Midwest Center for Structural Genomics

New York City, NY, United States

Midwest Center for Structural Genomics

New York City, NY, United States
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Zheng H.,University of Virginia | Zheng H.,Center for Structural Genomics of Infectious Diseases | Zheng H.,New York Structural Genomics Research Consortium NYSGRC | Zheng H.,Midwest Center for Structural Genomics | And 20 more authors.
Expert Opinion on Drug Discovery | Year: 2015

Introduction: Macromolecular X-ray crystallography has been the primary methodology for determining the three-dimensional structures of proteins, nucleic acids and viruses. Structural information has paved the way for structure-guided drug discovery and laid the foundations for structural bioinformatics. However, X-ray crystallography still has a few fundamental limitations, some of which may be overcome and complemented using emerging methods and technologies in other areas of structural biology.Areas covered: This review describes how structural knowledge gained from X-ray crystallography has been used to advance other biophysical methods for structure determination (and vice versa). This article also covers current practices for integrating data generated by other biochemical and biophysical methods with those obtained from X-ray crystallography. Finally, the authors articulate their vision about how a combination of structural and biochemical/biophysical methods may improve our understanding of biological processes and interactions.Expert opinion: X-ray crystallography has been, and will continue to serve as, the central source of experimental structural biology data used in the discovery of new drugs. However, other structural biology techniques are useful not only to overcome the major limitation of X-ray crystallography, but also to provide complementary structural data that is useful in drug discovery. The use of recent advancements in biochemical, spectroscopy and bioinformatics methods may revolutionize drug discovery, albeit only when these data are combined and analyzed with effective data management systems. Accurate and complete data management is crucial for developing experimental procedures that are robust and reproducible. © 2015 © Informa UK, Ltd.


Zheng H.,University of Virginia | Zheng H.,Center for Structural Genomics of Infectious Diseases | Zheng H.,Midwest Center for Structural Genomics | Zheng H.,New York Structural Genomics Research Consortium NYSGRC | And 15 more authors.
Expert Opinion on Drug Discovery | Year: 2014

Introduction: X-ray crystallography plays an important role in structure-based drug design (SBDD), and accurate analysis of crystal structures of target macromolecules and macromolecule-ligand complexes is critical at all stages. However, whereas there has been significant progress in improving methods of structural biology, particularly in X-ray crystallography, corresponding progress in the development of computational methods (such as in silico high-throughput screening) is still on the horizon. Crystal structures can be overinterpreted and thus bias hypotheses and follow-up experiments. As in any experimental science, the models of macromolecular structures derived from X-ray diffraction data have their limitations, which need to be critically evaluated and well understood for structure-based drug discovery. Areas covered: This review describes how the validity, accuracy and precision of a protein or nucleic acid structure determined by X-ray crystallography can be evaluated from three different perspectives: i) the nature of the diffraction experiment; ii) the interpretation of an electron density map; and iii) the interpretation of the structural model in terms of function and mechanism. The strategies to optimally exploit a macromolecular structure are also discussed in the context of 'Big Data' analysis, biochemical experimental design and structure-based drug discovery. Expert opinion: Although X-ray crystallography is one of the most detailed 'microscopes' available today for examining macromolecular structures, the authors would like to re-emphasize that such structures are only simplified models of the target macromolecules. The authors also wish to reinforce the idea that a structure should not be thought of as a set of precise coordinates but rather as a framework for generating hypotheses to be explored. Numerous biochemical and biophysical experiments, including new diffraction experiments, can and should be performed to verify or falsify these hypotheses. X-ray crystallography will find its future application in drug discovery by the development of specific tools that would allow realistic interpretation of the outcome coordinates and/or support testing of these hypotheses. © 2014 Informa UK, Ltd.


