Protein Structure Analysis Research Articles

Integrative structural analysis of disease-related proteins using SAXS and X-ray crystallography

Integrative structural analysis of disease-related proteins using SAXS and X-ray crystallography

All-in-One Pseudo-MS3 Method for the Analysis of Gas-Phase Cleavable Protein Crosslinking Reactions.

Crosslinking mass spectrometry (XL-MS) supports structure analysis of individual proteins and highly complex whole-cell interactomes. The identification of crosslinked peptides from enzymatic digests remains challenging, especially at the cell level. Empirical methods that use gas-phase cleavable crosslinkers can simplify the identification process by enabling an MS3-based strategy that turns crosslink identification into a simpler problem of detecting two separable peptides. However, the method is limited to select instrument platforms and is challenged by duty cycle constraints. Here, we revisit a pseudo-MS3 concept that incorporates in-source fragmentation, where a fast switch between gentle high-transmission source conditions and harsher in-source fragmentation settings liberates peptides for standard MS2-based peptide identification. We present an all-in-one method where retention time matches between the crosslink precursor and the liberated peptides establish linkage, and MS2 sequencing identifies the source-liberated peptides. We demonstrate that DC4, a very labile cleavable crosslinker, generates high-intensity peptides in-source. Crosslinks can be identified from these liberated peptides, as they are chromatographically well-resolved from monolinks. Using bovine serum albumin (BSA) as a crosslinking test case, we detect 27% more crosslinks with pseudo-MS3 over a best-in-class MS3 method. While performance is slightly lower for whole-cell lysates (generating two-thirds of the identifications of a standard method), we find that 60% of these hits are unique, highlighting the complementarity of the method.

Direct observation of the conformational states of PIEZO1

PIEZOs are mechanosensitive ion channels that convert force into chemoelectric signals1,2 and have essential roles in diverse physiological settings3. In vitro studies have proposed that PIEZO channels transduce mechanical force through the deformation of extensive blades of transmembrane domains emanating from a central ion-conducting pore4–8. However, little is known about how these channels interact with their native environment and which molecular movements underlie activation. Here we directly observe the conformational dynamics of the blades of individual PIEZO1 molecules in a cell using nanoscopic fluorescence imaging. Compared with previous structural models of PIEZO1, we show that the blades are significantly expanded at rest by the bending stress exerted by the plasma membrane. The degree of expansion varies dramatically along the length of the blade, where decreased binding strength between subdomains can explain increased flexibility of the distal blade. Using chemical and mechanical modulators of PIEZO1, we show that blade expansion and channel activation are correlated. Our findings begin to uncover how PIEZO1 is activated in a native environment. More generally, as we reliably detect conformational shifts of single nanometres from populations of channels, we expect that this approach will serve as a framework for the structural analysis of membrane proteins through nanoscopic imaging.

A Novel Mechanically Robust and Biodegradable Egg White Hydrogel Membrane by Combined Unidirectional Nanopore Dehydration and Annealing.

A homogeneous egg white obtained by high-speed shearing and centrifugation was dehydrated into a fragile and water-soluble egg white glass (EWG) by unidirectional nanopore dehydration (UND). After EWG annealing, it can become an egg white hydrogel membrane (EWHM) that is water-insoluble, flexible, biocompatible, and mechanically robust. Its tensile strength, elongation at break, and the swelling ratio are about 5.84 MPa, 50-110%, and 60-130%, respectively. Protein structure analysis showed that UND caused the rearrangement of the protein molecules to form EWG with random coil and α-helix structures. The thermal decomposition temperature of the EWG was 309.25 °C. After EWG annealing at over 100 or 110 °C for 1.0 h or 45 min, the porous network EWHM was mainly composed of β-sheet structures, and the thermal decomposition temperature increased to 317.25-318.43 °C. Their 12-day residues in five proteases ranged from 1% to 99%, and the order was pepsin > neutral protease > papain > trypsin > alkaline protease. Mouse fibroblast L929 cells can adhere, grow, and proliferate well on these EWHMs. Therefore, the combined technology of UND and annealing for green and novel processing of EWHM has potential applications in the field of biomimetic and biomedical materials.

Innovation in Cross-Linking Mass Spectrometry Workflows: Toward a Comprehensive, Flexible, and Customizable Data Analysis Platform.

