Poster Session #2

Conference: 2022: 72nd ACA Annual Meeting
07/31/2022: 5:30 PM - 7:30 PM
PS2 
Poster Session 
Portland Marriott Downtown Waterfront 
Room: Exhibit Hall 

Presentations

Characterization of a Trifunctional Sulfate-Activating Complex from Mycobacteria

The respiratory disease tuberculosis (TB), caused by the pathogen Mycobacterium tuberculosis (Mtb), is an ongoing worldwide epidemic which necessitates the identification of novel drug targets. Since sulfur is an essential element for the growth, virulence, and survival of Mtb, the disruption of sulfur metabolism may be a potential means of combating TB. To address this, we are studying CysDNC, a key sulfate-activating complex that lies at the beginning of the mycobacterial sulfur pathway. The first committed step of sulfur metabolism begins when intracellular sulfate is reacted with ATP to form the product adenosine-5'-phosphosulfate (APS) in a GTPase-coupled reaction. APS is then further phosphorylated into 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Both APS and PAPS serve as precursors for downstream sulfur-containing biomolecules. These three reactions--APS formation (CysD), GTP hydrolysis (CysN), and PAPS formation (CysC)--are catalyzed by the trifunctional complex CysDNC. This poster will feature ongoing research on this complex, including: in vitro biochemistry of CysDNC, characterization of a mutant strain of M. smegmatis which has been disrupted in the gene encoding CysD, as well as current attempts to solve the structure of CysDNC using cryogenic electron microscopy. 

View Abstract 1331

Poster Author

Samantha Hartanto, Department of Chemistry, University of California Davis Davis, CA 

Additional Author

Andrew Fisher, Depts of Chemistry & Mol Cell Bio, Univ of California Davis Davis, CA 

Cryo-EM SPA for Structural Understanding of A-to-I RNA Editing: Human Adenosine Deaminase Acting on RNA 2 (ADAR2) Complexed with dsRNA

Adenosine deaminases acting on RNA (ADARs) are enzymes which convert adenosine to inosine in the double-stranded RNA of humans and other animals. Double-stranded RNA-binding domains (dsRBDs) are present in all ADARs and are the main determinants of how many bases on a given RNA are edited. A healthy level of A-to-I RNA editing is necessary to produce mature RNA, but under- or over-editing leads to autoimmune and neurological diseases.

The goal of this work is to give insight into the structural basis of differential editing of RNAs by elucidating the binding sites of each dsRBD in a human ADAR:dsRNA complex. Despite the challenges inherent to small, asymmetric particles, 2D class averages with nominal resolutions as high as 5Å have been obtained. Work is ongoing to progress from 2D class averages into meaningful 3D reconstructions. 

View Abstract 1333

Poster Author

Melissa Matthews, Okinawa Institute of Science and Technology Yomitan, Japan 

Additional Author(s)

Alexander Thuy-Boun, University of California Davis, CA 
Peter Beal, University of California Davis, CA 
Andrew Fisher, Depts of Chemistry & Mol Cell Bio, Univ of California Davis Davis, CA 
Matthias Wolf, Okinawa Institute of Science and Technology Onna, Okinawa 

Crystal Structures of Polymerized LiCl and dimethyl sulfoxide in the form of {2LiCl•3DMSO}∞ and {LiCl•DMSO}∞

Two novel LiCl·DMSO polymer structures were created by combining dry LiCl salt with dimethyl sulfoxide (DMSO). The first phase that forms has very small block-shaped crystals (<0.08 mm) that are monoclinic with a 2 LiCl : 3 DMSO ratio. When the solution is placed on a laboratory glass slide, the DMSO evaporates and a second phase begins to form with a plate-shaped crystal morphology. After ~20 minutes, large (>0.20 mm) crystals form with an octahedron morphology. The plate crystals and the octahedron crystals are the same tetragonal structure with a 1 LiCl : 1 DMSO ratio. We report these structures and compare them to other known LiCl·solvent compounds.



Sandia National Laboratories is a multi-mission laboratory managed and operated by National Technology and Engineering Solutions of Sandia, LLC, a wholly owned subsidiary of Honeywell International, Inc., for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-NA0003525.

This paper describes objective technical results and analysis. Any subjective views or opinions that might be expressed in the paper do not necessarily represent the views of the U.S. Department of Energy or the United States Government. 

