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The results of tertiary structure assessment at CASP15 are reported. For the first time, recognizing the outstanding performance of AlphaFold 2 (AF2) at CASP14, all single-chain predictions were assessed together, irrespective of whether a template was available. At CASP15, there was no single stand-out group, with most of the best-scoring groups-led by PEZYFoldings, UM-TBM, and Yang Server-employing AF2 in one way or another. Many top groups paid special attention to generating deep Multiple Sequence Alignments (MSAs) and testing variant MSAs, thereby allowing them to successfully address some of the hardest targets. Such difficult targets, as well as lacking templates, were typically proteins with few homologues. Local divergence between prediction and target correlated with localization at crystal lattice or chain interfaces, and with regions exhibiting high B-factor factors in crystal structure targets, and should not necessarily be considered as representing error in the prediction. However, analysis of exposed and buried side chain accuracy showed room for improvement even in the latter. Nevertheless, a majority of groups produced high-quality predictions for most targets, which are valuable for experimental structure determination, functional analysis, and many other tasks across biology. These include those applying methods similar to those used to generate major resources such as the AlphaFold Protein Structure Database and the ESM Metagenomic atlas: the confidence estimates of the former were also notably accurate.

[Background]: Data management is fast becoming an essential part of scientific practice, driven by open science and FAIR (findable, accessible, interoperable, and reusable) data sharing requirements. Whilst data management plans (DMPs) are clear to data management experts and data stewards, understandings of their purpose and creation are often obscure to the producers of the data, which in academic environments are often PhD students., [Methods]: Within the RNAct EU Horizon 2020 ITN project, we engaged the 10 RNAct early-stage researchers (ESRs) in a training project aimed at formulating a DMP. To do so, we used the Data Stewardship Wizard (DSW) framework and modified the existing Life Sciences Knowledge Model into a simplified version aimed at training young scientists, with computational or experimental backgrounds, in core data management principles. We collected feedback from the ESRs during this exercise. Results: Here, we introduce our new life-sciences training DMP template for young scientists. We report and discuss our experiences as principal investigators (PIs) and ESRs during this project and address the typical difficulties that are encountered in developing and understanding a DMP., [Conclusions]: We found that the DS-wizard can also be an appropriate tool for DMP training, to get terminology and concepts across to researchers. A full training in addition requires an upstream step to present basic DMP concepts and a downstream step to publish a dataset in a (public) repository. Overall, the DS-Wizard tool was essential for our DMP training and we hope our efforts can be used in other projects.

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biochemistry and Biophysics, 2021., Non-coding (nc)RNAs demonstrate structural versatility to regulate genes at multiple levels. Many RNAs bind a variety of protein motifs to perform gene-regulatory activities at the transcriptional, post-transcriptional and translational levels. HIV-1 TAR RNA is one such structured RNA that recognizes the arginine-rich motif of the viral protein Tat, which is necessary to control viral mRNA transcription. During the latent stage of HIV infection, Tat is not produced appreciably, and inhibition of the TAR-Tat interaction represents a viable target for functional cure therapy. Numerous studies have determined the structures of HIV TAR in complex with BIV-Tat derived cyclic peptide inhibitors and small molecules, but they exhibit non-specific modes of molecular recognition and lack the potency needed for clinical efficacy. To address this shortcoming, I used rigorous structural (x-ray crystallography), biophysical (isothermal titration calorimetry and surface plasmon resonance) and computational (structural bioinformatics) methods to guide the development of new cyclic peptide inhibitors. I determined co-crystal structures of HIV-1 TAR RNA in complex with multiple lab-evolved TAR-binding proteins (TBPs) that possess a variety of arginine configurations. Using complementary ITC studies to relate structural observations to binding thermodynamics, I discovered how specific arginine configurations promote TAR binding, while others do not. I worked with the Fasan lab (University of Rochester) to develop a new library of TAR-binding cyclic peptides derived from lab-evolved proteins. I showed that several of these peptides retain TAR binding and that some also block binding of the HIV Tat RNA-binding domain to TAR. I observed that specific cyclization linkers can improve TAR binding and exhibit dose-dependent antiviral activity that protects cells against pseudotyped HIV-1 infection. In our structural characterization of TBP-TAR interactions, we observed a recurring structural

One of the major drawbacks of the industrial implementation of enzymatic processes is the low operational stability of the enzymes under tough industrial conditions. In this respect, the use of thermostable enzymes in the industry is gaining ground during the last decades. Herein, we report a structure-guided approach for the development of novel and thermostable 2'-deoxyribosyltransferases (NDTs) based on the computational design of disulfide bonds on hot spot positions. To this end, a small library of NDT variants from Lactobacillus delbrueckii (LdNDT) with introduced cysteine pairs was created. Among them, LdNDTS104C (100% retained activity) was chosen as the most thermostable variant, displaying a six- and two-fold enhanced long-term stability when stored at 55 °C (t1/255 °C ≈ 24 h) and 60 °C (t1/260 °C ≈ 4 h), respectively. Moreover, the biochemical characterization revealed that LdNDTS104C showed >60% relative activity across a broad range of temperature (30-90 °C) and pH (5-7). Finally, to study the potential application of LdNDTS104C as an industrial catalyst, the enzymatic synthesis of nelarabine was successfully carried out under different substrate conditions (1:1 and 3:1) at different reaction times. Under these experimental conditions, the production of nelarabine was increased up to 2.8-fold (72% conversion) compared with wild-type LdNDT.