In order to uncover the structure of these proteins, researchers used a technique called protein crystallography. Like a mosquito trapped in amber, compounds that are crystallized are placed in array in identical positions and ordered so that scientists can target them with X-ray beams and work backwards from the scattering patterns produced to recreate their three-dimensional structures atom by atom. In the first study, a group of researchers from the Structural Biology Center, which is funded by DOE's Office of Science, mapped out a protein responsible for breaking down organic compounds in soil bacteria, an important process for recycling carbon in the ecosystem. The bacteria used, called Acinetobacter, is located mostly in soil and water habitats, where it helps to change aromatic compounds (named for their ring shape) into forms that can be used as food. One of the sources of aromatic compounds found in soil is lignin, a tough polymer that is an essential part of all plants and that's hard for many organisms to digest. "But Acinetobacter can utilize these aromatic compounds as their sole source of carbon," said Andrzej Joachimiak, who co-authored both studies and is the director of the Structural Biology Center and the Midwest Center for Structural Genomics at Argonne. In order for Acinetobacter to break down the aromatic compounds, it needs to produce catabolic enzymes, molecular machines built from an organism's DNA that break down molecules into smaller parts that can be digested. Whether or not membrane transporters and catabolic enzymes are produced falls to the HcaR regulator, a sort of molecular policeman that controls when the genes that code for these enzymes can be activated. Joachimiak and his colleagues found that the regulator works in a cycle, activating genes when aromatic compounds are present and shutting genes down when the compounds are used up. "By nature it is very efficient," Joachimiak said. "If you don't have aromatic compounds inside a cell, the operon is shut down." The research team didn't stop at mapping out the regulator itself; to discover how the cycle worked, they crystalized the HcaR regulator during interactions with its two major inputs: the aromatic compounds and DNA. The group found that when aromatic compounds are not present in the cell, two wings found on either side of the HcaR regulator wrap around the DNA. This action is mirrored on both sides of the regulator, covering the DNA regulatory site and preventing genes from being activated. "This is something that has never been seen before," Joachimiak said. When the aromatic compounds are present, however, they attach themselves to the HcaR regulator, making it so stiff that it can no longer grapple with the DNA. Joachimiak said that this knowledge could help outside of the lab, with applications such as a sensor for harmful pesticides and as a template for converting more carbon in soil. "If we can train bacteria to better degrade lignin and other polymers produced by plants during photosynthesis, more natural carbon sources can be utilized for example for production of biofuels and bioproducts," Joachimiak said. The paper was published earlier this year by the Journal of Biological Chemistry under the title "How Aromatic Compounds Block DNA Binding of HcaR Catabolite Regulator." It was supported by the National Institutes of Health and the U.S. Department of Energy (Office of Biological and Environmental Research). A second paper focuses on a family of proteins identified as DUF89, which stands for "domain unknown function." This family is conserved across all three branches of the phylogenetic tree, which means that it is likely essential to many life forms. DUF89 has been identified as a type of enzyme called a phosphatase, which strips molecules of their phosphate groups. The paper's authors hypothesized that DUF89 proteins use this ability to save useful proteins in a cell from rogue molecules which could alter their structure, making them useless or destructive. The study found that DUF89 proteins use a metal ion, probably manganese, to lure in potentially harmful molecules and a water molecule to break off their phosphate group. DUF89 proteins could have an important role in breaking down a specific type of disruptive molecule: sugar. When the concentration of sugar in blood reaches high levels, simple sugars can have unwanted side reactions with proteins and DNA through a process called glycation. "We always have to deal with these side reactions that happen in our cells, and when we get older, we have an accumulation of these errors in our cells," Joachimiak said. Joachimiak said that this research could help scientists develop DUF89 treatments from non-human sources as a way to combat glycation in the bloodstream. The paper was published on the Nature Chemical Biology website on June 20 under the title "A family of metal-dependent phosphatases implicated in metabolite damage-control." Other authors on the paper were from the University of Florida, the University of Toronto, the University of California-Davis and Brookhaven National Laboratory. It was supported by the National Science Foundation, Genome Canada, the Ontario Genomics Institution, the Ontario Research Fund, the Natural Sciences and Engineering Research Council of Canada, the National Institutes of Health, the C.V. Griffin Sr. Foundation and the U.S. Department of Energy (Office of Basic Energy Sciences and Office of Biological and Environmental Research). Both studies used X-rays from the Advanced Photon Source, a DOE Office of Science User Facility, using beamlines 19-ID and 19-BM. Both also stem from the goal of the Midwest Center for Structural Genomics, which is to discover the structure and function of proteins potentially important to biomedicine. Joachimiak said that despite the new findings from these studies, when it comes to understanding what proteins do, we still have a long way to go. "When we sequence genomes, we can predict proteins, but when we predict those sequences we can only say something about function for about half of them," Joachimiak said. Explore further: New crystallization method to ease study of protein structures More information: Youngchang Kim et al. How Aromatic Compounds Block DNA Binding of HcaR Catabolite Regulator, Journal of Biological Chemistry (2016). DOI: 10.1074/jbc.M115.712067 Lili Huang et al. A family of metal-dependent phosphatases implicated in metabolite damage-control, Nature Chemical Biology (2016). DOI: 10.1038/nchembio.2108


Wlodawer A.,NCI Inc | Minor W.,University of Virginia | Minor W.,Midwest Center for Structural Genomics | Minor W.,Center for Structural Genomics of Infectious Diseases | And 2 more authors.
FEBS Journal | Year: 2013