View Abstract 1321

Poster Author

Nichole Valdez, Sandia National Laboratories Albuquerque, NM 

Additional Author(s)

David Herman, Sandia National Laboratories
Martin Nemer, Sandia National Laboratories
Mark Rodriguez, Sandia National Laboratories
Eric Allcorn, Sandia National Laboratories

Detailed mechanism of catalysis by tetrameric L-glutaminase-asparaginase from Pseudomonas 7A (PGA)

L-asparaginases (EC 3.5.1.1) are widely distributed enzymes among both bacterial and eukaryotic organisms. For over 40 years, L-asparaginases have played a critical role in the treatment of juvenile leukemias and lymphomas. Their primary biochemical function is to catalyze the hydrolysis of L-Asn to L-Asp. Most L-asparaginases also catalyze the hydrolysis of L-Gln to L-Glu, and those that exhibit glutaminase activity that is comparable or higher than asparaginase activity are often referred to as glutaminases-asparaginases. Based on extensive structural and functional studies of L-glutaminase-asparaginase from Pseudomonas 7A (PGA), we were able to show unequivocally that the reaction catalyzed by this enzyme proceeds through formation of a covalent intermediate and utilizes a common ping-pong catalytic mechanism consisting of two subsequent nucleophilic substitutions, as previously observed for EcAII. Additionally, by confirming that the same mechanism applies to L-Asn and L-Gln, we postulate that it is common for all these structurally related enzymes. Detailed structural studies of PGA and its complexes with substrates should create a foundation for rational development of L-asparaginases with modulated relative activities vs. L-Asn or L-Gln, which may be beneficial toward the development of improved anti-leukemia therapeutics. 

View Abstract 1310

Poster Author

Pawel Strzelczyk, Center for Structural Biology, Center for Cancer Research, National Cancer Institute Frederick, MD 

Additional Author(s)

Di Zhang, Center for Structural Biology, Center for Cancer Research, National Cancer Institute MD 
Marzena Dyba, Center for Structural Biology, Center for Cancer Research, National Cancer Institute MD 
Alexander Wlodawer, Center for Structural Biology, Center for Cancer Research, National Cancer Institute
Jacek Lubkowski, Center for Structural Biology, Center for Cancer Research, National Cancer Institute MD 

Development of Novel Chemical Probes for the Treatment of Lyme Disease

Development of Novel Chemical Probes for the Treatment of Lyme Disease
Mark Kowalewski, David Carlson, Philip Hughes, Timothy Haystead, Matthew Redinbo

Lyme disease, caused by the microbial pathogen Borrelia burgdorferi, affects nearly 500,000 people every year in the US, making it the most common vector borne illness. Current treatments for Lyme disease are lacking and it is critical that new therapeutics are developed. Here we examine High temperature protein G (HtpG), a molecular chaperone present in high abundance in Borrelia burgdorferi (Bb). BbHtpG is an ortholog of the 90 kDa Heat shock protein (Hsp90) present in humans, which is a target for several cancers. These proteins utilize ATP hydrolysis to power conformational changes required for client protein folding. The nucleotide binding site located within the N-terminal domain can be targeted by a ligand covalently linked to a photoactivable toxin, allowing for localized targeting of Bb. In this work, known Hsp90 inhibitors were examined using isothermal titration calorimetry to determine binding affinities for BbHtpG to identify promising ligands with selectivity for BbHtpG. Additionally, the co-crystal structure for the N-terminal domain of BbHtpG bound by a high affinity inhibitor, HS-289, in the ATP binding site was determined to 2.3 Å resolution, enabling key structural differences between the ATP binding sites of BbHtpG and Hsp90 to be identified. Chemical fragments that target BbHtpG were discovered by screening fragment libraries with surface plasmon resonance and 19F-NMR, strengthening our understanding of the ATP binding site of BbHtpG. Through this work, novel chemical scaffolds have been identified using fragment-based drug discovery and key structural differences between BbHtpG and Hsp90 have been elucidated by solving crystal structures of BbHtpG. Together these data may facilitate the development of chemical probes that are capable of targeting Bb for the treatment of Lyme disease. 