Homology modeling is a method for building protein 3D structures using protein primary sequence and utilizing prior knowledge gained from structural similarities with other proteins. The homology modeling process is done in sequential steps where sequence/structure alignment is optimized, then a backbone is built and later, side-chains are added. Once the low-homology loops are modeled, the whole 3D structure is optimized and validated. In the past three decades, a few collective and collaborative initiatives allowed for continuous progress in both homology and ab initio modeling. Critical Assessment of protein Structure Prediction (CASP) is a worldwide community experiment that has historically recorded the progress in this field. Folding@Home and Rosetta@Home are examples of crowd-sourcing initiatives where the community is sharing computational resources, whereas RosettaCommons is an example of an initiative where a community is sharing a codebase for the development of computational algorithms. Foldit is another initiative where participants compete with each other in a protein folding video game to predict 3D structure. In the past few years, contact maps deep machine learning was introduced to the 3D structure prediction process, adding more information and increasing the accuracy of models significantly. In this review, we will take the reader in a journey of exploration from the beginnings to the most recent turnabouts, which have revolutionized the field of homology modeling. Moreover, we discuss the new trends emerging in this rapidly growing field.

Whole genome sequencing approaches proved pivotal to modern medicine through their in-depth identification of variation within organisms, and subsequent variant association with disease. Ironically, as the scientific community refined the detection of variation, medical professionals faced the inverse problem in utilizing this information, as disease correlation does not imply its causation. Missense mutations present a notable challenge to variant prioritization, as despite only leading to a single amino acid change, they have been commonly implicated in different human diseases e.g., cancer and pathogen drug resistance. It is thought that the subtilty with which they affect protein structure permits mild changes in functional properties, enabling their retention within a population. However, not all missense mutations lead to functional effects, making the distinction of the causative variation from background an important avenue for research. To address this, my doctoral work focused on establishing the use of structural and functional consequences of missense mutations to gain insight into the protein fitness mechanisms underlying disease, and subsequently guide the development of clinically useful predictive tools. These consequences were estimated through in silico biophysical tools which described changes in protein stability, dynamics, and affinities to interacting molecules. Their analysis highlighted the main fitness effects imparted by disease- or resistance-causing mutations, which were ultimately used to predict the phenotype of novel variants through machine learning. Across different studies, I have applied this methodology specifically to variation leading to drug resistance in tuberculosis and that leading to different diseases within human gene PTEN. My work on tuberculosis resistance highlighted unique mechanisms for mutations in genes alr, katG and rpoB. Notably, resistance mutations were associated with a protein fitness penalty ranging from hig

Identification of Mycobacterium tuberculosis (Mtb) increasingly involves characterising large sections of genetic material, such as through whole genome sequencing. While some mutations identified through these techniques are well characterised and strongly associated with anti-tuberculous drug resistance, such molecular methods frequently identify mutations with unknown significance or limited understanding of associated functional biological pathways. In this PhD, I have developed computational protein structural tools and mathematical models of TB transmission, that use genomic data to understand the impact of genomic changes and predict the consequences with regards to transmissibility and drug susceptibility of Mtb. Drug resistant mutations often carry both a selective advantage and a fitness cost, which can be reflected by the changes in protein structure and function. I developed a pipeline that captured the molecular consequences of coding mutations on protein stability, dynamics and interactions. Using my pipeline to evaluate the mechanistic consequences of mutations, I applied it to the real-time genomic analysis of a Victorian tuberculosis patient. The analysis led to identification of a novel resistant strain and altered patient treatment – the first reported use of structural information to guide clinical resistance detection. The information was then used to inform a compartmental epidemiological model of Mtb transmission in order to understand the rise of drug resistance in two high TB-incidence setting. Using a adaptive metropolis algorithm, I estimated drug resistance amplification proportions for two first-line anti-tuberculosis drugs, and explored how structural changes may alter the fitness landscape and transmission dynamics. The work highlighted the power of combining genomic, epidemiological and structural information in the fight against tuberculosis, and presents examples of application across the spectrum from laboratory, clinical and progr

Bio3D is a family of R packages for the analysis of biomolecular sequence, structure, and dynamics. Major functionality includes biomolecular database searching and retrieval, sequence and structure conservation analysis, ensemble normal mode analysis, protein structure and correlation network analysis, principal component, and related multivariate analysis methods. Here, we review recent package developments, including a new underlying segregation into separate packages for distinct analysis, and introduce a new method for structure analysis named ensemble difference distance matrix analysis (eDDM). The eDDM approach calculates and compares atomic distance matrices across large sets of homologous atomic structures to help identify the residue wise determinants underlying specific functional processes. An eDDM workflow is detailed along with an example application to a large protein family. As a new member of the Bio3D family, the Bio3D-eddm package supports both experimental and theoretical simulation-generated structures, is integrated with other methods for dissecting sequence-structure-function relationships, and can be used in a highly automated and reproducible manner. Bio3D is distributed as an integrated set of platform independent open source R packages available from: http://thegrantlab.org/bio3d/.

This report describes the tertiary structure prediction assessment of difficult modeling targets in the 14th round of the Critical Assessment of Structure Prediction (CASP14). We implemented an official ranking scheme that used the same scores as the previous CASP topology-based assessment, but combined these scores with one that emphasized physically realistic models. The top performing AlphaFold2 group outperformed the rest of the prediction community on all but two of the difficult targets considered in this assessment. They provided high quality models for most of the targets (86% over GDT_TS 70), including larger targets above 150 residues, and they correctly predicted the topology of almost all the rest. AlphaFold2 performance was followed by two manual Baker methods, a Feig method that refined Zhang-server models, two notable automated Zhang server methods (QUARK and Zhang-server), and a Zhang manual group. Despite the remarkable progress in protein structure prediction of difficult targets, both the prediction community and AlphaFold2, to a lesser extent, faced challenges with flexible regions and obligate oligomeric assemblies. The official ranking of top-performing methods was supported by performance generated PCA and heatmap clusters that gave insight into target difficulties and the most successful state-of-the-art structure prediction methodologies.

