This document discusses using bioinformatics tools such as ProMol, PyMol, BLAST, and Dali to predict the function of protein 4EZI, which has an unknown function in the Protein Data Bank. Analysis with these tools indicates 4EZI has similarities to hydrolases and esterases. Kinetics assays were performed using p-nitrophenyl acetate, but little activity was observed. AutoDock was used to suggest inositol as a potential substrate, and future work is planned to test other potential substrates and confirm function through additional kinetics assays and structural analysis.
Homology modeling, docking and comparative study of the selectivity of some h...Alexander Decker
This document describes a study that used homology modeling to generate a 3D structural model of the Plasmodium falciparum histone deacetylase 1 (PfHDAC-1) enzyme, an important antimalarial drug target. Nine known HDAC inhibitors were docked into the PfHDAC-1 model as well as a human HDAC8 model to compare inhibitor selectivity. The PfHDAC-1 model was validated and found to have good geometry. Docking results revealed that the YC-II-88 inhibitor was most selective for PfHDAC-1 and showed no activity for human HDAC8, suggesting it may be a promising antimalarial lead.
This study examined DNA methylation at gametic differentially methylated regions (gDMRs) in mouse embryonic stem cells (ESCs) lacking DNA methyltransferases. The study aimed to determine: 1) if loss of methylation at imprinted gDMRs is equivalent when deleting Dnmt1 or Dnmt3a/3b, 2) if imprints can be restored in Dnmt3a/3b double knockout ESCs unlike Dnmt1 knockout ESCs, and 3) if loss of methylation dysregulates imprinted gene expression. The results showed that loss of methylation was equivalent when deleting either Dnmt1 or Dnmt3a/3b. Imprint
High Replayability in Browser-Based Games: How to Generate it and What You Ga...Jessica Tams
Delivered at Casual Connect Tel Aviv 2016. In this talk, Bytro Labs Managing Director Felix Faber will give an overview on how Bytro creates free-to-play games with high replayability and what the pros and cons of those games are. Most of the free to play online games rely heavily on content and lack replayability. Having board games as a role model, Bytro knows not only the benefits, but also the pitfalls and limitations which occur when creating F2P games that can be replayed for years.
The document discusses the benefits of an independent and decentralized internet (IndieWeb) compared to centralized platforms controlled by a single entity. It notes key issues with centralization like single points of failure, loss of content if a site shuts down, and lack of network effects. The document promotes using open protocols and standards to publish content across the independent web through personal websites and rolling your own solutions instead of relying on centralized platforms.
Dokumen ini berisi tabel penghitung alkalinitas phenolphthalein yang digunakan sebagai referensi pribadi untuk mendukung kegiatan kerja di kantor. Tabel tersebut berisi petunjuk pengisian data hasil treatment penelitian di lapangan mengenai volume asam sulfat, normalitasnya, volume sampel, berat ekivalen kapur, dan alkalinitas phenolphthalein.
El documento presenta información sobre dos tipos de presupuestos: presupuesto de producción y presupuesto de ventas. El presupuesto de producción determina los costos de materiales, mano de obra y gastos de fabricación para controlar inventario y asegurar la producción. El presupuesto de ventas proyecta las ventas futuras trimestralmente y es importante para derivar pronósticos, analizar tendencias de ventas y evaluar el potencial del mercado.
Como rentabilizar Periscope en tu estrategia Social SellingAlex Lopez Lopez
Te presentamos los aspectos clave para poner en marcha tu cuenta de Periscope y conseguir que tus retransmisiones lleguen a tu público final. Sin duda una herramienta fundamental en tu estrategia de social selling
Homology modeling, docking and comparative study of the selectivity of some h...Alexander Decker
This document describes a study that used homology modeling to generate a 3D structural model of the Plasmodium falciparum histone deacetylase 1 (PfHDAC-1) enzyme, an important antimalarial drug target. Nine known HDAC inhibitors were docked into the PfHDAC-1 model as well as a human HDAC8 model to compare inhibitor selectivity. The PfHDAC-1 model was validated and found to have good geometry. Docking results revealed that the YC-II-88 inhibitor was most selective for PfHDAC-1 and showed no activity for human HDAC8, suggesting it may be a promising antimalarial lead.