View Abstract 1266

Poster Author

Mark Kowalewski, UNC Chapel Hill Chapel Hill, NC 

Additional Author(s)

Matthew Redinbo, UNC Chapel Hill
Timothy Haystead, Duke University Durham, NC 
Philip Hughes, Duke University Durham, NC 
David Carlson, Duke University Durham, NC 

Development update of PIONEER, a single-crystal neutron diffractometer at the Second Target Station, ORNL

PIONEER is a single-crystal neutron diffractometer in the initial instrument suite at the second target station (STS) at the Spallation Neutron Source, Oak Ridge National Laboratory. By utilizing the high cold-neutron flux provided by the STS moderator and advanced neutron optics, PIONEER will enable scientists to study tiny crystals (0.001 mm3) and ultra-thin films (10 nm), which are comparable to those typically used for x-ray studies but not feasible at existing neutron diffractometers. PIONEER will have a high resolution to study crystals with a unit-cell size up to 200 angstroms, and has a polarized incident beam option for measuring weak magnetic signals. PIONEER will provide a variety of sample environments, including high/low temperatures, high magnetic fields, and high pressures, which aims at accelerating materials discovery for a wide range of research fields to address societal energy and environmental challenges.

Here we will report the instrument development progress, focusing on the beam transportation and collimation system to deliver a high-flux and highly uniform beam to study small volume samples. To achieve this, PIONEER will take a kinked beamline geometry with two sets of Montel mirrors (also known as nested KB mirrors) and a virtual source in between. This design helps reduce the background by moving the sample out of the direct line of sight to the moderator and provides a method to tune the beam size at the sample position. We will present Monte Carlo ray-tracing simulation results to show the performance of the beam transport system and use some virtual experiments to show PIONEER's capabilities for measuring small-volume samples.

This research used resources of the Spallation Neutron Source Second Target Station Project at Oak RidgeNational Laboratory (ORNL). ORNL is managed by UT-Battelle LLC for DOE's Office of Science, the single largest supporter of basic research in the physical sciences in the United States. 

View Abstract 1322

Poster Author

Yaohua Liu, Oak Ridge National Laboratory Oak Ridge, TN 

Additional Author

Peter Torres, Second Target Station Project

Glu445Ala in DesD Crystallization and ATP/AMP binding with Kinetics

Iron is vital for living organism since it plays a role to allow protein's biological activity in metabolic processes such as aerobic and anaerobic ATP biosynthesis. One of the many organisms that requires iron to keep themselves alive are bacteria specifically bacteria that cause diseases also known as pathogenic bacteria. The way pathogenic bacteria acquire iron is by a family of protein called NIS synthetases, the specific protein in the family being look at currently is DesD. The mechanism used by Pathogenic bacteria to acquire Iron and transport it back to the microorganism are call siderophores. NIS synthetases is plays a key role because one of its members of the family is present in many of the pathogenic bacteria so one model can be use for all. Current Pathogenic bacteria has become very resistance to antibiotics. This is concerning since eventually antibiotics will become useless leaving individuals vulnerable to common pathogenic bacteria. We predict that ATP binding association is diminished and will see that in the thermodynamics assay. As well it is predicted that this will have an impact on the kinetic rate, but mostly due to an increased KM (dissociation constant) when ATP is limiting while also finding new crystal structure subtracts binded to Glu445. It is expect significantly diminished kinetics and binding thermodynamics due to an increase in dissociation constants (KD or KM). Also testing the role of Glu445 in specifically coordinating ATP by comparing our ATP binding curves (both the wild type and the Glu445Ala) to AMP or adenine as well. Since the structure indicates contacts to the ribose ring and alpha phosphate, which AMP has but adenine does not, we expect these tests to bind poorly and well, respectively, compared to the same thermodynamics in the wild-type protein 

View Abstract 1311

Poster Author

Pablo Paredes Oxnard, CA 

Investigation of a Putative Polysaccharide Deacetylase from Gut Microbe Bacteroides ovatus

Bacteroides are often the most abundant species in the gut microbiome of industrialized human populations. Many have demonstrated beneficial properties to their host, for example gut microbe Bacteroides ovatus has been linked to the suppression of intestinal inflammation. A distinctive characteristic of gut-dwelling Bacteroides that may help confer these immunomodulatory effects is the production of cell surface components called capsular polysaccharides (CPS). Recent studies have begun to explore the diversity of CPS loci in Bacteroides, however there is still much unknown. Here we present characterization of a putative polysaccharide deacetylase (BACOVA_03992) encoded in a likely CPS loci from Bacteroides ovatus. Sequence analysis indicates the enzyme is a member of the carbohydrate esterase 4 (CE4) superfamily. However it is missing a key histidine present in other CE4 members that is known to coordinate a metal required for catalysis, and deacetylase activity against common carbohydrate substrates was not detected. Despite the absence of this conserved histidine, we obtained two high resolution crystal structures of the enzyme bound to either nickel or cobalt at 1.5 Å and 1.45 Å respectively. Our findings confirm that this putative polysaccharide deacetylase can bind metals and should be functional with the appropriate substrate. 

