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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. Source

Kuhn M.L.,Center for Structural Genomics of Infectious Diseases | Majorek K.A.,University of Virginia | Minor W.,University of Virginia | Anderson W.F.,Center for Structural Genomics of Infectious Diseases
Protein Science | Year: 2013

Due to a combination of efforts from individual laboratories and structural genomics centers, there has been a surge in the number of members of the Gcn5-related acetyltransferasesuperfamily that have been structurally determined within the past decade. Although the number of three-dimensional structures is increasing steadily, we know little about the individual functions of these enzymes. Part of the difficulty in assigning functions for members of this superfamily is the lack of information regarding how substrates bind to the active site of the protein. The majority of the structures do not show ligand bound in the active site, and since the substrate-binding domain is not strictly conserved, it is difficult to predict the function based on structure alone. Additionally, the enzymes are capable of acetylating a wide variety of metabolites and many may exhibit promiscuity regarding their ability to acetylate multiple classes of substrates, possibly having multiple functions for the same enzyme. Herein, we present an approach to identify potential substrates for previously uncharacterized members of the Gcn5-related acetyltransferase superfamily using a variety of metabolites including polyamines, amino acids, antibiotics, peptides, vitamins, catecholamines, and other metabolites. We have identified potential substrates for eight bacterial enzymes of this superfamily. This information will be used to further structurally and functionally characterize them. © 2012 The Protein Society. Source

News Article
Site: http://phys.org/chemistry-news/

Tuberculosis, caused by Mycobacterium tuberculosis bacteria, has proved incredibly stubborn even in the age of powerful antibiotics, infecting about one third of all people worldwide. Treatment can take up to nine months. It has stealth properties that protect it from antibiotics; it can hide inside human cells, avoiding the body's immune system while it waits for the opportune moment to multiply; and it's very resourceful at acquiring resistance. "What we have now may not work in a few years," said Andrzej Joachimiak, an Argonne Distinguished Fellow, head of the Structural Biology Center, co-principal investigator at the Center for Structural Genomics of Infectious Diseases and a corresponding author on the new study. In order to make new drugs, researchers need to search through the thousands of proteins in the bacterial world to find one that does something so important the bacterium can't live without it—and then make a little block to match. One such entry point might be IMPDH (inosine-5?-monophosphate dehydrogenase), which is part of a cellular process that controls the making of guanine nucleotides, one of the building blocks for DNA and RNA. It's so essential that virtually all living organisms, including human and bacterial pathogens, have versions of it. "What we discovered earlier this year is that the human and bacterial versions bind molecules differently," Joachimiak said. "This is very important for finding a molecule to build a drug around—you don't want to inhibit a human enzyme, just the pathogen one." Researchers have been interested in the mycobacterium IMPDH enzyme as a drug target for years, Joachimiak said, but haven't been able to produce it well enough to study it. The team observed that one part of the enzyme's structure was particularly wobbly, so they engineered a version without it using resources at the Advanced Protein Characterization Facility and then then determined the structure employing synchrotron protein crystallography at the Advanced Photon Source, a DOE Office of Science User Facility (both at Argonne). The modified version functions very similarly to the original, Joachimiak said, but is much easier to purify and crystallize for study. Brandeis University professor Lizbeth Hedstrom and University of Minnesota professor Courtney Aldrich, two of the study's other research collaborators, had identified several inhibitor molecules that bind to IMPDH, and thus might be a starting point for a drug—but they couldn't be imaged while interacting with the enzyme. The new engineered enzyme allowed them to capture the structures of Hedstrom's and Aldrich's inhibitors in action, locked with IMPDH. Helena Boshoff at the National Institute of Allergies and Infectious Diseases performed complementary studies showing that these inhibitors do in fact efficiently block mycobacterium growth. The new structures were deposited into the Protein Data Bank for continued study. Explore further: Cancer drug target is promising lead for new TB treatments More information: Magdalena Makowska-Grzyska et al. Mycobacterium tuberculosis IMPDH in Complexes with Substrates, Products and Antitubercular Compounds, PLOS ONE (2015). DOI: 10.1371/journal.pone.0138976

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. Source

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. Source

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