Produce Density Maps Research Articles

Automated multiconformer model building for X-ray crystallography and cryo-EM.

In their folded state, biomolecules exchange between multiple conformational states that are crucial for their function. Traditional structural biology methods, such as X-ray crystallography and cryogenic electron microscopy (cryo-EM), produce density maps that are ensemble averages, reflecting molecules in various conformations. Yet, most models derived from these maps explicitly represent only a single conformation, overlooking the complexity of biomolecular structures. To accurately reflect the diversity of biomolecular forms, there is a pressing need to shift toward modeling structural ensembles that mirror the experimental data. However, the challenge of distinguishing signal from noise complicates manual efforts to create these models. In response, we introduce the latest enhancements to qFit, an automated computational strategy designed to incorporate protein conformational heterogeneity into models built into density maps. These algorithmic improvements in qFit are substantiated by superior Rfree and geometry metrics across a wide range of proteins. Importantly, unlike more complex multicopy ensemble models, the multiconformer models produced by qFit can be manually modified in most major model building software (e.g., Coot) and fit can be further improved by refinement using standard pipelines (e.g., Phenix, Refmac, Buster). By reducing the barrier of creating multiconformer models, qFit can foster the development of new hypotheses about the relationship between macromolecular conformational dynamics and function.

Automated multiconformer model building for X-ray crystallography and cryo-EM

In their folded state, biomolecules exchange between multiple conformational states that are crucial for their function. Traditional structural biology methods, such as X-ray crystallography and cryogenic electron microscopy (cryo-EM), produce density maps that are ensemble averages, reflecting molecules in various conformations. Yet, most models derived from these maps explicitly represent only a single conformation, overlooking the complexity of biomolecular structures. To accurately reflect the diversity of biomolecular forms, there is a pressing need to shift toward modeling structural ensembles that mirror the experimental data. However, the challenge of distinguishing signal from noise complicates manual efforts to create these models. In response, we introduce the latest enhancements to qFit, an automated computational strategy designed to incorporate protein conformational heterogeneity into models built into density maps. These algorithmic improvements in qFit are substantiated by superior Rfree and geometry metrics across a wide range of proteins. Importantly, unlike more complex multicopy ensemble models, the multiconformer models produced by qFit can be manually modified in most major model building software (e.g., Coot) and fit can be further improved by refinement using standard pipelines (e.g., Phenix, Refmac, Buster). By reducing the barrier of creating multiconformer models, qFit can foster the development of new hypotheses about the relationship between macromolecular conformational dynamics and function.

Pampas fox spatial and temporal variation in Argentinean agroecosystems

Context Agricultural activities have reduced wildlife natural habitats and increased the spatial overlap between animals’ distribution and human activities. However, carnivores with a broad diet and flexible habitat requirements can tolerate human-induced environmental changes. Thus, identifying changes in their densities and spatial distribution are important factors to take into consideration when working towards minimising human–carnivore conflict. Aims Our aim was to identify the main environmental and human variables influencing Pampas fox density and to produce density maps in the reproductive and non-reproductive seasons. Methods We performed spotlight counts at night, following the line transect method in the non-reproductive and reproductive seasons during two consecutive years. We also obtained landscape, human-impact and environmental spatial variables using remote sensing data and Geographic Information Systems. We modelled and mapped the Pampas fox’s spatial density using Density Surface Models. Key results We found that disturbance by human activities and landscape configuration influenced the spatial variation of the Pampas fox density across time and space. We registered a positive association between the number of foxes and the proximity to urban areas and paved roads, and we also found higher densities near grasslands areas and less modified habitats varying with the season and year. A higher density of foxes was observed in the non-reproductive season compared with the reproductive season. Conclusions and Implications This study provides insight on the variation in Pampas fox densities across agroecosystems. It highlights the relevance of more naturalised and protected habitats to sustain the Pampas fox population in highly fragmented landscapes, but also shows a positive association with disturbed areas. The spatial information developed in this study is useful to identify areas where ecosystem services could be encouraged, helping to maintain the ecosystems’ equilibrium and biodiversity conservation, and develop new management programs creating human–wildlife coexistence.

Methylenecyclopropene: local vision of the first 1B2 excited state.

The 1A1 ground and the first 1B2 excited states of the methylenecyclopropene (triafulvene) are described by localized wave functions, based on 20 structures valence bond structures. The results are compared to CASSCF(4,4) calculations for both the energetics and the dipole moment. Additional calculations with partial electronic delocalization are presented, and it is shown that the dipole moment modification does not correspond to a situation where the antiaromatic situation prevails (with 4n electrons in the cycle). Part of the analysis uses a "trust factor" that helps to decide if a wave function is appropriate to describe a given state. The trust factor compares the VB wave function to the CASSCF's with their overlap. Finally, the valence bond density is used to produce density maps that illustrate the electron transfer upon excitation. Graphical Abstract A projector-based method compares CASSCF wave functions to local wave functions, including Lewis structures as shown in the picture. A "trust factor" (τ) is obtained. Both the ground state and the first excited state of the methylenecyclopropene are discussed.