This study examined DNA methylation at gametic differentially methylated regions (gDMRs) in mouse embryonic stem cells (ESCs) lacking DNA methyltransferases. The study aimed to determine: 1) if loss of methylation at imprinted gDMRs is equivalent when deleting Dnmt1 or Dnmt3a/3b, 2) if imprints can be restored in Dnmt3a/3b double knockout ESCs unlike Dnmt1 knockout ESCs, and 3) if loss of methylation dysregulates imprinted gene expression. The results showed that loss of methylation was equivalent when deleting either Dnmt1 or Dnmt3a/3b. Imprint
High Replayability in Browser-Based Games: How to Generate it and What You Ga...Jessica Tams
Delivered at Casual Connect Tel Aviv 2016. In this talk, Bytro Labs Managing Director Felix Faber will give an overview on how Bytro creates free-to-play games with high replayability and what the pros and cons of those games are. Most of the free to play online games rely heavily on content and lack replayability. Having board games as a role model, Bytro knows not only the benefits, but also the pitfalls and limitations which occur when creating F2P games that can be replayed for years.
The document discusses the benefits of an independent and decentralized internet (IndieWeb) compared to centralized platforms controlled by a single entity. It notes key issues with centralization like single points of failure, loss of content if a site shuts down, and lack of network effects. The document promotes using open protocols and standards to publish content across the independent web through personal websites and rolling your own solutions instead of relying on centralized platforms.
Dokumen ini berisi tabel penghitung alkalinitas phenolphthalein yang digunakan sebagai referensi pribadi untuk mendukung kegiatan kerja di kantor. Tabel tersebut berisi petunjuk pengisian data hasil treatment penelitian di lapangan mengenai volume asam sulfat, normalitasnya, volume sampel, berat ekivalen kapur, dan alkalinitas phenolphthalein.
El documento presenta información sobre dos tipos de presupuestos: presupuesto de producción y presupuesto de ventas. El presupuesto de producción determina los costos de materiales, mano de obra y gastos de fabricación para controlar inventario y asegurar la producción. El presupuesto de ventas proyecta las ventas futuras trimestralmente y es importante para derivar pronósticos, analizar tendencias de ventas y evaluar el potencial del mercado.
Como rentabilizar Periscope en tu estrategia Social SellingAlex Lopez Lopez
Te presentamos los aspectos clave para poner en marcha tu cuenta de Periscope y conseguir que tus retransmisiones lleguen a tu público final. Sin duda una herramienta fundamental en tu estrategia de social selling
This study used in silico and in vitro methods to characterize the unknown protein 4EZI from the Protein Data Bank. In silico screening with ProMOL, BLAST, and DALI suggested 4EZI was part of the esterase family. This was confirmed experimentally by expressing and purifying 4EZI, and using activity assays that showed 4EZI could hydrolyze short-chain but not long-chain p-nitrophenyl esters, characteristic of an esterase rather than a lipase. Therefore, through integrated computational and experimental analysis, 4EZI was determined to be an esterase.
20140613 Analysis of High Throughput DNA Methylation ProfilingYi-Feng Chang
This document provides an overview of analysis of high-throughput DNA methylation profiling using bisulfite sequencing (BS-Seq) technology. It discusses DNA methylation and the bisulfite conversion process. It also reviews current BS-Seq resources, information that can be presented in BS-Seq studies, and published tools for analyzing BS-Seq data, including alignment, calling methylation status, and identifying differential methylation regions. The document concludes by introducing MethPipe, a comprehensive tool for BS-Seq data analysis.