The number of macromolecular structures deposited in the Protein Data Bank now approaches 100 000, with the vast majority of them determined by crystallographic methods. Thousands of papers describing such structures have been published in the scientific literature, and 20 Nobel Prizes in chemistry or medicine have been awarded for discoveries based on macromolecular crystallography. New hardware and software tools have made crystallography appear to be an almost routine (but still far from being analytical) technique and many structures are now being determined by scientists with very limited experience in the practical aspects of the field. However, this apparent ease is sometimes illusory and proper procedures need to be followed to maintain high standards of structure quality. In addition, many noncrystallographers may have problems with the critical evaluation and interpretation of structural results published in the scientific literature. The present review provides an outline of the technical aspects of crystallography for less experienced practitioners, as well as information that might be useful for users of macromolecular structures, aiming to show them how to interpret (but not overinterpret) the information present in the coordinate files and in their description. A discussion of the extent of information that can be gleaned from the atomic coordinates of structures solved at different resolution is provided, as well as problems and pitfalls encountered in structure determination and interpretation. This review, addressed to the less experienced macromolecular crystallographers and to users of biological structures, provides an outline of the technical aspects of crystallography, and discusses common problems and pitfalls encountered during structure determination and interpretation. It includes tips on how to interpret, but not overinterpret, the information present in the coordinate files and in their description. © 2013 FEBS.


Chruszcz M.,University of Virginia | Chruszcz M.,Center for Structural Genomics of Infectious Diseases | Chruszcz M.,Midwest Center for Structural Genomics | Domagalski M.,University of Virginia | And 9 more authors.
Current Opinion in Structural Biology | Year: 2010

Structural genomics (SG) programs have developed during the last decade many novel methodologies for faster and more accurate structure determination. These new tools and approaches led to the determination of thousands of protein structures. The generation of enormous amounts of experimental data resulted in significant improvements in the understanding of many biological processes at molecular levels. However, the amount of data collected so far is so large that traditional analysis methods are limiting the rate of extraction of biological and biochemical information from 3D models. This situation has prompted us to review the challenges that remain unmet by SG, as well as the areas in which the potential impact of SG could exceed what has been achieved so far. © 2010 Elsevier Ltd.


Gront D.,University of Virginia | Gront D.,University of Warsaw | Gront D.,Midwest Center for Structural Genomics | Grabowski M.,University of Virginia | And 9 more authors.
Journal of Structural and Functional Genomics | Year: 2012

The explosion of the size of the universe of known protein sequences has stimulated two complementary approaches to structural mapping of these sequences: theoretical structure prediction and experimental determination by structural genomics (SG). In this work, we assess the accuracy of structure prediction by two automated template-based structure prediction metaservers (genesilico. pl and bioinfo.pl) by measuring the structural similarity of the predicted models to corresponding experimental models determined a posteriori. Of 199 targets chosen from SG programs, the metaservers predicted the structures of about a fourth of them "correctly." (In this case, "correct" was defined as placing more than 70 % of the alpha carbon atoms in the model within 2 A ° of the experimentally determined positions.) Almost all of the targets that could be modeled to this accuracy were those with an available template in the Protein Data Bank (PDB) with more than 25 % sequence identity. The majority of those SG targets with lower sequence identity to structures in the PDB were not predicted by the metaservers with this accuracy. We also compared metaserver results to CASP8 results, finding that the models obtained by participants in the CASP competition were significantly better than those produced by the metaservers. © Springer Science+Business Media B.V. 2012.


Majorek K.A.,University of Virginia | Majorek K.A.,Adam Mickiewicz University | Majorek K.A.,Midwest Center for Structural Genomics | Majorek K.A.,Center for Structural Genomics of Infectious Diseases | And 12 more authors.
Protein Science | Year: 2014

The availability of purified and active protein is the starting point for the majority of in vitro biomedical, biochemical, and drug discovery experiments. The use of polyhistidine affinity tags has resulted in great increases of the efficiency of the protein purification process, but can negatively affect structure and/or activity measurements. Similarly, buffer molecules may perturb the conformational stability of a protein or its activity. During the determination of the structure of a Gcn5-related N-acetyltransferase (GNAT) from Pseudomonas aeruginosa (PA4794), we found that both HEPES and the polyhistidine affinity tag bind (separately) in the substrate-binding site. In the case of HEPES, the molecule induces conformational changes in the active site, but does not significantly affect enzyme activity. In contrast, the uncleaved His-tag does not induce major conformational changes but acts as a weak competitive inhibitor of peptide substrate. In two other GNAT enzymes, we observed that the presence of the His-tag had a strong influence on the activity of these proteins. The influence of protein preparation on functional studies may affect the reproducibility of experiments in other laboratories, even when changes between protocols seem at first glance to be insignificant. Moreover, the results presented here show how critical it is to adjust the experimental conditions for each protein or family of proteins, and investigate the influence of these factors on protein activity and structure, as they may significantly alter the effectiveness of functional characterization and screening methods. Thus, we show that a polyhistidine tag and the buffer molecule HEPES bind in the substrate-binding site and influence the conformation of the active site and the activity of GNAT acetyltransferases. We believe that such discrepancies can influence the reproducibility of some experiments and therefore could have a significant "ripple effect" on subsequent studies. Interactive Figure 3 | PDB Code(s): 3KKW; 4M3S © 2014 The Protein Society.


