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Xu H.,State University of New York at Buffalo | Xu H.,Hauptman Woodward Medical Research Institute
Lecture Notes in Computer Science (including subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics) | Year: 2017

The direct-methods phasing program, Shake-and-Bake for single crystal structure determination, has been adapted and modified to solve microcrystal structures from X-ray powder diffraction data. © Springer International Publishing AG 2017.


Sutton K.A.,Hauptman Woodward Medical Research Institute | Black P.J.,University of Rochester | Black P.J.,Medical Center Boulevard | Mercer K.R.,University of Rochester | And 4 more authors.
Acta Crystallographica Section D: Biological Crystallography | Year: 2013

Electron paramagnetic resonance (EPR) and online UV-visible absorption microspectrophotometry with X-ray crystallography have been used in a complementary manner to follow X-ray-induced disulfide-bond cleavage. Online UV-visible spectroscopy showed that upon X-irradiation, disulfide radicalization appeared to saturate at an absorbed dose of approximately 0.5-0.8 MGy, in contrast to the saturating dose of ∼0.2 MGy observed using EPR at much lower dose rates. The observations suggest that a multi-track model involving product formation owing to the interaction of two separate tracks is a valid model for radiation damage in protein crystals. The saturation levels are remarkably consistent given the widely different experimental parameters and the range of total absorbed doses studied. The results indicate that even at the lowest doses used for structural investigations disulfide bonds are already radicalized. Multi-track considerations offer the first step in a comprehensive model of radiation damage that could potentially lead to a combined computational and experimental approach to identifying when damage is likely to be present, to quantitate it and to provide the ability to recover the native unperturbed structure. © 2013 International Union of Crystallography.


Thapar R.,Hauptman Woodward Medical Research Institute | Thapar R.,State University of New York at Buffalo | Thapar R.,Rice University | Denmon A.P.,Rice University
Cellular Signalling | Year: 2013

Cells regulate their genomes mainly at the level of transcription and at the level of mRNA decay. While regulation at the level of transcription is clearly important, the regulation of mRNA turnover by signaling networks is essential for a rapid response to external stimuli. Signaling pathways result in posttranslational modification of RNA binding proteins by phosphorylation, ubiquitination, methylation, acetylation etc. These modifications are important for rapid remodeling of dynamic ribonucleoprotein complexes and triggering mRNA decay. Understanding how these posttranslational modifications alter gene expression is therefore a fundamental question in biology. In this review we highlight recent findings on how signaling pathways and cell cycle checkpoints involving phosphorylation, ubiquitination, and arginine methylation affect mRNA turnover. © 2013 Elsevier Inc.


Gruner S.M.,Stanford University | Gruner S.M.,Cornell University | Lattman E.E.,Hauptman Woodward Medical Research Institute | Lattman E.E.,State University of New York at Buffalo | Lattman E.E.,Bio Center
Annual Review of Biophysics | Year: 2015

Next-generation synchrotron radiation sources, such as X-ray free-electron lasers, energy recovery linacs, and ultra-low-emittance storage rings, are catalyzing novel methods of biomolecular microcrystallography and solution scattering. These methods are described and future trends are predicted. Importantly, there is a growing realization that serial microcrystallography and certain cutting-edge solution scattering experiments can be performed at existing storage ring sources by utilizing new technology. In this sense, next-generation sources are serving two distinct functions, namely, provision of new capabilities that require the newer sources and inspiration of new methods that can be performed at existing sources. © Copyright ©2015 by Annual Reviews. All rights reserved.


Thapar R.,Hauptman Woodward Medical Research Institute | Thapar R.,State University of New York at Buffalo | Thapar R.,Rice University | Denmon A.P.,Rice University | Nikonowicz E.P.,Rice University
Wiley Interdisciplinary Reviews: RNA | Year: 2014