Integrative bioinformatics analysis of Parkinson's disease related omics dataEnrico Glaab
Presentation on statistical meta analysis of omics data from Parkinson's disease case-control studies. The results are used for a comparative analysis against aging-related omics alterations in the brain and a prioritization of new candidate disease genes using the phenologs approach.
This document provides an overview of DNA methylation analysis. It begins with background on DNA methylation functions and diseases. It then discusses methods for measuring DNA methylation status, including bisulfite sequencing. The document reviews steps for DNA methylation data analysis using tools like methylKit in R. It presents a case study example of analyzing DNA methylation data from human stem cells and fibroblasts. Alignment, quality control, differential methylation analysis and visualization are discussed.
The document discusses the integration of heterogeneous biological data and the development of computational tools and databases to analyze protein-protein interaction networks, phosphorylation signaling networks, and other molecular pathways. It describes several databases and web tools created by the author and other researchers, including NetworKIN, STRING, STITCH, NetPhorest, and Reflect, that combine data from diverse sources to build networks and gain new biological insights. It also addresses ongoing challenges in data integration like variable data quality, different data formats and identifiers, and the need for continued benchmarking and validation of computational predictions.
The document discusses the STRING database and related tools for exploring protein-protein association networks, gene neighborhoods, phylogenetic profiles, and other computational predictions and experimental data. It notes that individual databases cover different species and formats, and have variable quality. STRING aims to integrate these resources using common identifiers, quality scores, and text mining while calibrating scores against experimental data and curated knowledge. Resources discussed include STRING for protein networks, STITCH for chemical networks, and COMPARTMENTS and TISSUES for subcellular localization and tissue expression data.
'Stories that persuade with data' - talk at CENDI meeting January 9 2014Anita de Waard
Scientific articles tell stories with a structure similar to traditional stories. They introduce a topic, present a hypothesis and methods, and report results. As claims from papers are cited in subsequent work, they become established as facts. Networks of claims and data could improve knowledge by formally representing claims and connecting them to underlying data and other evidence. This would help address issues like reproducibility and data accessibility. Research data management is evolving to better preserve and share data through repositories, standardized workflows, and initiatives to develop sustainable data infrastructures.
Dissecting human brain development at high resolution using RNA-seq lcolladotor
L. Collado-Torres' ENAR 2015 presentation about using `derfinder` to identify differentially expressed regions in the DLPFC across the lifespan of normal individuals. Described in Jaffe et al, Nature Neuroscience, 2014
This document is a curriculum vitae for Hui Zhang, a professor at Johns Hopkins University School of Medicine. It lists his current appointments, education history, professional experience, and publications. Specifically, it details that he is currently a professor in the Department of Pathology at JHU and director of the Mass Spectrometry Core Facility. It provides information on his education from Beijing University and University of Pennsylvania. It also lists over 45 publications in peer-reviewed journals related to proteomics and mass spectrometry research.
dkNET Webinar: Illuminating The Druggable Genome With Pharos 10/23/2020dkNET
Abstract
Pharos (https://pharos.nih.gov/) is an integrated web-based informatics platform for the analysis of data aggregated by the Illuminating the Druggable Genome (IDG) Knowledge Management Center, an NIH Common Fund initiative. The current version of Pharos (as of October 2019) spans 20,244 proteins in the human proteome, 19,880 disease and phenotype associations, and 226,829 ChEMBL compounds. This resource not only collates and analyzes data from over 60 high-quality resources to generate these types, but also uses text indexing to find less apparent connections between targets, and has recently begun to collaborate with institutions that generate data and resources. Proteins are ranked according to a knowledge-based classification system, which can help researchers to identify less studied “dark” targets that could be potentially further illuminated. This is an important process for both drug discovery and target validation, as more knowledge can accelerate target identification, and previously understudied proteins can serve as novel targets in drug discovery. In this webinar, Dr. Tudor Oprea will introduce how to use Pharos to find targets of interest for drug discovery.
