Reference standards for accurate validation and optimization of assays that determine integrated lentiviral vector copy number in transduced cells.

Lentiviral vectors (LV) have emerged as a robust technology for therapeutic gene delivery into human cells as advanced medicinal products. As these products are increasingly commercialized, there are concomitant demands for their characterization to ensure safety, efficacy and consistency. Standards are essential for accurately measuring parameters for such product characterization.

The structure of a potassium-selective ion channel reveals a hydrophobic gate regulating ion permeation.

Protein dynamics are essential to function. One example of this is the various gating mechanisms within ion channels, which are transmembrane proteins that act as gateways into the cell. Typical ion channels switch between an open and closed state via a conformational transition which is often triggered by an external stimulus, such as ligand binding or pH and voltage differences.

Probing the role of the conserved residue Glu166 in a class A β-lactamase using neutron and X-ray protein crystallography.

The amino-acid sequence of the Toho-1 β-lactamase contains several conserved residues in the active site, including Ser70, Lys73, Ser130 and Glu166, some of which coordinate a catalytic water molecule. This catalytic water molecule is essential in the acylation and deacylation parts of the reaction mechanism through which Toho-1 inactivates specific antibiotics and provides resistance to its expressing bacterial strains. To investigate the function of Glu166 in the acylation part of the catalytic mechanism, neutron and X-ray crystallographic studies were performed on a Glu166Gln mutant.

Volumetric Segmentation Neural Networks Improves Neutron Crystallography Data Analysis.

Crystallography is the powerhouse technique for molecular structure determination, with applications in fields ranging from energy storage to drug design. Accurate structure determination, however, relies partly on determining the precise locations and integrated intensities of Bragg peaks in the resulting data. Here, we describe a method for Bragg peak integration that is accomplished using neural networks.

BraggNet: integrating Bragg peaks using neural networks.

Neutron crystallography offers enormous potential to complement structures from X-ray crystallography by clarifying the positions of low- elements, namely hydrogen. Macromolecular neutron crystallography, however, remains limited, in part owing to the challenge of integrating peak shapes from pulsed-source experiments. To advance existing software, this article demonstrates the use of machine learning to refine peak locations, predict peak shapes and yield more accurate integrated intensities when applied to whole data sets from a protein crystal.

Room-temperature photo-induced martensitic transformation in a protein crystal. Corrigendum.

[This retracts the article DOI: 10.1107/S2052252519005761.].

Room-temperature photo-induced martensitic transformation in a protein crystal.

Martensitic transformations are the first-order crystal-to-crystal phase transitions that occur mostly in materials such as steel, alloys and ceramics, thus having many technological applications. These phase transitions are rarely observed in molecular crystals and have not been detected in protein crystals. Reversibly switchable fluorescent proteins are widely used in biotechnology, including super-resolution molecular imaging, and hold promise as candidate biomaterials for future high-tech applications.

Crystallization of a potassium ion channel and X-ray and neutron data collection.

The mechanism by which potassium ions are transported through ion channels is currently being investigated by several groups using many different techniques. Clarification of the location of water molecules during transport is central to understanding how these integral membrane proteins function. Neutrons have a unique sensitivity to both hydrogen and potassium, rendering neutron crystallography capable of distinguishing waters from K ions.

Improving the accuracy and resolution of neutron crystallographic data by three-dimensional profile fitting of Bragg peaks in reciprocal space.

Neutron crystallography is a powerful technique for directly visualizing the locations of H atoms in biological macromolecules. This information has provided key new insights into enzyme mechanisms, ligand binding and hydration. However, despite the importance of this information, the application of neutron crystallography in biology has been limited by the relatively low flux of available neutron beams and the large incoherent neutron scattering from hydrogen, both of which contribute to weak diffraction data with relatively low signal-to-background ratios.

Anomalous X-ray diffraction studies of ion transport in K channels.

Potassium ion channels utilize a highly selective filter to rapidly transport K ions across cellular membranes. This selectivity filter is composed of four binding sites which display almost equal electron density in crystal structures with high potassium ion concentrations. This electron density can be interpreted to reflect a superposition of alternating potassium ion and water occupied states or as adjacent potassium ions.

The rise of neutron cryo-crystallography.

The use of boiled-off liquid nitrogen to maintain protein crystals at 100 K during X-ray data collection has become almost universal. Applying this to neutron protein crystallography offers the opportunity to significantly broaden the scope of biochemical problems that can be addressed, although care must be taken in assuming that direct extrapolation to room temperature is always valid. Here, the history to date of neutron protein cryo-crystallography and the particular problems and solutions associated with the mounting and cryocooling of the larger crystals needed for neutron crystallography are reviewed.

The structure of Toho1 β-lactamase in complex with penicillin reveals the role of Tyr105 in substrate recognition.

The role of the conserved residue Tyr105 in class A β-lactamases has been the subject of investigation using both structural studies and saturation mutagenesis. Both have shown that while it does not need to be strictly conserved for activity, it is important for substrate recognition. With this in mind we determined the crystal structure of Toho1 β-lactamase at 15 K to 1.

Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate.

The monobactam antibiotic aztreonam is used to treat cystic fibrosis patients with chronic pulmonary infections colonized by Pseudomonas aeruginosa strains expressing CTX-M extended-spectrum β-lactamases. The protonation states of active-site residues that are responsible for hydrolysis have been determined previously for the apo form of a CTX-M β-lactamase but not for a monobactam acyl-enzyme intermediate. Here we used neutron and high-resolution X-ray crystallography to probe the mechanism by which CTX-M extended-spectrum β-lactamases hydrolyze monobactam antibiotics.

Evolution and characterization of a new reversibly photoswitching chromogenic protein, Dathail.

We report the engineering of a new reversibly switching chromogenic protein, Dathail. Dathail was evolved from the extremely thermostable fluorescent proteins thermal green protein (TGP) and eCGP123 using directed evolution and ratiometric sorting. Dathail has two spectrally distinct chromogenic states with low quantum yields, corresponding to absorbance in a ground state with a maximum at 389nm, and a photo-induced metastable state with a maximum at 497nm.

Thermal green protein, an extremely stable, nonaggregating fluorescent protein created by structure-guided surface engineering.

In this article, we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP.