Solving protein structures by combining structure prediction, molecular replacement and direct-methods-aided model completion.

Highly accurate protein structure prediction can generate accurate models of protein and protein-protein complexes in X-ray crystallography. However, the question of how to make more effective use of predicted models for completing structure analysis, and which strategies should be employed for the more challenging cases such as multi-helical structures, multimeric structures and extremely large structures, both in the model preparation and in the completion steps, remains open for discussion. In this paper, a new strategy is proposed based on the framework of direct methods and dual-space iteration, which can greatly simplify the pre-processing steps of predicted models both in normal and in challenging cases. Following this strategy, full-length models or the conservative structural domains could be used directly as the starting model, and the phase error and the model bias between the starting model and the real structure would be modified in the direct-methods-based dual-space iteration. Many challenging cases (from CASP14) have been tested for the general applicability of this constructive strategy, and almost complete models have been generated with reasonable statistics. The hybrid strategy therefore provides a meaningful scheme for X-ray structure determination using a predicted model as the starting point.

Professor Dr Hendrik (Henk) Schenk (1939-2023).

Obituary for Professor Dr Hendrik (Henk) Schenk.

Facing the phase problem.

The marvel of X-ray crystallography is the beauty and precision of the atomic structures deduced from diffraction patterns. Since these patterns record only amplitudes, phases for the diffracted waves must also be evaluated for systematic structure determination. Thus, we have the phase problem as a central complication, both intellectually for the field and practically so for many analyses. Here, I discuss how we - myself, my laboratory and the diffraction community - have faced the phase problem, considering the evolution of methods for phase evaluation as structural biology developed to the present day. During the explosive growth of macromolecular crystallography, practice in diffraction analysis evolved from a universal reliance on isomorphous replacement to the eventual domination of anomalous diffraction for de novo structure determination. As the Protein Data Bank (PDB) grew and familial relationships among proteins became clear, molecular replacement overtook all other phasing methods; however, experimental phasing remained essential for molecules without obvious precedents, with multi- and single-wavelength anomalous diffraction (MAD and SAD) predominating. While the mathematics-based direct methods had proved to be inadequate for typical macromolecules, they returned to crack substantial selenium substructures in SAD analyses of selenomethionyl proteins. Native SAD, exploiting the intrinsic S and P atoms of biomolecules, has become routine. Selenomethionyl SAD and MAD were the mainstays of structural genomics efforts to populate the PDB with novel proteins. A recent dividend has been paid in the success of PDB-trained artificial intelligence approaches for protein structure prediction. Currently, molecular replacement with AlphaFold models often obviates the need for experimental phase evaluation. For multiple reasons, we are now unfazed by the phase problem. Cryo-EM analysis is an attractive alternative to crystallography for many applications faced by today's structural biologists. It simply finesses the phase problem; however, the principles and procedures of diffraction analysis remain pertinent and are adopted in single-particle cryo-EM studies of biomolecules.

Pattern and trends of the total and age-specific fertility rates during 1990-2018 in Pakistan.

BACKGROUND: Pakistan has an inadequate vital event registration system, leading to fewer than half of all births being registered, and this issue is further exacerbated by systematic recall errors and omission of births. This study aims to evaluate direct and indirect methods of fertility estimation to analyze the trends and patterns of fertility rates in Pakistan from 1990 to 2018. DESIGN/METHODOLOGY/APPROACH: Indirect methods are utilized in this study to evaluate the direction and extent of changes in total and age-specific fertility rates, and these findings are compared to direct estimates. The study draws data on livebirths from four waves of the Pakistan Demographic and Health Survey that took place between 1990 and 2018. To ensure the quality of data, graphical methods and Whipple and Myers indices are employed. Additionally, the Brass Relational Gompertz model was used to analyze the data. RESULTS: The Relational Gompertz model revealed that total fertility rates (TFRs) were higher than direct estimates by 0.4 children and age-specific fertility rates (ASFR) were higher for all age groups except the oldest. The difference was more significant among younger women aged 15-24, and less so for age groups 29 and above. The gap in estimated fertility between direct and indirect methods decreased with age. CONCLUSION: The indirect method is an invaluable tool in situations where direct measurement of fertility rates is challenging or impossible. By utilizing this method, policymakers can gain important insights into the fertility patterns and trends of a population, which is crucial for making informed decisions on fertility planning.

Multivariate estimation of substructure amplitudes for a single-wavelength anomalous diffraction experiment.

To determine a substructure from single-wavelength anomalous diffraction (SAD) data using Patterson or direct methods, the substructure-factor amplitude (|Fa|) is first estimated. Currently, the absolute value of the Bijvoet difference is widely used as an estimate of |Fa| values for SAD data. Here, an equation is derived from multivariate statistics and tested that takes into account the correlation between the observed positive (F+) and negative (F-) Friedel pairs and Fa along with measurement errors in the observed data. The multivariate estimation of |Fa| has been implemented in a new program, Afro. Results on over 180 test cases show that Afro provides a higher correlation to the final substructure-factor amplitudes (calculated from the refined, final substructures) than the Bijvoet differences and improves the robustness of direct-methods substructure detection.

Extending the novel |ρ|-based phasing algorithm to the solution of anomalous scattering substructures from SAD data of protein crystals.

Owing to the importance of the single-wavelength anomalous diffraction (SAD) technique, the recently developed |ρ|-based phasing algorithm (SM,|ρ|) incorporating the inner-pixel preservation (ipp) procedure [Rius & Torrelles (2021). Acta Cryst A77, 339-347] has been adapted to the determination of anomalous scattering substructures and its applicability tested on a series of 12 representative experimental data sets, mostly retrieved from the Protein Data Bank. To give an idea of the suitability of the data sets, the main indicators measuring their quality are also given. The dominant anomalous scatterers are either SeMet or S atoms, or metals/clusters incorporated by soaking. The resulting SAD-adapted algorithm solves the substructures of the test protein crystals quite efficiently.

Propulsive forces in human competitive swimming: a systematic review on direct assessment methods.

Human propulsive forces are a key-factor to enhance swimming performance, but there is scarce knowledge when using direct assessments. The aim of this review was to analyse the evidence about human propulsive forces in competitive swimming measured by direct assessment methods. A search up to 30 June 2020 was performed in Web of Science, PubMed, and Scopus databases. The Downs and Black Quality Assessment Checklist was used to assess the quality index (QI) of the included studies. Out of 2530 screened records, 35 articles met the inclusion criteria. Tethered-swimming and differential pressure sensors allow directly measure propulsive forces. Cross-sectional designs measured peak and mean propulsive force during the front crawl stroke and including men/boys (≥15 years-old) at different competitive levels were mostly reported. Men are more able to show higher propulsive forces than women counterparts. Short- and long-term effects were observed while using dry-land and in-water training programmes. The magnitude of propulsive force is dependent on the type of assessment method, swimming stroke, number of body limbs and gender. While the short-term effects supporting the different training programmes lead to an increase in propulsive force, there is a lack of long-term evidence.

A new density-modification procedure extending the application of the recent |ρ|-based phasing algorithm to larger crystal structures.

The incorporation of the new peakness-enhancing fast Fourier transform compatible ipp procedure (ipp = inner-pixel preservation) into the recently published SM algorithm based on |ρ| [Rius (2020). Acta Cryst A76, 489-493] improves its phasing efficiency for larger crystal structures with atomic resolution data. Its effectiveness is clearly demonstrated via a collection of test crystal structures (taken from the Protein Data Bank) either starting from random phase values or by using the randomly shifted modulus function (a Patterson-type synthesis) as initial ρ estimate. It has been found that in the presence of medium scatterers (e.g. S or Cl atoms) crystal structures with 1500 × c atoms in the unit cell (c = number of centerings) can be routinely solved. In the presence of strong scatterers like Fe, Cu or Zn atoms this number increases to around 5000 × c atoms. The implementation of this strengthened SM algorithm is simple, since it only includes a few easy-to-adjust parameters.

Determination and Environmental Implications of Aqueous-Phase Rate Constants in Radical Reactions.

Interests in the kinetics of radical-induced reactions in aqueous solution have grown remarkably due to their water engineering significance (e.g., advanced oxidation processes). Although compilations of the rate constants (k) for various radicals have been documented, surprisingly a systematic review has yet to be reported on the development of reliable methods for determining k values. A knowledge gap exists to critically evaluate and screen the various methods to measure them. In this review, we summarize the direct and indirect methods under steady-state and non-steady-state conditions, followed by critical evaluations on their advantages and disadvantages. The radicals of ·OH, [Formula: see text] , [Formula: see text] , and Cl· were chosen based on their significant aquatic environmental relevance. MS excel spreadsheets that demonstrate the determination processes were provided allowing one to reproduce the data and/or to analyze the unprocessed raw data as a "template". We formulated a standard operation procedure for the k determination, although there is simply no "versatile" method fitting for all radical reactions. Finally, existing challenges and future research focus are discussed. This is the first review covering methodological approaches and considerations, aiming to provide a holistic and fundamental basis to choose an appropriate method for determining the k values for bimolecular reactions between target compounds and radicals in the aqueous phase.

Ab Initio Determination of Peptide Structures by MicroED.