The top 3 key questions that Pharos can answer:
1. What are the novel drug targets that may play a role in a specific disease?
2. What are the diseases that are related directly or indirectly to a drug target?
3. Find researchers that are related directly or indirectly to a drug target.
Presenter: Tudor Oprea, MD, PhD, Professor of Medicine, Chief of Translational Informatics Division & Internal Medicine, University of New Mexico
dkNET Webinar Information: https://dknet.org/about/webinar
This document summarizes research on the structure-based design, synthesis, and evaluation of novel peptidic inhibitors of thrombin-induced platelet aggregation. Peptidic inhibitors were developed based on a known thrombin inhibitor scaffold. Analogs containing D-3,3-diphenylalanine in the P3 position showed improved binding to thrombin's active site and inhibition of platelet aggregation in the low nanomolar range. Surface plasmon resonance analysis revealed two lead compounds with dissociation constants in the 40-290 nanomolar range. The novel inhibitors show potential as scaffolds for developing anti-thrombotic drugs to treat acute coronary syndrome.
This document summarizes research on developing a human cancer coessentiality network using data from pooled shRNA screens across 107 cancer cell lines. Key points:
- A network of 866 genes and 1877 edges was constructed based on correlations in essentiality profiles across cell lines.
- Network clustering identified groups of genes essential for similar cell line subtypes (e.g. breast, ovarian, pancreatic cancers).
- One cluster involved in oxidative phosphorylation was particularly essential for luminal/HER2 breast cancers.
- The network provides a functional genomics resource, though opportunities exist to improve coverage and accuracy.
This document describes using computational methods to identify potential drug candidates that can inhibit breast cancer metastatic beta arrestin 2 (ARRB2). Ensemble-based virtual screening and pharmacophore modeling were used to screen drug molecules from the DrugBank database and identify top candidates. The 15 molecules with best binding were further analyzed with molecular dynamics simulations. The results suggest two molecules as the best ARRB2 inhibitor candidates based on their binding affinity and stability in simulations. The study provides a framework for discovering novel ARRB2 inhibitors using integrated computational approaches.
This document is a resume for Michael Berman summarizing his experience as a research scientist and lab manager with over 30 years of experience conducting molecular biology experiments. He has extensive experience in techniques such as PCR, cloning, purification, tissue/bacterial culture, and detection methods. Berman has worked at several institutions including Harvard Medical School and has authored several peer-reviewed publications.
Pharos: A Torch to Use in Your Journey in the Dark GenomeRajarshi Guha
This document summarizes Pharos, a knowledge management center and database for biomedical research targets developed by the National Center for Advancing Translational Sciences (NCATS). Key points:
- Pharos contains over 20,000 biomedical targets, drugs, diseases, and related publications to facilitate exploration and hypothesis generation.
- It provides search, visualization, and filtering capabilities to browse entity relationships and summaries. Target knowledge vectors also enable new methods like recommendations and clustering.
- The goals are to characterize understudied targets, identify small molecules/biologics, explore research landscapes, and support data mining. Pharos is intended for biologists, clinical researchers, and informatics scientists.
- Future work
This document discusses computational predictions of potential interactions and signaling crosstalk between CDK5 and other proteins involved in central nervous system pathways. Specifically, it predicts interactions between CDK5 and NogoR1 using online tools and molecular modeling software. Ten amino acid interactions between CDK5 and NogoR1 are identified. Pathway analysis with tools like STRING and Pathway Linker also predict potential crosstalk between CDK5, NogoR1, CDK5R1, GSK3β and RTN4. Molecular dynamics simulations of the CDK5-NogoR1 complex are performed and structural changes analyzed over 10,000 femtoseconds.