Homology modeling is a method for building protein 3D structures using protein primary sequence and utilizing prior knowledge gained from structural similarities with other proteins. The homology modeling process is done in sequential steps where sequence/structure alignment is optimized, then a backbone is built and later, side-chains are added. Once the low-homology loops are modeled, the whole 3D structure is optimized and validated. In the past three decades, a few collective and collaborative initiatives allowed for continuous progress in both homology and ab initio modeling. Critical Assessment of protein Structure Prediction (CASP) is a worldwide community experiment that has historically recorded the progress in this field. Folding@Home and Rosetta@Home are examples of crowd-sourcing initiatives where the community is sharing computational resources, whereas RosettaCommons is an example of an initiative where a community is sharing a codebase for the development of computational algorithms. Foldit is another initiative where participants compete with each other in a protein folding video game to predict 3D structure. In the past few years, contact maps deep machine learning was introduced to the 3D structure prediction process, adding more information and increasing the accuracy of models significantly. In this review, we will take the reader in a journey of exploration from the beginnings to the most recent turnabouts, which have revolutionized the field of homology modeling. Moreover, we discuss the new trends emerging in this rapidly growing field.

Homology modeling is a method for building protein 3D structures using protein primary sequence and utilizing prior knowledge gained from structural similarities with other proteins. The homology modeling process is done in sequential steps where sequence/structure alignment is optimized, then a backbone is built and later, side-chains are added. Once the low-homology loops are modeled, the whole 3D structure is optimized and validated. In the past three decades, a few collective and collaborative initiatives allowed for continuous progress in both homology and ab initio modeling. Critical Assessment of protein Structure Prediction (CASP) is a worldwide community experiment that has historically recorded the progress in this field. Folding@Home and Rosetta@Home are examples of crowd-sourcing initiatives where the community is sharing computational resources, whereas RosettaCommons is an example of an initiative where a community is sharing a codebase for the development of computational algorithms. Foldit is another initiative where participants compete with each other in a protein folding video game to predict 3D structure. In the past few years, contact maps deep machine learning was introduced to the 3D structure prediction process, adding more information and increasing the accuracy of models significantly. In this review, we will take the reader in a journey of exploration from the beginnings to the most recent turnabouts, which have revolutionized the field of homology modeling. Moreover, we discuss the new trends emerging in this rapidly growing field.

Homology modeling is a method for building protein 3D structures using protein primary sequence and utilizing prior knowledge gained from structural similarities with other proteins. The homology modeling process is done in sequential steps where sequence/structure alignment is optimized, then a backbone is built and later, side-chains are added. Once the low-homology loops are modeled, the whole 3D structure is optimized and validated. In the past three decades, a few collective and collaborative initiatives allowed for continuous progress in both homology and ab initio modeling. Critical Assessment of protein Structure Prediction (CASP) is a worldwide community experiment that has historically recorded the progress in this field. Folding@Home and Rosetta@Home are examples of crowd-sourcing initiatives where the community is sharing computational resources, whereas RosettaCommons is an example of an initiative where a community is sharing a codebase for the development of computational algorithms. Foldit is another initiative where participants compete with each other in a protein folding video game to predict 3D structure. In the past few years, contact maps deep machine learning was introduced to the 3D structure prediction process, adding more information and increasing the accuracy of models significantly. In this review, we will take the reader in a journey of exploration from the beginnings to the most recent turnabouts, which have revolutionized the field of homology modeling. Moreover, we discuss the new trends emerging in this rapidly growing field.

Coronavirus-like organisms have been previously identified in Arthropod ectoparasites (such as ticks and unfed cat flea). Yet, the question regarding the possible role of these arthropods as SARS-CoV-2 passive/biological transmission vectors is still poorly explored. In this study, we performed in silico structural and binding energy calculations to assess the risks associated with possible ectoparasite transmission. We found sufficient similarity between ectoparasite ACE and human ACE2 protein sequences to build good quality 3D-models of the SARS-CoV-2 Spike:ACE complex to assess the impacts of ectoparasite mutations on complex stability. For several species (e.g., water flea, deer tick, body louse), our analyses showed no significant destabilisation of the SARS-CoV-2 Spike:ACE complex, suggesting these species would bind the viral Spike protein. Our structural analyses also provide structural rationale for interactions between the viral Spike and the ectoparasite ACE proteins. Although we do not have experimental evidence of infection in these ectoparasites, the predicted stability of the complex suggests this is possible, raising concerns of a possible role in passive transmission of the virus to their human hosts

Structural bioinformatics provides the scientific methods and tools to analyse, archive, validate, and present the biomolecular structure data generated by the structural biology community. It also provides an important link with the genomics community, as structural bioinformaticians also use the extensive sequence data to predict protein structures and their functional sites. A very broad and active community of structural bioinformaticians exists across Europe, and 3D-Bioinfo will establish formal platforms to address their needs and better integrate their activities and initiatives. Our mission will be to strengthen the ties with the structural biology research communities in Europe covering life sciences, as well as chemistry and physics and to bridge the gap between these researchers in order to fully realize the potential of structural bioinformatics. Our Community will also undertake dedicated educational, training and outreach efforts to facilitate this, bringing new insights and thus facilitating the development of much needed innovative applications e.g. for human health, drug and protein design. Our combined efforts will be of critical importance to keep the European research efforts competitive in this respect.Here we highlight the major European contributions to the field of structural bioinformatics, the most pressing challenges remaining and how Europe-wide interactions, enabled by ELIXIR and its platforms, will help in addressing these challenges and in coordinating structural bioinformatics resources across Europe. In particular, we present recent activities and future plans to consolidate an ELIXIR 3D-Bioinfo Community in structural bioinformatics and propose means to develop better links across the community. These include building new consortia, organising workshops to establish data standards and seeking community agreement on benchmark data sets and strategies. We also highlight existing and planned collaborations with other ELIXIR Communities