Cross-linking mass spectrometry (XL-MS) is widely used in the analysis of protein structure and protein-protein interactions (PPIs). Throughout the entire workflow, the utilization of cross-linkers and the interpretation of cross-linking data are the core steps. In recent years, the development of cross-linkers and analytical software mostly follow up on the classical models of non-cleavable cross-linkers such as BS3/DSS and MS-cleavable cross-linkers such as DSSO. Although such a paradigm promotes the maturity and robustness of the XL-MS field, it confines the innovation and flexibility of new cross-linkers and analytical software. This critical insight will discuss the classification, advantages, and disadvantages of existing data analysis search engines. Take the new platinum-based metal cross-linker as an example, potential pitfalls in characterization of cross-linked peptides using existing software are discussed. Finally, ideas on developing more flexible, comprehensive, and user-friendly cross-linkers and software tools are proposed.

Identification and Functional Characterization of the Nonexpressor of Pathogenesis-Related Genes 1 (NPR1) Gene in the Tea Plant (Camellia sinensis)

Tea is one of the most popular non-alcoholic beverages globally. The leaves of the tea plants serve as the raw materials for tea production, making tea tree cultivation widespread as an economically significant tree species. Nonexpressor of pathogenesis-related genes 1 (NPR1) is a key regulator of the salicylic acid (SA) signaling pathway, playing a significant role in the plant’s response to biotic and abiotic stresses. However, the NPR1-like gene family in tea plants remains elusive. This study aims to systematically analyze the NPR1-like gene family in tea plants. Four CsNPR1-like genes were identified and categorized into three branches based on phylogenetic analysis. Collinearity analysis demonstrated conservation of the NPR1-like gene across different species. Analysis of cis-regulatory elements suggested that CsNPR1-like genes may be involved in various hormonal and stress responses. Protein structure analysis revealed that the CsNPR1-likes exhibited typical ANK and BTB/POZ structural domains. The protein interaction network identified various known and novel NPR1-interacting proteins, including the TGA transcription factor, which was further confirmed in planta. Meta-analysis of transcriptome data indicated that CsNPR1-like genes had spatiotemporal expression patterns and were induced by drought and cold stresses. Additionally, CsNPR1a activates the accumulation of the acidic SA-dependent pathogenic-associated protein PR1 but not the basic chitinase (PR3) in a transient expression assay. This study provides comprehensive information for investigating the NPR1-like gene family in tea plants.

Engagement of intrinsic disordered proteins in protein-protein interaction.

Proteins from the intrinsically disordered group (IDP) focus the attention of many researchers engaged in protein structure analysis. The main criteria used in their identification are lack of secondary structure and significant structural variability. This variability takes forms that cannot be identified in the X-ray technique. In the present study, different criteria were used to assess the status of IDP proteins and their fragments recognized as intrinsically disordered regions (IDRs). The status of the hydrophobic core in proteins identified as IDPs and in their complexes was assessed. The status of IDRs as components of the ordering structure resulting from the construction of the hydrophobic core was also assessed. The hydrophobic core is understood as a structure encompassing the entire molecule in the form of a centrally located high concentration of hydrophobicity and a shell with a gradually decreasing level of hydrophobicity until it reaches a level close to zero on the protein surface. It is a model assuming that the protein folding process follows a micellization pattern aiming at exposing polar residues on the surface, with the simultaneous isolation of hydrophobic amino acids from the polar aquatic environment. The use of the model of hydrophobicity distribution in proteins in the form of the 3D Gaussian distribution described on the protein particle introduces the possibility of assessing the degree of similarity to the assumed micelle-like distribution and also enables the identification of deviations and mismatch between the actual distribution and the idealized distribution. The FOD (fuzzy oil drop) model and its modified FOD-M version allow for the quantitative assessment of these differences and the assessment of the relationship of these areas to the protein function. In the present work, the sections of IDRs in protein complexes classified as IDPs are analyzed. The classification "disordered" in the structural sense (lack of secondary structure or high flexibility) does not always entail a mismatch with the structure of the hydrophobic core. Particularly, the interface area, often consisting of IDRs, in many analyzed complexes shows the compliance of the hydrophobicity distribution with the idealized distribution, which proves that matching to the structure of the hydrophobic core does not require secondary structure ordering.

Protein structuromics: A new method for protein structure-function crosstalk in glioma.

Glioma is a type of tumor that starts in the glial cells of the brain or spine. Since the 1800s, when the disease was first named, its survival rates have always been unsatisfactory. Despite great advances in molecular biology and traditional treatment methods, many questions regarding cancer occurrence and the underlying mechanism remain to be answered. In this study, we assessed the protein structural features of 20 oncogenes and 20 anti-oncogenes via protein structure and dynamic analysis methods and 3D structural and systematic analyses of the structure-function relationships of proteins. All of these results directly indicate that unfavorable group proteins show more complex structures than favorable group proteins. As the tumor cell microenvironment changes, the balance of oncogene-related and anti-oncogene-related proteins is disrupted, and most of the structures of the two groups of proteins will be disrupted. However, more unfavorable group proteins will maintain and refold to achieve their correct shape faster and perform their functions more quickly than favorable group proteins, and the former thus support cancer development. We hope that these analyses will help promote mechanistic research and the development of new treatments for glioma.

PROFASA-a web-based protein fragment and structure analysis workstation.

Introduction: In the field of bioinformatics and computational biology, protein structure modelling and analysis is a crucial aspect. However, most existing tools require a high degree of technical expertise and lack a user-friendly interface. To address this problem, we developed a protein workstation called PROFASA. Methods: PROFASA is an innovative protein workstation that combines state-of-the-art protein structure visualisation techniques with cutting-edge tools and algorithms for protein analysis. Our goal is to provide users with a comprehensive platform for all protein sequence and structure analyses. PROFASA is designed with the idea of simplifying complex protein analysis workflows into one-click operations, while providing powerful customisation options to meet the needs of professional users. Results: PROFASA provides a one-stop solution that enables users to perform protein structure evaluation, parametric analysis and protein visualisation. Users can use I-TASSER or AlphaFold2 to construct protein models with one click, generate new protein sequences, models, and calculate protein parameters. In addition, PROFASA offers features such as real-time collaboration, note sharing, and shared projects, making it an ideal tool for researchers and teaching professionals. Discussion: PROFASA's innovation lies in its user-friendly interface and one-stop solution. It not only lowers the barrier to entry for protein computation, analysis and visualisation tools, but also opens up new possibilities for protein research and education. We expect PROFASA to advance the study of protein design and engineering and open up new research areas.

Development of bright red-shifted miRFP704nano using structural analysis of miRFPnano proteins.

We recently converted the GAF domain of NpR3784 cyanobacteriochrome into near-infrared (NIR) fluorescent proteins (FPs). Unlike cyanobacterichrome, which incorporates phycocyanobilin tetrapyrrole, engineered NIR FPs bind biliverdin abundant in mammalian cells, thus being the smallest scaffold for it. Here, we determined the crystal structure of the brightest blue-shifted protein of the series, miRFP670nano3, at 1.8 Å resolution, characterized its chromophore environment and explained the molecular basis of its spectral properties. Using the determined structure, we have rationally designed a red-shifted NIR FP, termed miRFP704nano, with excitation at 680 nm and emission at 704 nm. miRFP704nano exhibits a small size of 17 kDa, enhanced molecular brightness, photostability and pH-stability. miRFP704nano performs well in various protein fusions in live mammalian cells and should become a versatile genetically-encoded NIR probe for multiplexed imaging across spatial scales in different modalities.

Determining the structure of the bacterial voltage-gated sodium channel NaChBac embedded in liposomes by cryo electron tomography and subtomogram averaging

Voltage-gated sodium channels shape action potentials that propagate signals along cells. When the membrane potential reaches a certain threshold, the channels open and allow sodium ions to flow through the membrane depolarizing it, followed by the deactivation of the channels. Opening and closing of the channels is important for cellular signalling and regulates various physiological processes in muscles, heart and brain. Mechanistic insights into the voltage-gated channels are difficult to achieve as the proteins are typically extracted from membranes for structural analysis which results in the loss of the transmembrane potential that regulates their activity. Here, we report the structural analysis of a bacterial voltage-gated sodium channel, NaChBac, reconstituted in liposomes under an electrochemical gradient by cryo electron tomography and subtomogram averaging. We show that the small channel, most of the residues of which are embedded in the membrane, can be localized using a genetically fused GFP. GFP can aid the initial alignment to an average resulting in a correct structure, but does not help for the final refinement. At a moderate resolution of ˜16 Å the structure of NaChBac in an unrestricted membrane bilayer is 10% wider than the structure of the purified protein previously solved in nanodiscs, suggesting the potential movement of the peripheral voltage-sensing domains. Our study explores the limits of structural analysis of membrane proteins in membranes.

Are the integrin binding motifs within SARS CoV-2 spike protein and MHC class II alleles playing the key role in COVID-19?

The previous studies on the RGD motif (aa403-405) within the SARS CoV-2 spike (S) protein receptor binding domain (RBD) suggest that the RGD motif binding integrin(s) may play an important role in infection of the host cells. We also discussed the possible role of two other integrin binding motifs that are present in S protein: LDI (aa585-587) and ECD (661-663), the motifs used by some other viruses in the course of infection. The MultiFOLD models for protein structure analysis have shown that the ECD motif is clearly accessible in the S protein, whereas the RGD and LDI motifs are partially accessible. Furthermore, the amino acids that are present in Epstein-Barr virus protein (EBV) gp42 playing very important role in binding to the HLA-DRB1 molecule and in the subsequent immune response evasion, are also present in the S protein heptad repeat-2. Our MultiFOLD model analyses have shown that these amino acids are clearly accessible on the surface in each S protein chain as monomers and in the homotrimer complex and bind to HLA-DRB1 β chain. Therefore, they may have the identical role in SARS CoV-2 immune evasion as in EBV infection. The prediction analyses of the MHC class II binding peptides within the S protein have shown that the RGD motif is present in the core 9-mer peptide IRGDEVRQI within the two HLA-DRB1*03:01 and HLA-DRB3*01.01 strong binding 15-mer peptides suggesting that RGD motif may be the potential immune epitope. Accordingly, infected HLA-DRB1*03:01 or HLA-DRB3*01.01 positive individuals may develop high affinity anti-RGD motif antibodies that react with the RGD motif in the host proteins, like fibrinogen, thrombin or von Willebrand factor, affecting haemostasis or participating in autoimmune disorders.

PH-induced egg white protein foaming properties enhancement: Insight into protein structure and quantitative proteomic analysis at protein adsorption layer

The foaming properties of egg white protein (EWP) after pH-induced unfolding (pH 2, pH 4, pH 10 and pH 12) and refolding treatment (pH 2–7, pH 4–7, pH 10–7 and pH 12-7 (adjusting the above pH back to 7)) were studied by structure changes and quantitative proteomic analysis. It was found that all pH treatment improved foaming capacity (FC) and foaming stability (FS) of EWP, especially at pH 2 and pH 12, where EWP was in a molten globule state with unfolded structure (especially at pH 12), high surface hydrophobicity (except for pH 12-treated EWP), more free sulfhydryl groups and up-regulated adsorption proteins. However, adjusting the pH back to 7 limited the foaming properties due to protein refolding. Meanwhile the presence of down-regulated proteins was another reason for relatively poor FC at pH 2–7. Notably, the firmest foam and high FS at pH 12-7 were attributed to the generation of aggregates and the long-term stability of adsorbed proteins. The results of quantitative proteomics implied that lysozyme, ovalbumin and protein TENP were identified as key proteins associated with excellent FC at pH 2, pH 12 and pH 12-7, while β-ovomucin contributed the most to FC at pH 2–7. Moreover, protein TENP and ovomucoid were more relevant to maintaining and reducing FS. Overall, this work elucidated the mechanism of pH treatment to enhance the foaming properties of EWP, which would provide the necessary theoretical support for the quality improvement and product development of EWP powder.

Identification of a xyloglucan beta-xylopyranosyltransferase from Vaccinium corymbosum.

Plant cell walls contain the hemicellulose xyloglucan, whose fine structure may vary depending on cell type, tissue, and/or plant species. Most but not all of the glycosyltransferases involved in the biosynthesis of xyloglucan sidechains have been identified. Here, we report the identification of several functional glycosyltransferases from blueberry (Vaccinium corymbosum bluecrop). Among those transferases is a hitherto elusive Xyloglucan:Beta-xylosylTransferase (XBT). Heterologous expression of VcXBT in the Arabidopsis thaliana double mutant mur3 xlt2, where xyloglucan consists only of an unsubstituted xylosylated glucan core structure, results in the production of the xylopyranose-containing "U" sidechain as characterized by mass spectrometry, glycosidic linkage, and NMR analysis. The introduction of the additional xylopyranosyl residue rescues the dwarfed phenotype of the untransformed Arabidopsis mur3 xlt2 mutant to wild-type height. Structural protein analysis using Alphafold of this and other related xyloglucan glycosyltransferase family 47 proteins not only identifies potential domains that might influence the regioselectivity of these enzymes but also gives hints to specific amino acids that might determine the donor-substrate specificity of these glycosyltransferases.

Synergism between x-ray crystallography and NMR residual dipolar couplings in characterizing protein dynamics

The important role of structural dynamics in protein function is widely recognized. Thermal or B-factors and their anisotropy, seen in x-ray analysis of protein structures, report on the presence of atomic coordinate heterogeneity that can be attributed to motion. However, their quantitative evaluation in terms of protein dynamics by x-ray ensemble refinement remains challenging. NMR spectroscopy provides quantitative information on the amplitudes and time scales of motional processes. Unfortunately, with a few exceptions, the NMR data do not provide direct insights into the atomic details of dynamic trajectories. Residual dipolar couplings, measured by solution NMR, are very precise parameters reporting on the time-averaged bond-vector orientations and may offer the opportunity to derive correctly weighted dynamic ensembles of structures for cases where multiple high-resolution x-ray structures are available. Applications to the SARS-CoV-2 main protease, Mpro, and ubiquitin highlight this complementarity of NMR and crystallography for quantitative assessment of internal motions.

Search of a genomic sequence database for potential novel blood group antigens: Investigation into why some amino acid substitutions are not immunogenic.

Polypeptide blood group antigens are typically identified through investigation of the antibodies they induce. Human genome sequence databases are a new tool to identify AA substitutions that potentially create blood group antigens. The Erythrogene genomic sequence database was searched for missense mutations not known to be blood group antigens in the extracellular domains of selected RBC proteins in European populations. Any mutations found with prevalence of 1%-90% and not known to have induced antibodies in transfusion practice were analyzed using protein structural analysis and epitope prediction programs to determine why they apparently lack immunogenicity. Thirteen missense mutations not known to create blood group antigens were identified in the extracellular domains of Kell, BCAM, and RhD proteins, but not in RhCE, Urea Transporter 1 (Kidd), Atypical Chemokine Receptor 1 (Duffy), glycophorin A or glycophorin B. While 11 of the 13 mutations had low prevalence (<1%), a Kell Ser726Pro substitution and a BCAM Val196Ile substitution had predicted phenotype prevalences of 43.2% and 5.7%, respectively. Ser726Pro had multiple properties of a linear B-cell epitope, but possible suboptimal protein location for B-cell receptor binding and limited T-cell epitope possibilities. Val196Ile was not predicted to be in a linear B-cell epitope. Multiple potential new blood group antigens of low prevalence were identified. Whether they are antigenic remains to be determined. Two higher prevalence variants in Kell and BCAM are unlikely antigens, otherwise their antibodies presumably would already have been identified. Possible reasons for their poor immunogenicity were identified.

Detection of Coronaviruses in Bats in Lebanon during 2020.

Bats are considered the main reservoir of coronaviruses (CoVs), and research evidence suggests the essential role of bats in the emergence of Severe Acute Respiratory Syndrome Coronaviruses (SARS-CoV) and SARS-CoV-2. SARS-CoV-like viruses have been recently detected in bats in different countries. In 2020, we conducted surveillance for CoVs among six different bat species in Lebanon. Of 622 swab specimens taken, 77 tested positive. Alpha- and Beta- CoVs were identified in samples collected from different species. Our results show that SARS-like coronaviruses circulate in bats in this region, and we provide new data on their genetic diversity. The interaction between the spike of the detected SARS-CoV-like viruses and the human angiotensin-converting enzyme 2 (hACE2) receptor could be crucial in understanding the origin of the epidemic. The 3D protein structure analysis revealed that the receptor-binding domains of the SARS-like virus identified in Lebanon bind to the hACE2 protein more efficiently than to the spike of the SARS-CoV-2 strain. The spike of the detected SARS-CoV-like viruses does not contain the recognition site of furin at the cleavage site. Thus, our study highlights the variety of bat coronaviruses in Lebanon and suggests the zoonotic potential for other SARS-CoV-like viruses.

Cross-Linking Mass Spectrometry on P-Glycoprotein.