RNA hairpins are the most commonly occurring secondary structural elements in RNAs and serve as nucleation sites for RNA folding, RNA-RNA, and RNA-protein interactions. RNA hairpins are frequently capped by tetraloops, and based on sequence similarity, three broad classes of RNA tetraloops have been defined: GNRA, UNCG, and CUYG. Other classes such as the UYUN tetraloop in histone mRNAs, the UGAA in 16S rRNA, the AUUA tetraloop from the MS2 bacteriophage, and the AGNN tetraloop that binds RNase III have also been characterized. The tetraloop structure is compact and is usually characterized by a paired interaction between the first and fourth nucleotides. The two unpaired nucleotides in the loop are usually involved in base-stacking or base-phosphate hydrogen bonding interactions. Several structures of RNA tetraloops, free and complexed to other RNAs or proteins, are now available and these studies have increased our understanding of the diverse mechanisms by which this motif is recognized. RNA tetraloops can mediate RNA-RNA contacts via the tetraloop-receptor motif, kissing hairpin loops, A-minor interactions, and pseudoknots. While these RNA-RNA interactions are fairly well understood, how RNA-binding proteins recognize RNA tetraloops and tetraloop-like motifs remains unclear. In this review, we summarize the structures of RNA tetraloop-protein complexes and the general themes that have emerged on sequence- and structure-specific recognition of RNA tetraloops. We highlight how proteins achieve molecular recognition of this nucleic acid motif, the structural adaptations observed in the tetraloop to accommodate the protein-binding partner, and the role of dynamics in recognition. © 2013 John Wiley & Sons, Ltd.


Cody V.,Hauptman Woodward Medical Research Institute | Cody V.,State University of New York at Buffalo | Pace J.,Hauptman Woodward Medical Research Institute | Nowak J.,Hauptman Woodward Medical Research Institute
Acta Crystallographica Section D: Biological Crystallography | Year: 2011

In order to understand the structure-activity profile observed for a series of substituted dibenz[b,f]azepine antifolates, the crystal structure of the binary complex of human dihydro-folate reductase (hDHFR) with the potent and selective inhibitor 2,4-diamino-6 - {2′-O-(3-carboxypropyl)oxydibenz[b,f]- azepin-5-yl}methylpteridine (PT684) was determined to 1.8 Å resolution. These data revealed that the carboxylate side chain of PT684 occupies two alternate positions, neither of which interacts with the conserved Arg70 in the active-site pocket, which in turn hydrogen bonds to water. These observations are in contrast to those reported for the ternary complex of mouse DHFR (mDHFR) with NADPH [Cody et al. (2008), Acta Cryst. D64, 977-984], in which the 3-carboxypropyl side chain of PT684 was hydrolyzed to its hydroxyl derivative, PT684a. The crystallization conditions differed for the human and mouse DHFR crystals (100 mM K2HPO4 pH 6.9, 30% ammonium sulfate for hDHFR; 15 mM Tris pH 8.3, 75 mM sodium cacodylate, PEG 4K for mDHFR). Additionally, the side chains of Phe31 and Gln35 in the hDHFR complex have a single conformation, whereas in the mDHFR complex they occupied two alternative conformations. These data show that the hDHFR complex has a decreased active-site volume compared with the mDHFR complex, as reflected in a relative shift of helix C (residues 59-64) of 1.2 Å, and a shift of 1.5 Å compared with the ternary complex of Pneumocystis carinii DHFR (pcDHFR) with the parent dibenz[b,f]azepine PT653. These data suggest that the greater inhibitory potency of PT684 against pcDHFR is consistent with the larger active-site volume of pcDHFR and the predicted interactions of the carboxylate side chain with Arg75. © 2011 International Union of Crystallography. Printed in Singapore - All rights reserved.


Cody V.,Hauptman Woodward Medical Research Institute | Cody V.,State University of New York at Buffalo | Pace J.,Hauptman Woodward Medical Research Institute
Acta Crystallographica Section D: Biological Crystallography | Year: 2011

Structural data are reported for five antifolates, namely 2,4-diamino-6-[5′-(5-carboxypentyloxy)-2′-methoxybenzyl] -5-methylpyrido[2,3-d]pyrimidine, (1), and the 5′-[3-(ethoxycarbonyl) propoxy]-, (2), 5′-[3-(ethoxycarbonyl)butoxy]-, (3), 5′-[3- (ethoxycarbonyl)pentyloxy]-, (4), and 5′-benzyloxy-, (5), derivatives, which are potent and selective for Pneumocystis carinii dihydrofolate reductase (pcDHFR). Crystal structures are reported for their ternary complexes with NADPH and pcDHFR refined to between 1.4 and 2.0 Å resolution and for that of 3 with human DHFR (hDHFR) to 1.8 Å resolution. These data reveal that the carboxylate of the-carboxyalkoxy side chain of 1, the most potent inhibitor in this series, forms ionic interactions with the conserved Arg75 in the substrate-binding pocket of pcDHFR, whereas the less potent ethyl esters of 2-4 bind with variable side-chain conformations. The benzyloxy side chain of 5 makes no contact with Arg75 and is the least active inhibitor in this series. These structural results suggest that the weaker binding of this series compared with that of their pyrimidine homologs in part arises from the flexibility observed in their side-chain conformations, which do not optimize intermolecular contact to Arg75. Structural data for the binding of 3 to both hDHFR and pcDHFR reveals that the inhibitor binds in two different conformations, one similar to each of the two conformations observed for the parent pyrido[2,3-d]pyrimidine, piritrexim (PTX), bound to hDHFR. The structure of the pcDHFR complex of 4 reveals disorder in the side-chain orientation; one orientation has the-carboxy-alkoxy side chain positioned in the folate-binding pocket similar to the others in this series, while the second orientation occupies a new site near the nicotinamide ring of NADPH. This alternate binding site has not been observed in other DHFR structures. Structural data for the pcDHFR complex of 5 show that its benzyl side chain forms intermolecular van der Waals interactions with Phe69 in the binding pocket that could account for its enhanced binding selectivity compared with the other analogs in this series. © 2011 International Union of Crystallography Printed in Singapore - all rights reserved.