This study used in silico and in vitro methods to characterize the unknown protein 4EZI from the Protein Data Bank. In silico screening with ProMOL, BLAST, and DALI suggested 4EZI was part of the esterase family. This was confirmed experimentally by expressing and purifying 4EZI, and using activity assays that showed 4EZI could hydrolyze short-chain but not long-chain p-nitrophenyl esters, characteristic of an esterase rather than a lipase. Therefore, through integrated computational and experimental analysis, 4EZI was determined to be an esterase.
20140613 Analysis of High Throughput DNA Methylation ProfilingYi-Feng Chang
This document provides an overview of analysis of high-throughput DNA methylation profiling using bisulfite sequencing (BS-Seq) technology. It discusses DNA methylation and the bisulfite conversion process. It also reviews current BS-Seq resources, information that can be presented in BS-Seq studies, and published tools for analyzing BS-Seq data, including alignment, calling methylation status, and identifying differential methylation regions. The document concludes by introducing MethPipe, a comprehensive tool for BS-Seq data analysis.
Integrative bioinformatics analysis of Parkinson's disease related omics dataEnrico Glaab
Presentation on statistical meta analysis of omics data from Parkinson's disease case-control studies. The results are used for a comparative analysis against aging-related omics alterations in the brain and a prioritization of new candidate disease genes using the phenologs approach.
This document provides an overview of DNA methylation analysis. It begins with background on DNA methylation functions and diseases. It then discusses methods for measuring DNA methylation status, including bisulfite sequencing. The document reviews steps for DNA methylation data analysis using tools like methylKit in R. It presents a case study example of analyzing DNA methylation data from human stem cells and fibroblasts. Alignment, quality control, differential methylation analysis and visualization are discussed.
The document discusses the integration of heterogeneous biological data and the development of computational tools and databases to analyze protein-protein interaction networks, phosphorylation signaling networks, and other molecular pathways. It describes several databases and web tools created by the author and other researchers, including NetworKIN, STRING, STITCH, NetPhorest, and Reflect, that combine data from diverse sources to build networks and gain new biological insights. It also addresses ongoing challenges in data integration like variable data quality, different data formats and identifiers, and the need for continued benchmarking and validation of computational predictions.
The document discusses the STRING database and related tools for exploring protein-protein association networks, gene neighborhoods, phylogenetic profiles, and other computational predictions and experimental data. It notes that individual databases cover different species and formats, and have variable quality. STRING aims to integrate these resources using common identifiers, quality scores, and text mining while calibrating scores against experimental data and curated knowledge. Resources discussed include STRING for protein networks, STITCH for chemical networks, and COMPARTMENTS and TISSUES for subcellular localization and tissue expression data.
'Stories that persuade with data' - talk at CENDI meeting January 9 2014Anita de Waard
Scientific articles tell stories with a structure similar to traditional stories. They introduce a topic, present a hypothesis and methods, and report results. As claims from papers are cited in subsequent work, they become established as facts. Networks of claims and data could improve knowledge by formally representing claims and connecting them to underlying data and other evidence. This would help address issues like reproducibility and data accessibility. Research data management is evolving to better preserve and share data through repositories, standardized workflows, and initiatives to develop sustainable data infrastructures.
Dissecting human brain development at high resolution using RNA-seq lcolladotor
L. Collado-Torres' ENAR 2015 presentation about using `derfinder` to identify differentially expressed regions in the DLPFC across the lifespan of normal individuals. Described in Jaffe et al, Nature Neuroscience, 2014
This document is a curriculum vitae for Hui Zhang, a professor at Johns Hopkins University School of Medicine. It lists his current appointments, education history, professional experience, and publications. Specifically, it details that he is currently a professor in the Department of Pathology at JHU and director of the Mass Spectrometry Core Facility. It provides information on his education from Beijing University and University of Pennsylvania. It also lists over 45 publications in peer-reviewed journals related to proteomics and mass spectrometry research.
