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. Here, we use single wavelength anomalous dispersion (SAD) X-ray diffraction data collected near the potassium absorption edge to show experimentally that all ion binding sites within the selectivity filter are fully occupied by K
ions. These data support the hypothesis that potassium ion transport occurs by direct Coulomb knock-on, and provide an example of solving the phase problem by K-SAD.
Neutron crystallography offers enormous potential to complement structures from X‐ray crystallography by clarifying the positions of low‐Z 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. The artificial neural network, based on the U‐Net architecture commonly used for image segmentation, is trained using about 100 000 simulated training peaks derived from strong peaks. After 100 training epochs (a round of training over the whole data set broken into smaller batches), training converges and achieves a Dice coefficient of around 65%, in contrast to just 15% for negative control data sets. Integrating whole peak sets using the neural network yields improved intensity statistics compared with other integration methods, including k‐nearest neighbours. These results demonstrate, for the first time, that neural networks can learn peak shapes and be used to integrate Bragg peaks. It is expected that integration using neural networks can be further developed to increase the quality of neutron, electron and X‐ray crystallography data.
It is shown that artificial neural networks can be used to predict the location and shape of neutron Bragg peaks, resulting in increased integration accuracy.
The rise of neutron cryo‐crystallography Kwon, Hanna; Langan, Patricia S.; Coates, Leighton ...
Acta crystallographica. Section D, Structural biology,
August 2018, Letnik:
74, Številka:
8
Journal Article
Recenzirano
Odprti dostop
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. Finally, the outlook for further cryogenic neutron studies using existing and future neutron instrumentation is discussed.
The application of the cryogenic data‐collection environments used in protein X‐ray crystallography to neutron protein crystallography is discussed.
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. The structure of this class A β‐lactamase mutant provides several insights into its previously reported reduced drug‐binding kinetic rates. A joint refinement of both X‐ray and neutron diffraction data was used to study the effects of the Glu166Gln mutation on the active site of Toho‐1. This structure reveals that while the Glu166Gln mutation has a somewhat limited impact on the positions of the conserved amino acids within the active site, it displaces the catalytic water molecule from the active site. These subtle changes offer a structural explanation for the previously observed decreases in the binding of non‐β‐lactam inhibitors such as the recently developed diazobicyclooctane inhibitor avibactam.
Neutron and X‐ray diffraction were used to investigate the effects of a Glu166Gln mutation on the active site of a class A β‐lactamase.
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. A method has been developed to fit weak data based on three‐dimensional profile fitting of Bragg peaks in reciprocal space by an Ikeda–Carpenter function with a bivariate Gaussian. When applied to data collected from three different proteins, three‐dimensional profile fitting yields intensities with higher correlation coefficients (CC1/2) at high resolutions, decreased Rfree factors, extended resolutions and improved nuclear density maps. Importantly, additional features are revealed in nuclear density maps that may provide additional scientific information. These results suggest that three‐dimensional profile fitting will help to extend the capabilities of neutron macromolecular crystallography.
It is demonstrated that using three‐dimensional profile fitting of Bragg peaks increases the accuracy and resolution of neutron crystallographic data collected from proteins and reveals new features in nuclear density maps calculated from these data.
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. A critical parameter is the vector copy number (VCN) which measures the genetic dose of a transgene present in gene-modified cells. Here we describe a set of clonal Jurkat cell lines with defined copy numbers of a reference lentiviral vector integrated into their genomes. Genomic DNA was characterized for copy number, genomic integrity and integration coordinates and showed uniform performance across independent quantitative PCR assays. Stability studies during continuous long-term culture demonstrated sustained renewability of the reference standard source material. DNA from the Jurkat VCN standards would be useful for control of quantitative PCR assays for VCN determination in LV gene-modified cellular products and clinical samples.
