Molecular Basis of Interchain Disulfide Bond Formation in BMP-9 and BMP-10.

BMP-9 and BMP-10 are TGF-β family signaling ligands naturally secreted into blood. They act on endothelial cells and are required for proper development and maintenance of the vasculature. In hereditary hemorrhagic telangiectasia, regulation is disrupted due to mutations in the BMP-9/10 pathway, namely in the type I receptor ALK1 or the co-receptor endoglin.

Structural basis of deoxynucleotide addition by HIV-1 RT during reverse transcription.

Reverse transcription of the retroviral RNA genome into DNA is an integral step during HIV-1 replication. Despite a wealth of structural information on reverse transcriptase (RT), we lack insight into the intermediate states of DNA synthesis. Using catalytically active substrates, and a blot/diffusion cryo-electron microscopy approach, we capture 11 structures encompassing reactant, intermediate and product states of dATP addition by RT at 2.

Molecular basis of interchain disulfide-bond formation in BMP-9 and BMP-10.

BMP-9 and BMP-10 are TGF-β family signaling ligands naturally secreted into blood. They act on endothelial cells and are required for proper development and maintenance of the vasculature. In hereditary hemorrhagic telangiectasia, regulation is disrupted due to mutations in the BMP-9/10 pathway, namely in the type I receptor ALK1 or the co-receptor endoglin.

Structural basis of transcription: RNA polymerase II substrate binding and metal coordination using a free-electron laser.

Catalysis and translocation of multisubunit DNA-directed RNA polymerases underlie all cellular mRNA synthesis. RNA polymerase II (Pol II) synthesizes eukaryotic pre-mRNAs from a DNA template strand buried in its active site. Structural details of catalysis at near-atomic resolution and precise arrangement of key active site components have been elusive.

Microwave-assisted acid treatment for the mineral transformation of chrysotile as an alternative for asbestos waste management.

In this work, the effect of microwave-assisted acid treatments on the morphological and crystallochemical characteristics of chrysotile fibers is investigated. A low concentration of nitric acid (0.2 N) is used to remove Mg-species located in the octahedral sheet of its structure, thereby causing a crystallo-chemical change forming a skeleton of non-crystalline amorphous silica.

Structures of kinetic intermediate states of HIV-1 reverse transcriptase DNA synthesis.

Reverse transcription of the retroviral single-stranded RNA into double-stranded DNA is an integral step during HIV-1 replication, and reverse transcriptase (RT) is a primary target for antiviral therapy. Despite a wealth of structural information on RT, we lack critical insight into the intermediate kinetic states of DNA synthesis. Using catalytically active substrates, and a novel blot/diffusion cryo-electron microscopy approach, we captured 11 structures that define the substrate binding, reactant, transition and product states of dATP addition by RT at 1.

Structural basis of transcription: RNA Polymerase II substrate binding and metal coordination at 3.0 Ã… using a free-electron laser.

Catalysis and translocation of multi-subunit DNA-directed RNA polymerases underlie all cellular mRNA synthesis. RNA polymerase II (Pol II) synthesizes eukaryotic pre-mRNAs from a DNA template strand buried in its active site. Structural details of catalysis at near atomic resolution and precise arrangement of key active site components have been elusive.

Preclinical assessment of a novel human antibody VH domain targeting mesothelin as an antibody-drug conjugate.

Mesothelin (MSLN) has been a validated tumor-associated antigen target for several solid tumors for over a decade, making it an attractive option for therapeutic interventions. Novel antibodies with high affinity and better therapeutic properties are needed. In the current study, we have isolated and characterized a novel heavy chain variable (VH) domain 3C9 from a large-size human immunoglobulin VH domain library.

Structure of HIV-1 Vpr in complex with the human nucleotide excision repair protein hHR23A.

HIV-1 Vpr is a prototypic member of a large family of structurally related lentiviral virulence factors that antagonize various aspects of innate antiviral immunity. It subverts host cell DNA repair and protein degradation machineries by binding and inhibiting specific post-replication repair enzymes, linking them via the DCAF1 substrate adaptor to the Cullin 4 RING E3 ligase (CRL4). HIV-1 Vpr also binds to the multi-domain protein hHR23A, which interacts with the nucleotide excision repair protein XPC and shuttles ubiquitinated proteins to the proteasome.

A highly-specific fully-human antibody and CAR-T cells targeting CD66e/CEACAM5 are cytotoxic for CD66e-expressing cancer cells in vitro and in vivo.

Neuro-endocrine prostate cancer (NEPC) accounts for about 20% of lethal metastatic castration-resistant prostate cancer (CRPC). NEPC has the most aggressive biologic behavior of all prostate cancers and is associated with poor patient outcome. Effective treatment for NEPC is not available because NEPC exhibit distinct cell-surface expression profiles compared to other types of prostate cancer.

A resource of high-quality and versatile nanobodies for drug delivery.

Therapeutic and diagnostic efficacies of small biomolecules and chemical compounds are hampered by suboptimal pharmacokinetics. Here, we developed a repertoire of robust and high-affinity antihuman serum albumin nanobodies (Nb) that can be readily fused to small biologics for half-life extension. We characterized the thermostability, binding kinetics, and cross-species reactivity of Nbs, mapped their epitopes, and structurally resolved a tetrameric HSA-Nb complex.

Detection of Microcrystals for CryoEM.

Here, we present a strategy to identify microcrystals from initial protein crystallization screen experiments and to optimize diffraction quality of those crystals using negative stain transmission electron microscopy (TEM) as a guiding technique. The use of negative stain TEM allows visualization along the process and thus enables optimization of crystal diffraction by monitoring the lattice quality of crystallization conditions. Nanocrystals bearing perfect lattices are seeded and can be used for MicroED as well as growing larger crystals for X-ray and free electron laser (FEL) data collection.

Transcription with a laser: Radiation-damage-free diffraction of RNA Polymerase II crystals.

Well-diffracting crystals are essential to obtain relevant structural data that will lead to understanding of RNA Polymerase II (Pol II) transcriptional processes at a molecular level. Here we present a strategy to study Pol II crystals using negative stain transmission electron microscopy (TEM) and a methodology to optimize radiation damage free data collection using free electron laser (FEL) at the Linac Coherent Light Source (LCLS). The use of negative stain TEM allowed visualization and optimization of crystal diffraction by monitoring the lattice quality of crystallization conditions.

Is the largest aqueous gold cluster a superatom complex? Electronic structure & optical response of the structurally determined Au(p-MBA).

The new water-soluble gold cluster Au(p-MBA), the structure of which has been recently determined at sub-atomic resolution by Vergara et al., is the largest aqueous gold cluster ever structurally determined and likewise the smallest cluster with a stacking fault. The core presents a twinned truncated octahedron, while additional peripheral gold atoms follow a C rotational symmetry.

MicroED Structure of Au(p-MBA) at Subatomic Resolution Reveals a Twinned FCC Cluster.

Solving the atomic structure of metallic clusters is fundamental to understanding their optical, electronic, and chemical properties. Herein we present the structure of the largest aqueous gold cluster, Au(p-MBA) (p-MBA: para-mercaptobenzoic acid), solved by electron micro-diffraction (MicroED) to subatomic resolution (0.85 Ã…) and by X-ray diffraction at atomic resolution (1.

An engineered transforming growth factor β (TGF-β) monomer that functions as a dominant negative to block TGF-β signaling.

The transforming growth factor β isoforms, TGF-β1, -β2, and -β3, are small secreted homodimeric signaling proteins with essential roles in regulating the adaptive immune system and maintaining the extracellular matrix. However, dysregulation of the TGF-β pathway is responsible for promoting the progression of several human diseases, including cancer and fibrosis. Despite the known importance of TGF-βs in promoting disease progression, no inhibitors have been approved for use in humans.

Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED.

Traditionally, crystallographic analysis of macromolecules has depended on large, well-ordered crystals, which often require significant effort to obtain. Even sizable crystals sometimes suffer from pathologies that render them inappropriate for high-resolution structure determination. Here we show that fragmentation of large, imperfect crystals into microcrystals or nanocrystals can provide a simple path for high-resolution structure determination by the cryoEM method MicroED and potentially by serial femtosecond crystallography.

The DDB1-DCAF1-Vpr-UNG2 crystal structure reveals how HIV-1 Vpr steers human UNG2 toward destruction.

The HIV-1 accessory protein Vpr is required for efficient viral infection of macrophages and promotion of viral replication in T cells. Vpr's biological activities are closely linked to the interaction with human DCAF1, a cellular substrate receptor of the Cullin4-RING E3 ubiquitin ligase (CRL4) of the host ubiquitin-proteasome-mediated protein degradation pathway. The molecular details of how Vpr usurps the protein degradation pathway have not been delineated.

Transmission electron microscopy for the evaluation and optimization of crystal growth.

The crystallization of protein samples remains the most significant challenge in structure determination by X-ray crystallography. Here, the effectiveness of transmission electron microscopy (TEM) analysis to aid in the crystallization of biological macromolecules is demonstrated. It was found that the presence of well ordered lattices with higher order Bragg spots, revealed by Fourier analysis of TEM images, is a good predictor of diffraction-quality crystals.

The collection of MicroED data for macromolecular crystallography.

The formation of large, well-ordered crystals for crystallographic experiments remains a crucial bottleneck to the structural understanding of many important biological systems. To help alleviate this problem in crystallography, we have developed the MicroED method for the collection of electron diffraction data from 3D microcrystals and nanocrystals of radiation-sensitive biological material. In this approach, liquid solutions containing protein microcrystals are deposited on carbon-coated electron microscopy grids and are vitrified by plunging them into liquid ethane.

Assessment of microcrystal quality by transmission electron microscopy for efficient serial femtosecond crystallography.

Serial femtosecond crystallography (SFX) employing high-intensity X-ray free-electron laser (XFEL) sources has enabled structural studies on microcrystalline protein samples at non-cryogenic temperatures. However, the identification and optimization of conditions that produce well diffracting microcrystals remains an experimental challenge. Here, we report parallel SFX and transmission electron microscopy (TEM) experiments using fragmented microcrystals of wild type (WT) homoprotocatechuate 2,3-dioxygenase (HPCD) and an active site variant (H200Q).

High-density grids for efficient data collection from multiple crystals.

Higher throughput methods to mount and collect data from multiple small and radiation-sensitive crystals are important to support challenging structural investigations using microfocus synchrotron beamlines. Furthermore, efficient sample-delivery methods are essential to carry out productive femtosecond crystallography experiments at X-ray free-electron laser (XFEL) sources such as the Linac Coherent Light Source (LCLS). To address these needs, a high-density sample grid useful as a scaffold for both crystal growth and diffraction data collection has been developed and utilized for efficient goniometer-based sample delivery at synchrotron and XFEL sources.

Microfluidic sorting of protein nanocrystals by size for X-ray free-electron laser diffraction.

The advent and application of the X-ray free-electron laser (XFEL) has uncovered the structures of proteins that could not previously be solved using traditional crystallography. While this new technology is powerful, optimization of the process is still needed to improve data quality and analysis efficiency. One area is sample heterogeneity, where variations in crystal size (among other factors) lead to the requirement of large data sets (and thus 10-100 mg of protein) for determining accurate structure factors.