dkNET Webinar: Illuminating The Druggable Genome With Pharos 10/23/2020dkNET
Abstract
Pharos (https://pharos.nih.gov/) is an integrated web-based informatics platform for the analysis of data aggregated by the Illuminating the Druggable Genome (IDG) Knowledge Management Center, an NIH Common Fund initiative. The current version of Pharos (as of October 2019) spans 20,244 proteins in the human proteome, 19,880 disease and phenotype associations, and 226,829 ChEMBL compounds. This resource not only collates and analyzes data from over 60 high-quality resources to generate these types, but also uses text indexing to find less apparent connections between targets, and has recently begun to collaborate with institutions that generate data and resources. Proteins are ranked according to a knowledge-based classification system, which can help researchers to identify less studied “dark” targets that could be potentially further illuminated. This is an important process for both drug discovery and target validation, as more knowledge can accelerate target identification, and previously understudied proteins can serve as novel targets in drug discovery. In this webinar, Dr. Tudor Oprea will introduce how to use Pharos to find targets of interest for drug discovery.
The top 3 key questions that Pharos can answer:
1. What are the novel drug targets that may play a role in a specific disease?
2. What are the diseases that are related directly or indirectly to a drug target?
3. Find researchers that are related directly or indirectly to a drug target.
Presenter: Tudor Oprea, MD, PhD, Professor of Medicine, Chief of Translational Informatics Division & Internal Medicine, University of New Mexico
dkNET Webinar Information: https://dknet.org/about/webinar
This document summarizes research on the structure-based design, synthesis, and evaluation of novel peptidic inhibitors of thrombin-induced platelet aggregation. Peptidic inhibitors were developed based on a known thrombin inhibitor scaffold. Analogs containing D-3,3-diphenylalanine in the P3 position showed improved binding to thrombin's active site and inhibition of platelet aggregation in the low nanomolar range. Surface plasmon resonance analysis revealed two lead compounds with dissociation constants in the 40-290 nanomolar range. The novel inhibitors show potential as scaffolds for developing anti-thrombotic drugs to treat acute coronary syndrome.
This document summarizes research on developing a human cancer coessentiality network using data from pooled shRNA screens across 107 cancer cell lines. Key points:
- A network of 866 genes and 1877 edges was constructed based on correlations in essentiality profiles across cell lines.
- Network clustering identified groups of genes essential for similar cell line subtypes (e.g. breast, ovarian, pancreatic cancers).
- One cluster involved in oxidative phosphorylation was particularly essential for luminal/HER2 breast cancers.
- The network provides a functional genomics resource, though opportunities exist to improve coverage and accuracy.
This document describes using computational methods to identify potential drug candidates that can inhibit breast cancer metastatic beta arrestin 2 (ARRB2). Ensemble-based virtual screening and pharmacophore modeling were used to screen drug molecules from the DrugBank database and identify top candidates. The 15 molecules with best binding were further analyzed with molecular dynamics simulations. The results suggest two molecules as the best ARRB2 inhibitor candidates based on their binding affinity and stability in simulations. The study provides a framework for discovering novel ARRB2 inhibitors using integrated computational approaches.
This document is a resume for Michael Berman summarizing his experience as a research scientist and lab manager with over 30 years of experience conducting molecular biology experiments. He has extensive experience in techniques such as PCR, cloning, purification, tissue/bacterial culture, and detection methods. Berman has worked at several institutions including Harvard Medical School and has authored several peer-reviewed publications.
Pharos: A Torch to Use in Your Journey in the Dark GenomeRajarshi Guha
This document summarizes Pharos, a knowledge management center and database for biomedical research targets developed by the National Center for Advancing Translational Sciences (NCATS). Key points:
- Pharos contains over 20,000 biomedical targets, drugs, diseases, and related publications to facilitate exploration and hypothesis generation.
- It provides search, visualization, and filtering capabilities to browse entity relationships and summaries. Target knowledge vectors also enable new methods like recommendations and clustering.