Modeling Beta-Traces for Beta-Barrels from Cryo-EM Density Maps.

Cryo-electron microscopy (cryo-EM) has produced density maps of various resolutions. Although α-helices can be detected from density maps at 5–8 Å resolutions, β-strands are challenging to detect at such density maps due to close-spacing of β-strands. The variety of shapes of β-sheets adds the complexity of β-strands detection from density maps. We propose a new approach to model traces of β-strands for β-barrel density regions that are extracted from cryo-EM density maps. In the test containing eight β-barrels extracted from experimental cryo-EM density maps at 5.5 Å–8.25 Å resolution, StrandRoller detected about 74.26% of the amino acids in the β-strands with an overall 2.05 Å 2-way distance between the detected β-traces and the observed ones, if the best of the fifteen detection cases is considered.

Resolution and Probabilistic Structural Models of Subcomponents Derived from CryoEM Maps of Mature P22 Bacteriophage

CryoEM continues to produce density maps of larger and more complex assemblies with multiple protein components of mixed symmetries. Resolution is not always uniform throughout a cryoEM map, and thus it can be very useful to be able to estimate resolution in specific components. Also, measuring the uncertainty in structural models built based on density maps is becoming increasingly important especially at medium to low resolutions. Here, we present procedures to 1) estimate resolution in subcomponents by gold-standard FSC, 2) validate modeling procedures, particularly at medium resolutions, which can include loop modeling and flexible fitting, and 3) build probabilistic models that merge high-accuracy priors (such as crystallographic structures) with medium resolution information (the cryoEM densities they are fitted to). The main goal of such procedures is to better understand what information and insights can be obtained from cryoEM density maps, particularly at medium resolution. As an example, we apply these methods to new cryoEM maps of the mature bacteriophage P22 virion, reconstructed without imposing icosahedral symmetry. Resolution estimates based on gold-stadard FSC show the highest accuracies are in the coat region (7.6A), while portal, hub, adhesin, and needle components are seen at lower resolutions (9.2A, 8.5A, 10.9A, and 10.5A respectively). These differences are indicative of inherent structural heterogeneity and/or reconstruction accuracy in different subcomponents of the map. Using loop modeling and flexible fitting to augment and mold crystallographic models based on observed density, we then build probabilistic models of some of the subcomponents including the portal, hub, and adhesin proteins, leading to some new insights into the structure and composition of the P22 virion.

Resolution and Probabilistic Models of Components in CryoEM Maps of Mature P22 Bacteriophage

CryoEM continues to produce density maps of larger and more complex assemblies with multiple protein components of mixed symmetries. Resolution is not always uniform throughout a cryoEM map, and it can be useful to estimate the resolution in specific molecular components of a large assembly. In this study, we present procedures to 1) estimate the resolution in subcomponents by gold-standard Fourier shell correlation (FSC); 2) validate modeling procedures, particularly at medium resolutions, which can include loop modeling and flexible fitting; and 3) build probabilistic models that combine high-accuracy priors (such as crystallographic structures) with medium-resolution cryoEM densities. As an example, we apply these methods to new cryoEM maps of the mature bacteriophage P22, reconstructed without imposing icosahedral symmetry. Resolution estimates based on gold-standard FSC show the highest resolution in the coat region (7.6 Å), whereas other components are at slightly lower resolutions: portal (9.2 Å), hub (8.5 Å), tailspike (10.9 Å), and needle (10.5 Å). These differences are indicative of inherent structural heterogeneity and/or reconstruction accuracy in different subcomponents of the map. Probabilistic models for these subcomponents provide new insights, to our knowledge, and structural information when taking into account uncertainty given the limitations of the observed density.

Cryo-EM Reveals Promoter DNA Binding and Conformational Flexibility of the General Transcription Factor TFIID

The general transcription factor IID (TFIID) is required for initiation of RNA polymerase II-dependent transcription at many eukaryotic promoters. TFIID comprises the TATA-binding protein (TBP) and several conserved TBP-associated factors (TAFs). Recognition of the core promoter by TFIID assists assembly of the preinitiation complex. Using cryo-electron microscopy in combination with methods for ab initio single-particle reconstruction and heterogeneity analysis, we have produced density maps of two conformational states of Schizosaccharomyces pombe TFIID, containing and lacking TBP. We report that TBP-binding is coupled to a massive histone-fold domain rearrangement. Moreover, docking of the TBP-TAF1(N-terminus) atomic structure to the TFIID map and reconstruction of a TAF-promoter DNA complex helps to account for TAF-dependent regulation of promoter-TBP and promoter-TAF interactions.