The ABC transporter P-glycoprotein (Pgp) has been found to be involved in multidrug resistance in tumor cells. Lipids and cholesterol have a pivotal role in Pgp's conformations; however, it is often difficult to investigate it with conventional structural biology techniques. Here, we applied robust approaches coupled with cross-linking mass spectrometry (XL-MS), where the natural lipid environment remains quasi-intact. Two experimental approaches were carried out using different cross-linkers (i) on living cells, followed by membrane preparation and immunoprecipitation enrichment of Pgp, and (ii) on-bead, subsequent to membrane preparation and immunoprecipitation. Pgp-containing complexes were enriched employing extracellular monoclonal anti-Pgp antibodies on magnetic beads, followed by on-bead enzymatic digestion. The LC-MS/MS results revealed mono-links on Pgp's solvent-accessible residues, while intraprotein cross-links confirmed a complex interplay between extracellular, transmembrane, and intracellular segments of the protein, of which several have been reported to be connected to cholesterol. Harnessing the MS results and those of molecular docking, we suggest an epitope for the 15D3 cholesterol-dependent mouse monoclonal antibody. Additionally, enriched neighbors of Pgp prove the strong connection of Pgp to the cytoskeleton and other cholesterol-regulated proteins. These findings suggest that XL-MS may be utilized for protein structure and network analyses in such convoluted systems as membrane proteins.

Identification and genome-wide analysis provide insights into the genetic diversity and biotechnological potentials of novel cold-adapted Acinetobacter strain.

Extreme cold environments, such as polar regions or high-altitude mountains, are known for their challenging conditions including low temperatures, high salinity, and limited nutrient availability. Microbes that thrive in these environments have evolved specialized strategies to survive and function under such harsh conditions. The study aims to identify, sequence the genome, perform genome assembly, and conduct a comparative genome-wide analysis of Acinetobacter sp. strain P1, which was isolated from the Batura glacier regions of Pakistan. A basic local alignment search tool of NCBI using 16s RNA gene sequence confirmed the strain Acinetobacter following phylogenetic analysis revealed that strain P1 clustered with Acinetobacter sp. strain AcBz01. The high-throughput Genome sequencing was done by the NovaSeq 6000 sequencing system following de novo genome assembly reported 23 contigs, a genome size of 3,732,502bp containing approximately 3489 genes and 63 RNAs (60 tRNA, 3 rRNA). The comparative genome analysis revealed thatAcinetobacter sp. strain P1 exhibited the highest homology with theAcinetobacter baumannii ATCC 17978 genome and encompassed 1668 indispensable genes, 1280 conserved genes 1821 specific genes suggesting high genomic plasticity and evolutionary diversity. The genes with functional assignments include exopolysaccharide phosphotransferase enzyme, cold-shock proteins, T6SS, membrane modifications, antibiotic resistance, and set of genes related to a wide range of metabolic characteristics such as exopolysaccharides were also present. Moreover, the structural prediction analysis of EPS proteins reveals that structural flexibility allows for conformational modifications during catalysis, which boosts or increases the catalytic effectiveness at lower temperatures. Overall, the identification of Acinetobacter, a cold-adapted bacterium, offers promising applications in bioremediation, enzyme production, food preservation, pharmaceutical development, and astrobiology. Further research and exploration of these microorganisms can unlock their full biotechnological potential and contribute to various industries and scientific endeavors.

Network integration and protein structural binding analysis of neurodegeneration-related interactome.

Neurodegenerative diseases (NDs) usually connect with aggregation and molecular interactions of pathological proteins. The integration of accumulative data from clinical and biomedical research will allow for the excavation of pathological proteins and related interactors. It is also important to systematically study their interacting proteins in order to find more related proteins and potential therapeutic targets. Understanding binding regions in protein interactions will help functional proteomics and provide an alternative method for predicting novel interactions. This study integrated data from biomedical research to achieve systematic mining and analysis of pathogenic proteins and their interaction network. A workflow has been built as a solution for the collective information of proteins involved in NDs, related protein-protein interactions (PPIs) and interactive visualizations. It also included protein isoforms and mapped them in a disease-related PPI network to illuminate the impact of alternative splicing on protein binding. The interacting proteins enriched by diseases and biological processes (BPs) revealed possible regulatory modules. A high-resolution network with structural affinity information was generated. Finally, Neurodegenerative Disease Atlas (NDAtlas) was constructed with an interactive and intuitive view of protein docking with 3D molecular graphics beyond the traditional 2D network. NDAtlas is available at http://bis.zju.edu.cn/ndatlas.