- The goals are to characterize understudied targets, identify small molecules/biologics, explore research landscapes, and support data mining. Pharos is intended for biologists, clinical researchers, and informatics scientists.
- Future work
This document discusses computational predictions of potential interactions and signaling crosstalk between CDK5 and other proteins involved in central nervous system pathways. Specifically, it predicts interactions between CDK5 and NogoR1 using online tools and molecular modeling software. Ten amino acid interactions between CDK5 and NogoR1 are identified. Pathway analysis with tools like STRING and Pathway Linker also predict potential crosstalk between CDK5, NogoR1, CDK5R1, GSK3β and RTN4. Molecular dynamics simulations of the CDK5-NogoR1 complex are performed and structural changes analyzed over 10,000 femtoseconds.
1. Kaitlin Hart
Protein Function Prediction Using ProMol,
PyMol, and other Bioinformatics Tools
Rochester Institute of Technology
2. Introduction
● 3,454 (as of 7/25/2014) PDB entries with unknown function in
the Protein Data Bank
● The purpose of this project is to determine the hypothetical
function of proteins in a timely and cost effective manner
● In silico methods allow us to rapidly narrow candidate functions
● In vitro characterization alone is expensive and time consuming
9. AutoDock Vina
O. Trott, A. J. Olson, AutoDock Vina: improving the speed and accuracy of docking with a
new scoring function, efficient optimization and multithreading, Journal of
Computational Chemistry 31 (2010) 455-461.
11. Conclusion and Future Plans
● 4EZI has been hypothesized as possibly hydrolase, or
more specifically, an esterase
● 4EZI shows little activity with p-nitrophenyl acetate
● AutoDock suggests that inositol would be a good
substrate candidate
Conclusions:
Future Plans:
● Continue work with AutoDock to find other possible
substrates
● Test substates with kinetics assays
12. Acknowledgements
Thank you to all of the present and past members of the SBEVSL (Structural Biology Extensible
Visualization Scripting Language) project.
This project was funded by RIT, Dowling College, the National Institute of Health
(2R15GM078077 & 3R15GM078077-02S1), and the National Science Foundation (NSF ATE
040208).
Dr. Paul Craig
Dr. Lea
Michel
Greg Dodge
Dr. Herbert
Bernstein
Dr. Jeff Mills
13. References
Altschul, S. F., T. L. Madden, A. A. Schäffer, J Zhang, Z Zhang, W Miller, and D J Lipman. “Gapped BLAST and
PSI-BLAST: a New Generation of Protein Database Search Programs.” Nucleic Acids Research 25, no. 17
(September 1, 1997): 3389–3402.
Bernstein, F. C., T. F. Koetzle, G. J.Williams, E. F. Meyer Jr, M. D. Brice, J. R. Rodgers, O. Kennard, T. Shimanouchi,
and M. Tasumi. “The Protein Data Bank: a Computer-based Archival File for Macromolecular Structures.” Journal
of Molecular Biology 112, no. 3 (May 25, 1977): 535–542.
Furnham N., G.L Holliday, T.A. de Beer, J.O. Jacobsen, W.R. Pearson, J.M. Thornton. “The Catalytic Site Atlas 2.0:
cataloging catalytic sites and residues identified in enzymes.” Nucleic Acids Research 42. (January
2014):D485-D489. doi: 10.1093/nar/gkt1243.
Hanson, B., C. Westin, M. Rosa, A. Grier, M. Osipovitch, M. MacDonald, G. Dodge, P. Boli, C. Corwin, H. Kessler, T.
McKay, H. Bernstein, P. Craig. “Estimation of Protein Function Using Template-Based Alignment of Enzyme
Active Sites.” BMC Bioinformatics 15, no. 87. (March 27, 2014): doi:10.1186/1471-2105-15-87
Holm, L., and P. Rosenström. “Dali Server: Conservation Mapping in 3D.” Nucleic Acids Research 38, no. suppl 2
(July 1, 2010): W545–W549. doi:10.1093/nar/gkq366.