Structural elucidation of small macromolecules such as peptides has recently been facilitated by a growing number of technological advances to existing crystallographic methods. The emergence of electron micro-diffraction (MicroED) of protein nanocrystals under cryogenic conditions has enabled the interrogation of crystalline peptide assemblies only hundreds of nanometers thick. Collection of atomic or near-atomic resolution data by these methods has permitted the ab initio determination of structures of various amyloid-forming peptides, including segments derived from prions and ice-nucleating proteins. This chapter focuses on the process of ab initio structural determination from nano-scale peptide assemblies and other similar molecules.

Novel phasing method using the origin-free modulus sum function expressed in terms of the absolute electron density.

The origin-free modulus sum function SM refines the set Φ of phases of the structure factors by maximizing the coincidence between the experimental origin-free modulus function and the calculated one in terms of the ρ(Φ)2 density function [Rius, J. (1993). Acta Cryst. A49, 406-409]. Maximization is normally achieved through the recursive application of a Fourier-based algorithm. The purpose of the present study is: (i) to show that ρ(Φ)2 can be replaced by |ρ(Φ)| in SM; (ii) to illustrate the viability of the corresponding phasing algorithm with experimental data.

Low-dose electron diffraction tomography (LD-EDT).

A new electron diffraction tomography method is presented that is aimed to enable high performance electron crystallography experiments on beam sensitive materials using a standard TEM without any special equipment. Low-dose electron diffraction tomography minimizes the electron dose necessary for obtaining data sets suitable for structure solution by irradiating the crystal exclusively during the acquisition of the diffraction patterns. The performance of the method is successfully tested on two model structures, a complex oxide Sr5CuGe9O24 and the beam sensitive metal-organic framework (MOF) of manganese formiate. Even when the limited lifetime of a beam sensitive material only allows obtaining a data set of rather low completeness, the data quality is high enough for the structures to be solved to a high precision. Low-dose electron diffraction tomography is easy to implement and doesn't require any special equipment or lengthy calibration processes, making it accessible to a large number of scientists.

Updating direct methods.

The standard method of joint probability distribution functions, so crucial for the development of direct methods, has been revisited and updated. It consists of three steps: identification of the reflections which may contribute to the estimation of a given structure invariant or seminvariant, calculation of the corresponding joint probability distribution, and derivation of the conditional distribution of the invariant or seminvariant phase given the values of some diffracted amplitudes. In this article the conditional distributions are derived directly without passing through the second step. A good feature of direct methods is that they may work in the absence of any prior information: that is also their weakness. Different types of prior information have been taken into consideration: interatomic distances, interatomic vectors, Patterson peaks, structural model. The method of directly deriving the conditional distributions has been applied to those cases. Some new formulas have been obtained estimating two-, three- and four-phase invariants. Special attention has been dedicated to the practical aspects of the new formulas, in order to simplify their possible use in direct phasing procedures.

An introduction to experimental phasing of macromolecules illustrated by SHELX; new autotracing features.

For the purpose of this article, experimental phasing is understood to mean the determination of macromolecular structures by exploiting small intensity differences of Friedel opposites and possibly of reflections measured at different wavelengths or for heavy-atom derivatives, without the use of specific structural models. The SHELX programs provide a robust and efficient route for routine structure solution by the SAD, MAD and related methods, but involve a number of simplifying assumptions that may limit their applicability in borderline cases. The substructure atoms (i.e. those with significant anomalous scattering) are first located by direct methods, and the experimental data are then used to estimate phase shifts that are added to the substructure phases to obtain starting phases for the native reflections. These are then improved by density modification and, if the resolution of the data and the type of structure permit, polyalanine tracing. A number of extensions to the tracing algorithm are discussed; these are designed to improve its performance at low resolution. Given native data to 2.5 Šresolution or better, a correlation coefficient greater than 25% between the structure factors calculated from such a trace and the native data is usually a good indication that the structure has been solved.

Multiplex polymerase chain reaction assays for the detection of the zearalenone chemotype of Fusarium species in white and brown rice.

Early detection of the zearalenone (ZEA) chemotype of Fusarium species could be a precautionary measure for preventing ZEA contamination in rice. In this study, a multiplex polymerase chain reaction (mPCR) assay for detecting ZEA-producing fungi in rice was established using a set of four primers targeting the ZEA biosynthesis genes PKS3, PKS13, ZEB1, and ZEB2. Two mPCR approaches were used: one that amplified the DNA obtained from Fusarium isolates (conventional method) and another that directly amplified the target DNA from rice samples without time-consuming DNA isolation (direct method). The two mPCR methods showed high sensitivity in detecting ZEA-producing species, with a detection limit of 1.25 pg/µL of genomic DNA and 102 and 103 spores/g of white and brown rice, respectively. Both methods were specific for ZEA-producing species and gave four band patterns. The application of the two mPCR methods to 51 Fusarium isolates and 41 rice samples revealed that 31% (16 of 51) and 24% (10 of 41) of the samples were contaminated with ZEA-producing species, respectively. The mPCR results were further evaluated using high-performance liquid chromatography; in general, the two methods yielded similar results. These findings indicate that both mPCR methods are suitable for the detection of ZEA-producing Fusarium species in white and brown rice; however, the direct method yielded more rapid results.