The study of enzyme binding sites is an essential but rather demanding process of increased complexity since the amino acids lining these areas are not rigid. At the same time, the minimization of side effects and the specificity of new ligands is a great challenge in the structure-based drug design approach. Using glycogen phosphorylase - a validated target for the development of new antidiabetic agents - as a case study, this project focuses on the examination of side-chain conformations of amino acids that play a key role in the catalytic site of the enzyme. Specifically, different rotamers of each amino acid were collected to build a dataset of different conformations of the catalytic site. The rotamers were filtered by their probability of occurrence and subsequently, all rotamers that create steric clashes were rejected. Then, these conformations were clustered based on their similarity. Three different clustering algorithms and multiple numbers of clusters were tested using the silhouette scores evaluation for the clustering process. In order to measure the similarity, the Euclidean metric was used which due to the correspondence of the coordinates between the conformations was very similar to the cRMSD metric. Two-level clustering was applied to the dataset for more in-depth observations. According to the clustering results, specific aminoacids with major geometrical variations in their rotamers play the most important role in the separation of the clusters. Additionally, all rotamers of an amino acid can be grouped based on their structure, something that was confirmed using “Chimera” software as a visualization tool. To this end, the ultimate aim of this study is to examine whether the clustering of conformations produces clusters with points geometrically similar to each other, in order to identify near neighbors, i.e. conformations that are quite similar in structure but do not play a determinant role in the function and those that are quite diverse and

Homology modeling is a method for building protein 3D structures using protein primary sequence and utilizing prior knowledge gained from structural similarities with other proteins. The homology modeling process is done in sequential steps where sequence/structure alignment is optimized, then a backbone is built and later, side-chains are added. Once the low-homology loops are modeled, the whole 3D structure is optimized and validated. In the past three decades, a few collective and collaborative initiatives allowed for continuous progress in both homology and ab initio modeling. Critical Assessment of protein Structure Prediction (CASP) is a worldwide community experiment that has historically recorded the progress in this field. Folding@Home and Rosetta@Home are examples of crowd-sourcing initiatives where the community is sharing computational resources, whereas RosettaCommons is an example of an initiative where a community is sharing a codebase for the development of computational algorithms. Foldit is another initiative where participants compete with each other in a protein folding video game to predict 3D structure. In the past few years, contact maps deep machine learning was introduced to the 3D structure prediction process, adding more information and increasing the accuracy of models significantly. In this review, we will take the reader in a journey of exploration from the beginnings to the most recent turnabouts, which have revolutionized the field of homology modeling. Moreover, we discuss the new trends emerging in this rapidly growing field.

Structural bioinformatics provides the scientific methods and tools to analyse, archive, validate, and present the biomolecular structure data generated by the structural biology community. It also provides an important link with the genomics community, as structural bioinformaticians also use the extensive sequence data to predict protein structures and their functional sites. A very broad and active community of structural bioinformaticians exists across Europe, and 3D-Bioinfo will establish formal platforms to address their needs and better integrate their activities and initiatives. Our mission will be to strengthen the ties with the structural biology research communities in Europe covering life sciences, as well as chemistry and physics and to bridge the gap between these researchers in order to fully realize the potential of structural bioinformatics. Our Community will also undertake dedicated educational, training and outreach efforts to facilitate this, bringing new insights and thus facilitating the development of much needed innovative applications e.g. for human health, drug and protein design. Our combined efforts will be of critical importance to keep the European research efforts competitive in this respect. Here we highlight the major European contributions to the field of structural bioinformatics, the most pressing challenges remaining and how Europe-wide interactions, enabled by ELIXIR and its platforms, will help in addressing these challenges and in coordinating structural bioinformatics resources across Europe. In particular, we present recent activities and future plans to consolidate an ELIXIR 3D-Bioinfo Community in structural bioinformatics and propose means to develop better links across the community. These include building new consortia, organising workshops to establish data standards and seeking community agreement on benchmark data sets and strategies. We also highlight existing and planned collaborations with other ELIXIR Communi

Mutation of a single amino acid in a protein often has consequences on the interaction with other proteins, which may affect other interaction networks and pathways and ultimately lead to pathological phenotypes. A detailed structural analysis of these altered protein–protein complexes is essential to interpret the impact of a given mutation at the molecular level, which may facilitate intervention with therapeutic purposes. Given current limitations in the structural coverage of the human interactome, computational docking is emerging as a complementary source of information. Structural analysis can help to locate a given mutation at a protein–protein interface, but further characterisation of its impact on binding affinity is needed for a full interpretation. The integration of computational docking methods and energy‐based descriptors is facilitating the characterisation of an increasing number of disease‐related mutations, thus improving our understanding of the consequences of such mutations at the phenotypic level.

Motivation: Simultaneous alignment and folding (SA&F) of RNAs is the indispensable gold standard for inferring the structure of non-coding RNAs and their general analysis. The original algorithm, proposed by Sankoff, solves the theoretical problem exactly with a complexity of O(n^6) in the full energy model. Over the last two decades, several variants and improvements of the Sankoff algorithm have been proposed to reduce its extreme complexity by proposing simplified energy models or imposing restrictions on the predicted alignments. Results: Here we introduce a novel variant of Sankoff’s algorithm that reconciles the simplifications of PMcomp, namely moving from the full energy model to a simpler base pair-based model, with the accuracy of the loop-based full energy model. Instead of estimating pseudo-energies from unconditional base pair probabilities, our model calculates energies from conditional base pair probabilities that allow to accurately capture structure probabilities, which obey a conditional dependency. Supporting modifications with surgical precision, this model gives rise to the fast and highly accurate novel algorithm Pankov (Probabilistic Sankoff-like simultaneous alignment and folding of RNAs inspired by Markov chains). Pankov benefits from the speed-up of excluding unreliable base-pairing without compromising the loop-based free energy model of the Sankoff’s algorithm. We show that Pankov outperforms its predecessors LocARNA and SPARSE in folding quality and is faster than LocARNA. Pankov is developed as a branch of the LocARNA package and available at https://github.com/mmiladi/Pankov.

