the principal cells of the collecting duct, aquaporin-2 (AQP2) is responsible for the regulated water reabsorption in the kidney and is indispensable for the maintenance of body water balance. Disregulation or malfunctioning of AQP2 can lead to severe diseases such as nephrogenic diabetes insipidus, congestive heart failure, liver cirrhosis and pre-eclampsia. Here we present the crystallization of recombinantly expressed human AQP2 into two-dimensional protein-lipid arrays and their structural characterization by atomic force microscopy and electron crystallography.