Eschenfeldt W.H.,Midwest Center for Structural Genomics | Maltseva N.,University of Chicago | Stols L.,Midwest Center for Structural Genomics | Donnelly M.I.,Midwest Center for Structural Genomics | And 8 more authors.
Journal of Structural and Functional Genomics | Year: 2010

High-throughput structural genomics projects seek to delineate protein structure space by determining the structure of representatives of all major protein families. Generally this is accomplished by processing numerous proteins through standardized protocols, for the most part involving purification of N-terminally His-tagged proteins. Often proteins that fail this approach are abandoned, but in many cases further effort is warranted because of a protein's intrinsic value. In addition, failure often occurs relatively far into the path to structure determination, and many failed proteins passed the first critical step, expression as a soluble protein. Salvage pathways seek to recoup the investment in this subset of failed proteins through alternative cloning, nested truncations, chemical modification, mutagenesis, screening buffers, ligands and modifying processing steps. To this end we have developed a series of ligation-independent cloning expression vectors that append various cleavable C-terminal tags instead of the conventional N-terminal tags. In an initial set of 16 proteins that failed with an N-terminal appendage, structures were obtained for C-terminally tagged derivatives of five proteins, including an example for which several alternative salvaging steps had failed. The new vectors allow appending C-terminal His6-tag and His6- and MBP-tags, and are cleavable with TEV or with both TEV and TVMV proteases. © 2010 US Government.


Goral A.M.,University of Virginia | Goral A.M.,Midwest Center for Structural Genomics | Goral A.M.,University of Warsaw | Tkaczuk K.L.,University of Virginia | And 9 more authors.
Journal of Structural and Functional Genomics | Year: 2012

Isochorismatase-like hydrolases (IHL) constitute a large family of enzymes divided into five structural families (by SCOP). IHLs are crucial for siderophore-mediated ferric iron acquisition by cells. Knowledge of the structural characteristics of these molecules will enhance the understanding of the molecular basis of iron transport, and perhaps resolve which of the mechanisms previously proposed in the literature is the correct one. We determined the crystal structure of the apo-form of a putative isochorismatase hydrolase OaIHL (PDB code: 3LQY) from the antarctic γ-proteobacterium Oleispira antarctica, and did comparative sequential and structural analysis of its closest homologs. The characteristic features of all analyzed structures were identified and discussed. We also docked isochorismate to the determined crystal structure by in silico methods, to highlight the interactions of the active center with the substrate. The putative isochorismate hydrolase OaIHL from O. antarctica possesses the typical catalytic triad for IHL proteins. Its active center resembles those IHLs with a D-K-C catalytic triad, rather than those variants with a D-K-X triad. OaIHL shares some structural and sequential features with other members of the IHL superfamily. In silico docking results showed that despite small differences in active site composition, isochorismate binds to in the structure of OaIHL in a similar mode to its binding in phenazine biosynthesis protein PhzD (PDB code 1NF8). © 2012 Springer Science+Business Media B.V.


Tkaczuk K.L.,University of Virginia | Tkaczuk K.L.,Midwest Center for Structural Genomics | Shumilin A.I.,University of Virginia | Shumilin A.I.,Midwest Center for Structural Genomics | And 9 more authors.
Evolutionary Applications | Year: 2013

We present the crystal structures of two universal stress proteins (USP) from Archaeoglobus fulgidus and Nitrosomonas europaea in both apo- and ligand-bound forms. This work is the first complete synthesis of the structural properties of 26 USP available in the Protein Data Bank, over 75% of which were determined by structure genomics centers with no additional information provided. The results of bioinformatic analyses of all available USP structures and their sequence homologs revealed that these two new USP structures share overall structural similarity with structures of USPs previously determined. Clustering and cladogram analyses, however, show how they diverge from other members of the USP superfamily and show greater similarity to USPs from organisms inhabiting extreme environments. We compared them with other archaeal and bacterial USPs and discuss their similarities and differences in context of structure, sequential motifs, and potential function. We also attempted to group all analyzed USPs into families, so that assignment of the potential function to those with no experimental data available would be possible by extrapolation. © 2013 The Authors. Published by Blackwell Publishing Ltd.

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