Improving sampling of crystallographic disorder in ensemble refinement

Ensemble refinement, the application of molecular dynamics to crystallographic refinement, explicitly models the disorder inherent in macromolecular structures. These ensemble models have been shown to produce more accurate structures than traditional single-model structures. However, suboptimal sampling of the molecular-dynamics simulation and modelling of crystallographic disorder has limited the utility of the method, and can lead to unphysical and strained models. Here, two improvements to the ensemble refinement method implemented within Phenix are presented: DEN restraints, which guide the local sampling of conformations and allow a more robust exploration of local conformational landscapes, and ECHT disorder models, which allow the selection of more physically meaningful and effective disorder models for parameterizing the continuous disorder components within a crystal. These improvements lead to more consistent and physically interpretable simulations of macromolecules in crystals, and allow structural heterogeneity and disorder to be systematically explored on different scales. The new approach is demonstrated on several case studies and the SARS-CoV-2 main protease, and demonstrates how the choice of disorder model affects the type of disorder that is sampled by the restrained molecular-dynamics simulation

Cryo-EM single-particle structure refinement and map calculation using Servalcat.

In 2020, cryo-EM single-particle analysis achieved true atomic resolution thanks to technological developments in hardware and software. The number of high-resolution reconstructions continues to grow, increasing the importance of the accurate determination of atomic coordinates. Here, a new Python package and program called Servalcat is presented that is designed to facilitate atomic model refinement. Servalcat implements a refinement pipeline using the program REFMAC5 from the CCP4 package. After the refinement, Servalcat calculates a weighted Fo - Fc difference map, which is derived from Bayesian statistics. This map helps manual and automatic model building in real space, as is common practice in crystallography. The Fo - Fc map helps in the visualization of weak features including hydrogen densities. Although hydrogen densities are weak, they are stronger than in the electron-density maps produced by X-ray crystallography, and some H atoms are even visible at ∼1.8 Å resolution. Servalcat also facilitates atomic model refinement under symmetry constraints. If point-group symmetry has been applied to the map during reconstruction, the asymmetric unit model is refined with the appropriate symmetry constraints

Правове регулювання безоплатної приватизації земель запасу

Досліджуються правові підстави безоплатної приватизації земель запасу. Автор виокремлює два її різновиди – гарантовану і негарантовану. Розкриваються особли­вості багатоетапної процедури реалізації права на безоплатну приватизацію земель запасу в Україні.Исследуются правовые основания бесплатной приватизации земель запаса. Автор разделяет два ее разновидности – гарантированную и негарантированную. Раскрыва­ются особенности многоэтапной процедуры реализации права на бесплатную прива­тизацию земель в Украине.Legal rules about free of payment land privatization are investigated. The author pro­poses dividing free of payment land privatization into two varieties: guaranteed and non­guaranteed ones. Legal peculiarities of multistoried procedure of free of payment land priva­tization in Ukraine are disclosed

A Structural Study of the Cytoplasmic Chaperone Effect of 14-3-3 Proteins on Ataxin-1

Expansion of the polyglutamine tract in the N terminus of Ataxin-1 is the main cause of the neurodegenerative disease, spinocerebellar ataxia type 1 (SCA1). However, the C-terminal part of the protein - including its AXH domain and a phosphorylation on residue serine 776 - also plays a crucial role in disease development. This phosphorylation event is known to be crucial for the interaction of Ataxin-1 with the 14-3-3 adaptor proteins and has been shown to indirectly contribute to Ataxin-1 stability. Here we show that 14-3-3 also has a direct anti-aggregation or chaperone effect on Ataxin-1. Furthermore, we provide structural and biophysical information revealing how phosphorylated S776 in the intrinsically disordered C terminus of Ataxin-1 mediates the cytoplasmic interaction with 14-3-3 proteins. Based on these findings, we propose that 14-3-3 exerts the observed chaperone effect by interfering with Ataxin-1 dimerization through its AXH domain, reducing further self-association. The chaperone effect is particularly important in the context of SCA1, as it was previously shown that a soluble form of mutant Ataxin-1 is the major driver of pathology

Cryo-EM model validation recommendations based on outcomes of the 2019 EMDataResource challenge

This paper describes outcomes of the 2019 Cryo-EM Model Challenge. The goals were to (1) assess the quality of models that can be produced from cryogenic electron microscopy (cryo-EM) maps using current modeling software, (2) evaluate reproducibility of modeling results from different software developers and users and (3) compare performance of current metrics used for model evaluation, particularly Fit-to-Map metrics, with focus on near-atomic resolution. Our findings demonstrate the relatively high accuracy and reproducibility of cryo-EM models derived by 13 participating teams from four benchmark maps, including three forming a resolution series (1.8 to 3.1 Å). The results permit specific recommendations to be made about validating near-atomic cryo-EM structures both in the context of individual experiments and structure data archives such as the Protein Data Bank. We recommend the adoption of multiple scoring parameters to provide full and objective annotation and assessment of the model, reflective of the observed cryo-EM map density

A life cycle assessment of the closed-loop recycling and thermal recovery of post-consumer PET

Waste polyethylene terephthalate (PET) bottles collected by source-segregation recycling schemes can be treated by mechanical and chemical means to remove any contaminants. The cleaned PET can then be melted and formed into pellets with the same physical properties as virgin PET. Full-scale trials have shown that this reprocessed material can be used with virgin PET to make new bottles. An alternative recovery option is to collect the waste PET with the non-recyclable waste and burn it in an energy-from-waste plant. A life cycle inventory was produced for these two recovery options to compare the atmospheric emissions of key pollutants and the overall environmental impacts. The recycling option resulted in an overall reduction in the emission of each key pollutant and in the overall environmental impact. This was due to the reduction in emissions from displacing virgin PET. The energy-from-waste route also leads to a reduction in the emissions of several of the pollutants, depending on the assumptions made about the thermal efficiency of the process and the pollutants generated by burning the PET