View Abstract 1324

Poster Author

Krystle McLaughlin, Vassar College Poughkeepsie, NY 

Additional Author(s)

Lilith Schwartz, Vassar College Poughkeepsie, NY 
Sharika Hasan, Vassar College Poughkeepsie, NY 
Banumathi Sankaran, Lawrence Berkeley National Lab Berkeley, CA 

New and Updated Phenix features

Phenix is a comprehensive software package for macromolecular structure determination that handles data from diffraction experiments and electron cryo-microscopy. Tasks performed with Phenix include data-quality assessment, map improvement, model building, the validation/rebuilding/refinement cycle and deposition. Each tool caters to the type of experimental data. The design of Phenix emphasizes the automation of procedures, where possible, to minimize repetitive and time-consuming manual tasks, while default parameters are chosen to encourage best practice. A graphical user interface provides access to many command-line features of Phenix and streamlines the transition between programs, project tracking and re-running of previous tasks. Some of the new features in Phenix, like utilizing predicted models from AlphaFold and a viewer for reciprocal space data, are highlighted in this poster. 

View Abstract 1317

Poster Author

Billy Poon, Lawrence Berkeley National Laboratory Berkeley, CA 

Additional Author(s)

Paul Adams, Lawrence Berkeley Laboratory
Pavel Afonine
Dorothee Liebschner, Lawrence Berkeley National Laboratory Berkeley, CA 
Nigel Moriarty, Lawrence Berkeley National Laboratory Berkeley, CA 
Oleg Sobolev, Lawrence Berkeley National Laboratory Berkeley, CA 
Christopher Schlicksup, Lawrence Berkeley National Laboratory Berkeley, CA 

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Poster Author

Kristin Stevens, here North Tonanwada, NY 

Predicting Target Binding Sites in Plant Immune Receptors

Plants rely on repertoires of hundreds of NLR immune receptors to defend against diverse and rapidly evolving pathogens. Based on Arabidopsis and Brachypodium data, we have observed that a subset of highly variable NLRs (hvNLRs) show a high degree of intra-species diversity making them the likely source of novel specificities and, by extension, a species-wide pool of potentially adaptive variants. Recent expansion of available crop pan-genomes allowed extending this observation to rice, soy, and maize. Coupled with computational protein structure prediction, the observed sequence diversity is sufficient to predict target-binding sites in the hvNLRs of model species and crops. The resulting understanding will guide immune receptor engineering efforts aimed at improving plant health. It also provides clues to how our own innate immune receptors arose prior to the evolution of the adaptive immunity. 

View Abstract 1177

Poster Author

Daniil Prigozhin, Lawrence Berkeley National Laboratory Berkeley, CA 

Psilocybin: Crystal Structure Solutions Enable Phase Analysis of Prior Art and Recently Patented Examples

Psilocybin (systematic name: 3-[2-(dimethylamino)ethyl]-1H-indol-4-yl dihydrogen phosphate) is a zwitterionic tryptamine natural product found in numerous species of fungi known for their psychoactive properties. Following its structural elucidation and chemical synthesis in 1959, purified synthetic psilocybin has been evaluated in clinical trials, and has shown promise in its utility for the alleviation of suffering associated with various mental health disorders. In a recent process-scale crystallization investigation, three crystalline forms of psilocybin were repeatedly observed: Hydrate A, Polymorph A, and Polymorph B. The crystal structure for Hydrate A was previously solved by single crystal X-ray diffraction. This report presents new crystal structure solutions for the two anhydrates, Polymorph A and Polymorph B, based on Rietveld refinement using laboratory and synchrotron X-ray diffraction data and density functional theory (DFT) optimizations. Utilizing the three solved structures, investigation was conducted via Rietveld method (RM) based quantitative phase analysis (QPA) to estimate the contribution of the three different forms in powder X-ray diffraction (PXRD) patterns of different sources of bulk psilocybin produced between 1963 and 2021. Over the last 57 years, each of these samples quantitatively reflect one or more of the hydrate and anhydrate polymorphs. In addition to quantitatively evaluating the composition of each sample, this paper evaluates correlations between the crystal forms present, corresponding process methods, sample age, and storage conditions. Further, revision is recommended on characterizations in recently granted patents that include descriptions of crystalline psilocybin inappropriately reported as a single phase "isostructural variant". Rietveld refinement demonstrated that the claimed material was composed of approximately 81% Polymorph A and 19% Polymorph B, both of which have been identified in historical samples. We show conclusively that all published data can be explained in terms of three well defined forms of psilocybin and that no additional forms are needed to explain the diffraction patterns. 