One of the known potential effects of disease-causing amino acid substitutions in proteins is to modulate protein-protein interactions (PPIs). To interpret such variants at the molecular level and to obtain useful information for prediction purposes, it is important to determine whether they are located at protein-protein interfaces, which are composed of two main regions, core and rim, with different evolutionary conservation and physicochemical properties. Here we have performed a structural, energetics and computational analysis of interactions between proteins hosting mutations related to diseases detected in newborn screening. Interface residues were classified as core or rim, showing that the core residues contribute the most to the binding free energy of the PPI. Disease-causing variants are more likely to occur at the interface core region rather than at the interface rim (p < 0.0001). In contrast, neutral variants are more often found at the interface rim or at the non-interacting surface rather than at the interface core region. We also found that arginine, tryptophan, and tyrosine are over-represented among mutated residues leading to disease. These results can enhance our understanding of disease at molecular level and thus contribute towards personalized medicine by helping clinicians to provide adequate diagnosis and treatments.

Using computational methods to identify protein-protein interactions (PPIs) supports experimental techniques by using less time and less resources. Identifying PPIs can be made through a template-based approach that describes how unstudied proteins interact by aligning a common structural template that exists in both interacting proteins. A pipeline that uses this is InterPred, that combines homology modelling and massive template comparison to construct coarse interaction models. These models are reviewed by a machine learning classifier that classifies models that shows traits of being true, which can be further refined with a docking technique. However, InterPred is dependent on using complex structural information, that might not be available from unstudied proteins, while it is suggested that PPIs are dependent of the shape and interface of proteins. A method that aligns structures based on the interface attributes is InterComp, which uses topological and sequence-order independent structural comparison. Implementing this method into InterPred will lead to restricting structural information to the interface of proteins, which could lead to discovery of undetected PPI models. The result showed that the modified pipeline was not comparable based on the receiver operating characteristic (ROC) performance. However, the modified pipeline could identify new potential PPIs that were undetected by InterPred.

Tras un breve repaso de conceptos bioquímicos, este material presenta y analiza unos cuantos algoritmos básicos para el estudio de la estructura molecular de proteínas y ácidos nucleicos. Al igual que en muchos libros de texto, iremos introduciendo problemas (y estrategias para resolverlos) desde el nivel de estructura primaria al nivel de estructura cuaternaria. A lo largo del curso encontraréis referencias a artículos que profundizan sobre aspectos particulares de cada tema, junto con ejemplos y ejercicios que a veces requieren de ciertos conocimientos de programación.

Many protein structure prediction programs exist and they can efficiently generate a number of protein models of a varying quality. One of the problems is that it is difficult to know which model is the best one for a given target sequence. Selecting the best model is one of the major tasks of Model Quality Assessment Programs (MQAPs). These programs are able to predict model accuracy before the native structure is determined. The accuracy estimation can be divided into two parts: global (the whole model accuracy) and local (the accuracy of each residue). ProQ2 is one of the most successful MQAPs for prediction of both local and global model accuracy and is based on a Machine Learning approach. In this thesis, I present my own contribution to Model Quality Assessment (MQA) and the newest developments of ProQ program series. Firstly, I describe a new ProQ2 implementation in the protein modelling software package Rosetta. This new implementation allows use of ProQ2 as a scoring function for conformational sampling inside Rosetta, which was not possible before. Moreover, I present two new methods, ProQ3 and ProQ3D that both outperform their predecessor. ProQ3 introduces new training features that are calculated from Rosetta energy functions and ProQ3D introduces a new machine learning approach based on deep learning. ProQ3 program participated in the 12th Community Wide Experiment on the Critical Assessment of Techniques for Protein Structure Prediction (CASP12) and was one of the best methods in the MQA category. Finally, an important issue in model quality assessment is how to select a target function that the predictor is trying to learn. In the fourth manuscript, I show that MQA results can be improved by selecting a contact-based target function instead of more conventional superposition based functions., At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.

Steroid hormones play clinically important and specific regulatory roles in the development, growth, metabolism, reproduction and brain function in human. The type 1 and 2 11-beta hydroxysteroid dehydrogenase enzymes (11beta-HSD1 and 2) have key roles in the pre-receptor modification of glucocorticoids allowing aldosterone regulation of blood pressure, control of systemic fluid and electrolyte homeostasis and modulation of integrated metabolism and brain function. Although the activity and function of 11beta-HSDs is thought to be understood, there exists an open reading frame for a distinct 11betaHSD-like gene; HSD11B1L, which is present in human, non-human primate, sheep, pig and many other higher organisms, whereas an orthologue is absent in the genomes of mouse, rat and rabbit. We have now characterised this novel HSD11B1L gene as encoded by 9 exons and analysis of EST library transcripts indicated the use of two alternate ATG start sites in exons 2 and 3, and alternate splicing in exon 9. Relatively strong HSD11B1L gene expression was detected in human, non-human primate and sheep tissue samples from the brain, ovary and testis. Analysis in non-human primates and sheep by immunohistochemistry localised HSD11B1L protein to the cytoplasm of ovarian granulosa cells, testis Leydig cells, and gonadatroph cells in the anterior pituitary. Intracellular localisation analysis in transfected human HEK293 cells showed HSD1L protein within the endoplasmic reticulum and sequence analysis suggests that similar to 11betaHSD1 it is membrane bound. The endogenous substrate of this third HSD enzyme remains elusive with localisation and expression data suggesting a reproductive hormone as a likely substrate.Copyright © 2017 The authors.