Gulick A.M.,Hauptman Woodward Medical Research Institute | Gulick A.M.,State University of New York at Buffalo
Current Opinion in Chemical Biology | Year: 2016

Nonribosomal peptide synthetases (NRPSs) catalyze the assembly line biosynthesis of peptide natural products that play important roles in microbial signaling and communication. These multidomain enzymes use an integrated carrier protein that delivers the growing peptide to the catalytic domains, requiring coordinated conformational changes that allow the proper sequence of synthetic steps. Recent structural studies of NRPSs have described important conformational states and illustrate the critical role of a small subdomain within the adenylation domains. This subdomain alternates between catalytic conformations and also serves as a linker domain, providing further conformational flexibility to enable the carrier to project from the core of NRPS. These studies are described along with remaining questions in the study of the structural dynamics of NRPSs. © 2016 Elsevier Ltd


Meilleur F.,North Carolina State University | Meilleur F.,Oak Ridge National Laboratory | Munshi P.,Oak Ridge National Laboratory | Robertson L.,Oak Ridge National Laboratory | And 7 more authors.
Acta Crystallographica Section D: Biological Crystallography | Year: 2013

The first high-resolution neutron protein structure of perdeuterated rubredoxin from Pyrococcus furiosus (PfRd) determined using the new IMAGINE macromolecular neutron crystallography instrument at the Oak Ridge National Laboratory is reported. Neutron diffraction data extending to 1.6514;Å resolution were collected from a relatively small 0.714;mm3 PfRd crystal using 2.514;d (6014;h) of beam time. The refined structure contains 371 out of 391, or 95%, of the D atoms of the protein and 58 solvent molecules. The IMAGINE instrument is designed to provide neutron data at or near atomic resolution (1.514;Å) from crystals with volume <1.014;mm3 and with unit-cell edges <10014;Å. Beamline features include novel elliptical focusing mirrors that deliver neutrons into a 2.0 × 3.214;mm focal spot at the sample position with full-width vertical and horizontal divergences of 0.5 and 0.6°, respectively. Variable short- and long-wavelength cutoff optics provide automated exchange between multiple-wavelength configurations (λmin = 2.0, 2.8, 3.314;Å to λmax = 3.0, 4.0, 4.5, ∼2014;Å). These optics produce a more than 20-fold increase in the flux density at the sample and should help to enable more routine collection of high-resolution data from submillimetre-cubed crystals. Notably, the crystal used to collect these PfRd data was 5-10 times smaller than those previously reported. © 2013 International Union of Crystallography Printed in Singapore - all rights reserved. © 2013.


Park K.-T.,University of Kansas Medical Center | Wu W.,University of Kansas Medical Center | Battaile K.P.,Hauptman Woodward Medical Research Institute | Lovell S.,University of Kansas | And 2 more authors.
Cell | Year: 2011

In E. coli, MinD recruits MinE to the membrane, leading to a coupled oscillation required for spatial regulation of the cytokinetic Z ring. How these proteins interact, however, is not clear because the MinD-binding regions of MinE are sequestered within a six-stranded β sheet and masked by N-terminal helices. minE mutations that restore interaction between some MinD and MinE mutants were isolated. These mutations alter the MinE structure leading to release of the MinD-binding regions and the N-terminal helices that bind the membrane. Crystallization of MinD-MinE complexes revealed a four-stranded β sheet MinE dimer with the released β strands (MinD-binding regions) converted to α helices bound to MinD dimers. These results identify the MinD-dependent conformational changes in MinE that convert it from a latent to an active form and lead to a model of how MinE persists at the MinD-membrane surface. PaperFlick: © 2011 Elsevier Inc.

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