O. Trott, A. J. Olson, AutoDock Vina: improving the speed and accuracy of docking with a new scoring function,
efficient optimization and multithreading, Journal of Computational Chemistry 31 (2010) 455-461
Punta M., P.C. Coggill, R.Y. Eberhardt, J. Mistry, J. Tate, C. Boursnell, N. Pang, K. Forslund, G. Ceric, J. Clements, A.
Heger, L. Holm, E.L.L. Sonnhammer, S.R. Eddy, A. Bateman, R.D. Finn. “The Pfam Protein Families
Database.” Nucleic Acids Research 40, no. D1 (November 29, 2011): D290-D301. doi:10.1093/nar/gk
-Protein Data Bank is a repository of protein structures. All publicly relseased structures are published there
-There are almost 3,500 proteins in the PDB with no assigned function; basically, we know the structure, but we do not know what these proteins actually do
-purpose of our project was to analyze these proteins in an attempt to find their function
- we can do this via in silico (meaning using computers), which allows us to rapidly narrow candidate functions
-can do this via in vitro methods (testing in the wet lab), but this is costly and time consuming, but accurate
-we need to use both methods when characterizing proteins
-we analyzed 4EZI in silico using three additional bioinformatics tools (BLAST, Dali, and Pfam), which categorize proteins based on entire sequence and/or 3D structure
-after we compared the results of all for computer programs, we performed literature searches to determine the best methods for testing the function of the protein in the wet lab
-In vitro (meaning in the wet lab) characterization involved mixing the enzyme with different substrates. The rate at which the enzyme breaks down the different substrates was measured.
-ProMOL is a computer program used to visualize structural alignment among proteins
- developed by RIT students in 2008
-ProMol looks at specific parts of proteins that we know the function for, called the active site. This is where substrates bind to the protein to catalyze chemical reactions (hand demo)
-ran most of the 3,000+ proteins of unknown function through ProMOL
-found the promising hit, 4EZI, which we decided to pursue
-started with running 4ezi through promol
-best match was 1orv, a hydrolase. Hydrolases are enzymes which cut bonds by adding water molecules
-in this image, 4EZI is red and 1orv is blue. In ProMOL, this is considered an excellent match
-BLAST compares sequences of proteins. When we ran the sequence of 4EZI through BLAST, the best hit was 3H2H, which is a specific type of hydrolase, called an esterase. Esterases cut molecules with carbon chains with less than 10 carbons.
-Dali compares 3D structures of proteins. When we ran 4EZI through the Dali database, the best hit was again the esterase 3H2H
-query cover: percent of 3h2h that is aligned with 4ezi: 3h2h has an extra loop that might have a different function
-e value: expecation value: probability that this alignment will randomly occur. more negative, the better the value (it wouldn’t normally be expected to happen)
-identical: percent that they are identical
-the image on the left is from a previous slide; remember that 1orv is a hydrolase
-The image on the right was generated by running the esterase (a type of hydrolase) 3h2h (the best hit from BLAST and Dali) through ProMOL.
-as you can see, the two alignments are very similar
4EZI and 1ORV
RMSD: 0.36RMSD Alpha: 0.19RMSD:Alpha & Beta: 0.21
4EZI and 3H2H
RMSD All: 0.24RMSD Alpha: 0.17RMSD Alpha & Beta: 0.21
acetate
y = 0.0011ln(x) + 0.0089
R² = 0.924
PNP is a chromogenic substrate
slope: 0.02692
Dr. Oleg Trott
Figure 7. Binding of ITP in 4EZI active site. Serine-195 is highlighted in lime green within the active site of 4EZI. The Phosphate group of the ITP interacts with the hydroxy group of the serine with a distance of 2.9 Å. ITP bound to 4EZI with an affinity of -6.9 kcal/mol.