Different methods to determine the encapsulation efficiency of protein in PLGA nanoparticles.

BACKGROUND: Effective encapsulation of drugs into the delivery systems could increase the efficiency of nanoparticles in prevention and treatment of diseases. OBJECTIVE: The purpose of this study was to compare the different methods for determination of encapsulation efficiency of a model protein in the PLGA nanoparticles. METHODS: The various direct methods include dichloromethane, acetonitrile, modified acetonitrile and NaOH based extraction and radioactive methods were used to directly calculate the encapsulation efficiency of the loaded protein in the PLGA nanoparticles. Furthermore, indirect methods include BCA, Fluorescent and radioactive methods were compared. RESULTS: The encapsulation efficiencies determined by indirect methods include dichloromethane, acetonitrile, modified acetonitrile, NaOH based extraction and radioactive methods were 12.62% ± 1.97, 17.43% ± 2.51, 64.69% ± 4.31, 86.36% ± 2.25 and 90.15% ± 1.78, respectively. Moreover, the encapsulation efficiencies determined by indirect methods include BCA, fluorescent and radioactive methods were 81.46% ± 1.92, 88.23% ± 1.15 and 89.6% ± 1.9, respectively. CONCLUSIONS: Among the results obtained by indirect methods, radioactive and fluorescent methods showed more reliable. Moreover, NaOH and radioactive methods were the most reliable methods among the direct methods.

Experimental Phasing: Substructure Solution and Density Modification as Implemented in SHELX.

This chapter describes experimental phasing methods as implemented in SHELX. After introducing fundamental concepts underlying all experimental phasing approaches, the methods used by SHELXC/D/E are described in greater detail, such as dual-space direct methods, Patterson seeding and density modification with the sphere of influence algorithm. Intensity differences from data for experimental phasing can also be used for the generation and usage of difference maps with ANODE for validation and phasing purposes. A short section describes how molecular replacement can be combined with experimental phasing methods. The second half covers practical challenges, such as prerequisites for successful experimental phasing, evaluation of potential solutions, and what to do if substructure search or density modification fails. It is also shown how auto-tracing in SHELXE can improve automation and how it ties in with automatic model building after phasing.

MPF, a multipurpose figure of merit for phasing procedures.

The efficient multipurpose figure of merit MPF has been defined and characterized. It may be very helpful in phasing procedures. Indeed, it might be used for establishing the centric or acentric nature of an unknown structure, for identifying the presence of some pseudotranslational symmetry, for recognizing the correct solution in multisolution approaches and for estimating the quality of structure models as they become available during the phasing process. Thus, phase improvement or deterioration may be monitored and useless models may be discarded to save computing time. It is also shown that MPF may be applied in different phasing approaches, no matter if ab initio or non ab initio.

Accuracy assessment methods of tissue marker clip placement after 11-gauge vacuum-assisted stereotactic breast biopsy: comparison of measurements using direct and conventional methods.

BACKGROUND: The objective of the study was to compare direct measurement with a conventional method for evaluation of clip placement in stereotactic vacuum-assisted breast biopsy (ST-VAB) and to evaluate the accuracy of clip placement using the direct method. METHODS: Accuracy of clip placement was assessed by measuring the distance from a residual calcification of a targeted calcification clustered to a clip on a mammogram after ST-VAB. Distances in the craniocaudal (CC) and mediolateral oblique (MLO) views were measured in 28 subjects with mammograms recorded twice or more after ST-VAB. The difference in the distance between the first and second measurements was defined as the reproducibility and was compared with that from a conventional method using a mask system with overlap of transparent film on the mammogram. The 3D clip-to-calcification distance was measured using the direct method in 71 subjects. RESULTS: The reproducibility of the direct method was higher than that of the conventional method in CC and MLO views (P = 0.002, P < 0.001). The median 3D clip-to-calcification distance was 2.8 mm, with an interquartile range of 2.0-4.8 mm and a range of 1.1-36.3 mm. CONCLUSION: The direct method used in this study was more accurate than the conventional method, and gave a median 3D distance of 2.8 mm between the calcification and clip.