Most proteins carry out their functions through interactions with other molecules. Thus, proteins taking part in similar interactions are likely to carry out related functions. One way to determine whether two proteins do take part in similar interactions is by quantifying the likeness of their structures. This work focuses on the development of methods for the comparison of protein-protein and protein-ligand interactions, as well as their application to structure-based classification schemes. A method based on the MultiMer-align (or MM-align) program was developed and used to compare all known dimeric protein complexes. The results of the comparison demonstrates that the method improves over MM-align in a significant number of cases. The data was employed to classify the complexes, resulting in 1,761 different protein-protein interaction types. Through a statistical model, the number of existing protein-protein interaction types in nature was estimated at around 4,000. The model allowed the establishment of a relationship between the number of quaternary families (sequence-based groups of protein-protein complexes) and quaternary folds (structure-based groups). The interactions between proteins and small organic ligands were studied using sequence-independent methodologies. A new method was introduced to test three similarity metrics. The best of these metrics was subsequently employed, together with five other existing methodologies, to conduct an all-to-all comparison of all the known protein-FAD (Flavin-Adenine Dinucleotide) complexes. The results demonstrates that the new methodology captures the best the similarities between complexes in terms of protein-ligand contacts. Based on the all-to-all comparison, the protein-FAD complexes were subsequently separated into 237 groups. In the majority of cases, the classification divided the complexes according to their annotated function. Using a graph-based description of the FAD-binding sites, each group cou, Tiivistelmä Suurin osa proteiinien toiminnasta tapahtuu vuorovaikutuksessa muiden molekyylien kanssa. Proteiinit, jotka osallistuvat samanlaisiin vuorovaikutuksiin todennäköisesti toimivat samalla tavalla. Kahden proteiinin todennäköisyys esiintyä samanlaisissa vuorovaikutustilanteissa voidaan määrittää tutkimalla niiden rakenteellista samankaltaisuutta. Tämä väitöskirjatyö käsittelee proteiini-proteiini- ja proteiini-ligandi -vuorovaikutusten vertailuun käytettyjen menetelmien kehitystä, ja niiden soveltamista rakenteeseen perustuvissa luokittelujärjestelmissä. Tunnettuja dimeerisiä proteiinikomplekseja tutkittiin uudella MultiMer-align-ohjelmaan (MM-align) perustuvalla menetelmällä. Vertailun tulokset osoittavat, että uusi menetelmä suoriutui MM-alignia paremmin merkittävässä osassa tapauksista. Tuloksia käytettiin myös kompleksien luokitteluun, jonka tuloksena oli 1761 erilaista proteiinien välistä vuorovaikutustyyppiä. Luonnossa esiintyvien proteiinien välisten vuorovaikutusten määrän arvioitiin tilastollisen mallin avulla olevan noin 4000. Tilastollisen mallin avulla saatiin vertailtua sekä sekvenssin (”quaternary families”) sekä rakenteen (”quaternary folds”) mukaan ryhmiteltyjen proteiinikompleksien määriä. Proteiinien ja pienien orgaanisten ligandien välisiä vuorovaikutuksia tutkittiin sekvenssistä riippumattomilla menetelmillä. Uudella menetelmällä testattiin kolmea eri samankaltaisuutta mittaavaa metriikkaa. Näistä parasta käytettiin viiden muun tunnetun menetelmän kanssa vertailemaan kaikkia tunnettuja proteiini-FAD (Flavin-Adenine-Dinucleotide, flaviiniadeniinidinukleotidi) -komplekseja. Proteiini-ligandikontaktien osalta uusi menetelmä kuvasi kompleksien samankaltaisuutta muita menetelmiä paremmin. Vertailun tuloksia hyödyntäen proteiini-FAD-kompleksit luokiteltiin edelleen 237 ryhmään. Suurimmassa osassa tapauksista luokittelujärjestelmä oli onnistunut jakamaan kompleksit ryhmiin niiden toiminnallisuuden mukaisesti. Ryhmät voitiin määritellä yksikäsitt

Computational prediction of protein–protein complex structure by docking can provide structural and mechanistic insights for protein interactions of biomedical interest. However, current methods struggle with difficult cases, such as those involving flexible proteins, low-affinity complexes or transient interactions. A major challenge is how to efficiently sample the structural and energetic landscape of the association at different resolution levels, given that each scoring function is often highly coupled to a specific type of search method. Thus, new methodologies capable of accommodating multi-scale conformational flexibility and scoring are strongly needed. We describe here a new multi-scale protein–protein docking methodology, LightDock, capable of accommodating conformational flexibility and a variety of scoring functions at different resolution levels. Implicit use of normal modes during the search and atomic/coarse-grained combined scoring functions yielded improved predictive results with respect to state-of-the-art rigid-body docking, especially in flexible cases., B.J-G was supported by a FPI fellowship from the Spanish Ministry of Economy and Competitiveness. This work was supported by I+D+I Research Project grants BIO2013-48213-R and BIO2016-79930-R from the Spanish Ministry of Economy and Competitiveness. This work is partially supported by the European Union H2020 program through HiPEAC (GA 687698), by the Spanish Government through Programa Severo Ochoa (SEV-2015-0493), by the Spanish Ministry of Science and Technology (TIN2015-65316-P) and the Departament d’Innovació, Universitats i Empresa de la Generalitat de Catalunya, under project MPEXPAR: Models de Programaciói Entorns d’Execució Paral·lels (2014-SGR-1051)., Peer Reviewed, Postprint (author's final draft)

We present an in silico method to estimate the contribution of each residue in a protein to its overall stability using three database-derived statistical potentials that are based on inter-residue distances, backbone torsion angles and solvent accessibility, respectively. Residues that contribute very unfavorably to the folding free energy are defined as stability weaknesses, whereas residues that show a highly stabilizing contribution are called stability strengths. Strengths and/or weaknesses on residues that are in spatial contact are clustered into 3-dimensional (3D) stability patches. The identification and analysis of strength- and weakness-containing regions in a protein may reveal structural or functional characteristics, and/or interesting spots to introduce mutations. To illustrate the power of our method, we apply it to bovine seminal ribonuclease. This enzyme catalyzes the degradation of RNA strands, and has the peculiarity of undergoing 3D domain swapping in physiological conditions. The weaknesses and strengths were compared among the monomeric, dimeric and swapped dimeric forms. We identified weaknesses among the catalytic residues and a mixture of weaknesses and strengths among the substrate-binding residues in the three forms. In the regions involved in 3D swapping, we observed an accumulation of weaknesses in the monomer, which disappear in the dimer and especially in the swapped dimer. Moreover, monomeric homologous proteins were found to exhibit less weaknesses in these regions, whereas mutants known to favor unswapped dimerization appear stabilized in this form. Our method has several perspectives for functional annotation, rational prediction of targeted mutations, and mapping of stability changes upon conformational rearrangements., SCOPUS: ar.j, FLWIN, info:eu-repo/semantics/published