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. In these first reported structures of a class A β-lactamase in an acyl-enzyme complex with aztreonam, we directly observed most of the hydrogen atoms (as deuterium) within the active site. Although Lys 234 is fully protonated in the acyl intermediate, we found that Lys 73 is neutral. These findings are consistent with Lys 73 being able to serve as a general base during the acylation part of the catalytic mechanism, as previously proposed.
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. In contrast to all previously described photoswitchable proteins, both spectral states of Dathail are non-fluorescent. The photo-induced chromogenic state of Dathail has a lifetime of ~50min at 293K and pH7.5 as measured by UV–Vis spectrophotometry, returning to the ground state through thermal relaxation. X-ray crystallography provided structural insights supporting a change in conformation and coordination in the chromophore pocket as being responsible for Dathail's photoswitching. Neutron crystallography, carried out for the first time on a protein from the green fluorescent protein family, showed a distribution of hydrogen atoms revealing protonation of the chromophore 4-hydroxybenzyl group in the ground state. The neutron structure also supports the hypothesis that the photo-induced proton transfer from the chromophore occurs through water-mediated proton relay into the bulk solvent. Beyond its spectroscopic curiosity, Dathail has several characteristics that are improvements for applications, including low background fluorescence, large spectral separation, rapid switching time, and the ability to switch many times. Therefore, Dathail is likely to be extremely useful in the quickly developing fields of imaging and biosensors, including photochromic Förster resonance energy transfer, high-resolution microscopy, and live tracking within the cell.
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•Dathail will be useful in imaging and biosensors, particularly pcFRET, high-resolution microscopy, and live cell tracking.•Dathail is a reversibly switching chromoprotein with two spectrally distinct chromogenic states with low quantum yields.•X-ray crystallography shows changes in conformation and coordination of the chromophore during Dathail's photoswitching.•Crystallography suggests photo-induced proton transfer from the chromophore with water-mediated proton relay to bulk solvent.•The first time that neutron crystallography has been used for a protein in the GFP family provides surprising new insights.
The emergence and dissemination of bacterial resistance to β-lactam antibiotics via β-lactamase enzymes is a serious problem in clinical settings, often leaving few treatment options for infections ...resulting from multidrug-resistant superbugs. Understanding the catalytic mechanism of β-lactamases is important for developing strategies to overcome resistance. Binding of a substrate in the active site of an enzyme can alter the conformations and pK as of catalytic residues, thereby contributing to enzyme catalysis. Here we report X-ray and neutron crystal structures of the class A Toho-1 β-lactamase in the apo form and an X-ray structure of a Michaelis-like complex with the cephalosporin antibiotic cefotaxime in the active site. Comparison of these structures reveals that substrate binding induces a series of changes. The side chains of conserved residues important in catalysis, Lys73 and Tyr105, and the main chain of Ser130 alter their conformations, with Nζ of Lys73 moving closer to the position of the conserved catalytic nucleophile Ser70. This movement of Lys73 closer to Ser70 is consistent with proton transfer between the two residues prior to acylation. In combination with the tightly bound catalytic water molecule located between Glu166 and the position of Ser70, the enzyme is primed for catalysis when Ser70 is activated for nucleophilic attack of the β-lactam ring. Quantum mechanical/molecular mechanical (QM/MM) free energy simulations of models of the wild-type enzyme show that proton transfer from the Nζ of Lys73 to the Oε2 atom of Glu166 is more thermodynamically favorable than when it is absent. Taken together, our findings indicate that substrate binding enhances the favorability of the initial proton transfer steps that precede the formation of the acyl-enzyme intermediate.
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. The atomic resolution structure of a potassium-selective ion channel named NaK2K has allowed us to observe that a hydrophobic residue at the bottom of the selectivity filter, Phe92, appears in dual conformations. One of the two conformations of Phe92 restricts the diameter of the exit pore around the selectivity filter, limiting ion flow through the channel, while the other conformation of Phe92 provides a larger-diameter exit pore from the selectivity filter. Thus, it can be concluded that Phe92 acts as a hydrophobic gate, regulating the flow of ions through the selectivity filter.
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