New tools to study the conformational dynamics of large proteins: A study of FBP-aldolase and other enzymes

Proteins are inherently flexible molecules, both in structure and in function. Although multiple conformational changes appear vital for enzyme catalysis, these essential . motions are poorly understood for the majority of systems at present. Escherichia coli Class II fructose 1,6-bisphosphate aldolase (FBP-aldolase) provides an excellent model system for the study of enzyme dynamics as it has been extensively characterised and adopts the (a1~)8 barrel fold. This ubiquitous and versatile architecture represents -10% of all known enzyme structures. Multiple structures of FBP-aldolase have been solved previously, and reveal significant conformational changes. In addition;· sitedirected mutagenesis studies restricting loop flexibility have been shown to reduce catalytic rate, further highlighting the potential importance of conformational dynamics. The work presented here expands these studies by using a novel suite of NMR relaxation experiments to present the first quantified description of the su19-nanosecond dynamics of a protein that exhibits the (a/~)8 barrel.morphology. Experimental studies of this important fold have been restricted due to the size and high helical content that cause highly overlapped 20 spectra. This critically hinders NMR relaxation techniques, preventing the observation of molecular motion at atomic resolution. 20 HSQC based NMR ,sp~troscopy experiments only produce unambiguous rate measurement of less than 40% of residues in FBP-aldolase. Here, a novel sUit~ of Hadamard CO' encoded T1, T2 and NOE pulse sequences is presented· which dramatically reduce spectral crowding to allow the measurement of over 80% of the cross-peaks in FBP-aldolase spectra. The general applicability of these new methods will allow NMR relaxation studies to be conducted on significantly larger protein systems. To apply these Hadamard encoded pulse sequences, and also to assign the backbone amide chemical shifts, 2H, 13C and 15N isotopically enriched FBP-aldolase samples were prepared. Expression and purification protocols were optimised to provide suffiCient material to maximise resonance signal. These samples, in conjunction with multidimensional TROSY experiments, yielded chemical shift assignment for 55% of backbone resonances. This assignment process was aided by the production of seven samples of FBP-aldolase selectiVity labelled for specific amino' acid types. The T10 Tz and NOE relaxation rates were measured for FBP~aldolase at two high-field strengths, 750MHz and 900MHz. The data were quantified using an adapted version ·of the model-free protocol, and revealed important dynamic contributions from the major catalytic loop and the catalytic divalent cation. These experiments were repeated in the presence of the first binding substrate DHAp, which illustrated that the major loop remains flexible to accommodate the binding of the second substrate, G3P. In addition, a new protocol for the comparison of crystal structure pairs based on the distance difference matrix (DDM) is presented. An extensive database of enzyme structure pairs was generated, and the DDM-based methods were used to seek and describe structural deviation. This provided a comprehensive description of the prevalence, magnitude and manner of substrate-induced conformational changes. This study also demonstrates the conformational fluctuations present in enzyme pairs when both structures are solved either in the presence or absence of substrate. The methods developed in this work provide a detailed description of sub-nanosecond dynamics in FBP-aldolase and the conformational changes obseNed for enzymes in general. It is hoped that these novel tools will advance future investigations to further delineate the role of dynamics in enzyme catalysis.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Improving sampling of crystallographic disorder in ensemble refinement

Ensemble refinement, the application of molecular dynamics to crystallographic refinement, explicitly models the disorder inherent in macromolecular structures. These ensemble models have been shown to produce more accurate structures than traditional single-model structures. However, suboptimal sampling of the molecular-dynamics simulation and modelling of crystallo­graphic disorder has limited the utility of the method, and can lead to unphysical and strained models. Here, two improvements to the ensemble refinement method implemented within Phenix are presented: DEN restraints, which guide the local sampling of conformations and allow a more robust exploration of local conformational landscapes, and ECHT disorder models, which allow the selection of more physically meaningful and effective disorder models for parameterizing the continuous disorder components within a crystal. These improvements lead to more consistent and physically interpretable simulations of macromolecules in crystals, and allow structural heterogeneity and disorder to be systematically explored on different scales. The new approach is demonstrated on several case studies and the SARS-CoV-2 main protease, and demonstrates how the choice of disorder model affects the type of disorder that is sampled by the restrained molecular-dynamics simulation

Data from: Modelling dynamics in protein crystal structures by ensemble refinement

Single-structure models derived from X-ray data do not adequately account for the inherent, functionally important dynamics of protein molecules. We generated ensembles of structures by time-averaged refinement, where local molecular vibrations were sampled by molecular-dynamics (MD) simulation whilst global disorder was partitioned into an underlying overall translation–libration–screw (TLS) model. Modeling of 20 protein datasets at 1.1–3.1 Å resolution reduced cross-validated R_free values by 0.3–4.9%, indicating that ensemble models fit the X-ray data better than single structures. The ensembles revealed that, while most proteins display a well-ordered core, some proteins exhibit a ‘molten core’ likely supporting functionally important dynamics in ligand binding, enzyme activity and protomer assembly. Order–disorder changes in HIV protease indicate a mechanism of entropy compensation for ordering the catalytic residues upon ligand binding by disordering specific core residues. Thus, ensemble refinement extracts dynamical details from the X-ray data that allow a more comprehensive understanding of structure–dynamics–function relationships