The antibody crystallizable fragment (Fc) is recognized by effector proteins as part of the immune system. Pathogens produce proteins that bind Fc in order to subvert or evade the immune response. The structural characterization of the determinants of Fc–protein association is essential to improve our understanding of the immune system at the molecular level and to develop new therapeutic agents. Furthermore, Fc-binding peptides and proteins are frequently used to purify therapeutic antibodies. Although several structures of Fc–protein complexes are available, numerous others have not yet been determined. Protein–protein docking could be used to investigate Fc–protein complexes; however, improved approaches are necessary to efficiently model such cases. In this study, a docking-based structural bioinformatics approach is developed for predicting the structures of Fc–protein complexes. Based on the available set of X-ray structures of Fc–protein complexes, three regions of the Fc, loosely corresponding to three turns within the structure, were defined as containing the essential features for protein recognition and used as restraints to filter the initial docking search. Rescoring the filtered poses with an optimal scoring strategy provided a success rate of approximately 80% of the test cases examined within the top ranked 20 poses, compared to approximately 20% by the initial unrestrained docking. The developed docking protocol provides a significant improvement over the initial unrestrained docking and will be valuable for predicting the structures of currently undetermined Fc–protein complexes, as well as in the design of peptides and proteins that target Fc., This work was supported by grant number BIO2013‐48213‐R from Spanish Government. M.A. is a recipient of an NHMRC Early Career Fellowship (GNT1054245). We acknowledge the computational resources provided by the Australian Government through the Victorian Life Sciences Computational Initiative under the National Computational Merit Allocation Scheme (project dq3). The authors gratefully acknowledge the contribution toward this study fromthe VictorianOperational Infrastructure Support Program received by the Burnet Institute., Peer Reviewed, Postprint (author's final draft)

Proteins play an essential role in virtually all of life's processes. Their function is tightly coupled to the three-dimensional structure they adopt. Solving protein structures experimentally is a complicated, time- and resource-consuming endeavor. With the rapid growth of the amount of protein sequences known, it is very likely that only a small fraction of known proteins will ever have their structures solved experimentally. Recently, computational methods for protein structure prediction have become increasingly accurate and offer a promise for bridging this gap. In this work, we show the ways the rapidly growing amounts of available biological data can be used to improve the accuracy of protein structure prediction. We discuss the use of multiple sources of structural information to improve the quality of predicted models. The methods for assigning the estimated quality scores for predicted models are discussed as well. In particular we present a novel, successful approach to the clustering-based quality assessment, which runs nearly 50 times faster than other methods of comparable accuracy, allowing to tackle much larger problems. Additionally, this thesis discusses the impact the recent breakthroughs in sequencing and the consequent rapid growth of sequence data have on the prediction of residue-residue contacts. We propose a novel methodology, which allows for predicting such contacts with astonishing, previously unheard-of accuracy. These contacts in turn can be used to guide protein modeling, allowing for discovering protein structures that have been unattainable by conventional prediction methods. Finally, a considerable part of this dissertation discusses the community efforts in protein structure prediction, as embodied by CASP (Critical Assessment of protein Structure Prediction) experiments., At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Submitted. Paper 4: Submitted.

Structural alignment is the process of finding similarities between a pair of proteins based on their three-dimensional shape. Accurate detection of such similarities could reveal evolutionary relationships and predict the functions of different proteins. The algorithmic solutions proposed so far rely on heuristics or approximation methods, but these could provably deliver poor results in general. Moreover, exact solutions tend to be very slow in practice. In this thesis we consider a graph-theoretic approach, which consists of representing tertiary protein structures as labeled graphs, and interpreting the structural alignment problem as a particular case of the Maximum Common Subgraph problem. We discuss the utility of employing a recent algorithmic technique for solving Maximum Common Subgraph. When applied to pairs of proteins, obtained from the Protein Data Bank (PDB), the proposed algorithm is shown to deliver reasonably accurate results. In order to improve the time-efficiency of this approach, a scalable parallel version is presented.

Protein structure alignment methods are essential for many different challenges in protein science, such as the determination of relations between proteins in the fold space or the analysis and prediction of their biological function. A number of different pairwise and multiple structure alignment (MStA) programs have been developed and provided to the community. Prior knowledge of the expected alignment accuracy is desirable for the user of such tools. To retrieve an estimate of the performance of current structure alignment methods, we compiled a test suite taken from literature and the SISYPHUS database consisting of proteins that are difficult to align. Subsequently, different MStA programs were evaluated regarding alignment correctness and general limitations. The analysis shows that there are large differences in the success between the methods in terms of applicability and correctness. The latter ranges from 44 to 75% correct core positions. Taking only the best method result per test case this number increases to 84%. We conclude that the methods available are applicable to difficult cases, but also that there is still room for improvements in both, practicability and alignment correctness. An approach that combines the currently available methods supported by a proper score would be useful. Until then, a user should not rely on just a single program., authorCount :3

The complexity of protein structures calls for simplified representations of their topology. The simplest possible mathematical description of a protein structure is a onedimensional profile representing, for instance, buriedness or secondary structure. This kind of representation has been introduced for studying the sequence to structure relationship, with applications to fold recognition. Here we define the effective connectivity profile (EC), a network theoretical profile that self-consistently represents the network structure of the protein contact matrix. The EC profile makes mathematically explicit the relationship between protein structure and protein sequence, because it allows predicting the average hydrophobicity profile (HP) and the distributions of amino acids at each site for families of homologous proteins sharing the same structure. In this sense, the EC provides an analytic solution to the statistical inverse folding problem, which consists in finding the statistical properties of the set of sequences compatible with a given structure. We tested these predictions with simulations of the structurally constrained neutral (SCN) model of protein evolution with structure conservation, for singleand multi-domain proteins, and for a wide range of mutation processes, the latter producing sequences with very different hydrophobicity profiles, finding that the EC-based predictions are accurate even when only one sequence of the family is known. The EC profile is very significantly correlated with the HP for sequence-structure pairs in the PDB as well. The EC profile generalizes the properties of previously introduced structural profiles to modular proteins such as multidomain chains, and its correlation with the sequence profile is substantially improved with respect to the previously defined profiles, particularly for long proteins. Furthermore, the EC profile has a dynamic interpretation, since the EC components are strongly inversely related with the temper

Structural bioinformatics deals with the analysis, classification and prediction of three-dimensional structures of biomacromolecules. It is becoming increasingly important as the number of structures is growing rapidly. This thesis describes three studies concerned with protein-function prediction and two studies about protein structure validation. New protein structures are often compared to known structures to find out if they have a known fold, which may provide hints about their function. The functionality and performance of eleven fold-comparison servers were evaluated. None of the tested servers achieved perfect recall, so in practise a combination of servers should be used. If fold comparison does not provide any hints about the function of a protein, structural motif searches can be employed. A survey of left-handed helices in known protein structures was carried out. The results show that left-handed helices are rare motifs, but most of them occur in active or ligand-binding sites. Their identification can therefore help to pinpoint potentially important residues. Sometimes all available methods fail to provide hints about the function of a protein. Therefore, the potential of using docking techniques to predict which ligands are likely to bind to a particular protein has been investigated. Initial results show that it will be difficult to build a reliable automated docking protocol that will suit all proteins. The effect of various phenomena on the precision of accessible surface area calculations was also investigated. The results suggest that it is prudent to report such values with a precision of 50 to 100 Å2. Finally, a survey of register shifts in known protein structures was carried out. The identified potential register shifts were analysed and classified. A machine-learning approach ("rough sets") was used in an attempt to diagnose register errors in structures.

L'objet de cette thèse de bioinformatique est de mieux comprendre l’ensemble des systèmes de régulation génique chez les bactéries. La disponibilité de centaines de génomes complets chez les bactéries ouvre la voie aux approches de génomique comparative et donc à l’étude de l’évolution des réseaux transcriptionnels bactériens. Dans un premier temps, nous avons implémenté et validé plusieurs méthodes de prédiction d’opérons sur base des génomes bactériens séquencés. Suite à cette étude, nous avons décidé d’utiliser un algorithme qui se base simplement sur un seuil sur la distance intergénique, à savoir la distance en paires de bases entre deux gènes adjacents. Notre évaluation sur base d’opérons annotés chez Escherichia coli et Bacillus subtilis nous permet de définir un seuil optimal de 55pb pour lequel nous obtenons respectivement 78 et 79% de précision. Deuxièmement, l’identification des motifs de régulation transcriptionnelle, tels les sites de liaison des facteurs de transcription, donne des indications de l’organisation de la régulation. Nous avons développé une méthode de recherche d’empreintes phylogénétiques qui consiste à découvrir des paires de mots espacés (dyades) statistiquement sur-représentées en amont de gènes orthologues bactériens. Notre méthode est particulièrement adaptée à la recherche de motifs chez les bactéries puisqu’elle profite d’une part des centaines de génomes bactériens séquencés et d’autre part les facteurs de transcription bactériens présentent des domaines Hélice-Tour-Hélice qui reconnaissent spécifiquement des dyades. Une évaluation systématique sur 368 gènes de E.coli a permis d’évaluer les performances de notre méthode et de tester l’influence de plus de 40 combinaisons de paramètres concernant le niveau taxonomique, l’inférence d’opérons, le filtrage des dyades spécifiques de E.coli, le choix des modèles de fond pour le calcul du score de significativité, et enfin un seuil sur ce score. L’analyse détaillée pour un cas d’étude, l, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Molecular Biology has allowed the characterization and manipulation of the molecules of life in the wet lab. Also the structures of those macromolecules are being continuously elucidated. During the last decades of the past century, there was an increasing interest to study how the different genes are organized into different organisms (‘genomes’) and how those genes are expressed into proteins to achieve their functions. Currently the sequences for many genes over several genomes have been determined. In parallel, the efforts to have the structure of the proteins coded by those genes go on. However it is experimentally much harder to obtain the structure of a protein, rather than just its sequence. For this reason, the number of protein structures available in databases is an order of magnitude or so lower than protein sequences. Furthermore, in order to understand how living organisms work at molecular level we need the information about the interaction of those proteins. Elucidating the structure of protein macromolecular assemblies is still more difficult. To that end, the use of computers to predict the structure of these complexes has gained interest over the last decades.The main subject of this thesis is the evaluation of current available computational methods to predict protein – protein interactions and build an atomic model of the complex. The core of the thesis is the evaluation protocol I have developed at Service de Conformation des Macromolécules Biologiques et de Bioinformatique, Université Libre de Bruxelles, and its computer implementation. This method has been massively used to evaluate the results on blind protein – protein interaction prediction in the context of the world-wide experiment CAPRI, which have been thoroughly reviewed in several publications [1-3]. In this experiment the structure of a protein complex (‘the target’) had to be modeled starting from the coordinates of the isolated molecules, prior to the release of the structure o, Doctorat en Sciences, info:eu-repo/semantics/nonPublished