View Abstract 1287

Poster Author

James Kaduk, Chemistry, Illinois Inst of Technology Naperville, IL 

Additional Author(s)

Alexander Sherwood, Usona Institute Madison, WI 
Robert Kargbo, Usona Institute Madison, WI 
Kristi Kaylo, Usona Institute Madison, WI 
Poncho Meisenheimer, Usona Institute Madison, WI 
Nicholas Cozzi, University of Wisconsin School of Medicine and Public Health Madison, WI 

Reading the message: A structural and functional comparison of the ATAD2/B bromodomain binding activity

Bromodomains are conserved epigenetic reader domains known to recognize acetylated lysine residues on the N-terminal histone tails protruding from the nucleosome. The 61 human bromodomains are found in 41 different proteins and have been classified into eight different subfamilies based on sequence conservation. These subfamilies often share unique sequence variations that drive differences in histone ligand preference for specific patterns of post-translational modifications on core and variant histone proteins. The BET bromodomains, which are an attractive drug target in multiple disease states, have been the focus of many studies to elucidate the molecular mechanism of histone recognition. However, much less is known about non-BET bromodomain-containing proteins. In this study, we examined the ligand specificity of the ATAD2 bromodomain and compared it to its closely related paralog in ATAD2B. We show that the ATAD2/B bromodomains recognize mono- and di-acetyllysine modifications on histones H4 and H2A. A structure-function approach using X-ray crystallography, isothermal titration calorimetry experiments, and site-directed mutagenesis coupled to ligand binding assays identified key residues in the acetyllysine binding pocket that dictate the molecular recognition process. Furthermore, our results demonstrate how cross-talk between multiple modifications alters the binding activity of the ATAD2/B bromodomains. The structure of the ATAD2B bromodomain in complex with a small molecule inhibitor of the ATAD2 bromodomain revealed that critical contacts required for coordination are conserved between the ATAD2/B bromodomains, and many of these residues play a dual role in acetyllysine recognition. We further characterized an alternative splice variant of ATAD2B that results in a loss of bromodomain function. Our comparative analysis of the structural and functional features of the ATAD2 and ATAD2B bromodomains highlights features that contribute to their unique binding specificities driving histone recognition in the epigenetic landscape. 

View Abstract 1046

Poster Author

Karen Glass, University of Vermont, Pharmacology Department Burlington, VT 

Additional Author(s)

Kiera Malone, University of Vermont Burlington, VT 
Margaret Phillips, Univeristy of Vermont Burlington, VT 
Jonathan Lloyd, Albany College of Pharmacy and Health Sciences Colchester, VT 
Kyle McLaughlin, University of Vermont Burlington, VT 
Seth Frietze, University of Vermont Burlington, VT 

Structural insight on ubiquitination of NEMO for canonical activation of NF-кB

Activation of canonical NF-кB signaling is mediated through the ubiquitination of the NF-кB essential modulator (NEMO). LUBAC is a E3 ubiquitin ligase that catalyzes the linear ubiquitination of NEMO. LUBAC is a multisubunit enzyme which consists of HOIP, HOIL-1L and SHARPIN. HOIP is the catalytic subunit of LUBAC which consists of a catalytic RING-in between-RING domain followed by the linear chain determining domain (RBR-LDD). RBR-LDD is known to synthesize linear ubiquitin chains. HOIP also consists of a NZF1 domain which has been identified to recognize NEMO as a substrate for ubiquitination. To get an insight on the LUBAC-mediated ubiquitination of NEMO, we crystallized NEMO in complex with linear diubiquitin and HOIP-NZF1. To obtain the crystal structure, the proteins were individually expressed as GST-fusion proteins in E.coli and purified using chromatography techniques. The three proteins were mixed in equimolar ratio for crystallization. The crystals were obtained after six weeks in sitting drops containing 0.1 M Tris-HCl (pH 8.5), and 22 % v/v PEG 11 Smear Broad. In this heterotrimeric structure, HOIP NZF1 binds to NEMO and ubiquitin, simultaneously. The C-terminal tail of the ubiquitin is oriented towards the ubiquitination site on NEMO. This suggests that the LUBAC recognizes the monoubiquitinated NEMO and recruits the catalytic domain, thereby facilitating linear ubiquitin chain elongation. We also determined the binding affinities of HOIP NZF1 with NEMO and ubiquitin using Surface Plasmon Resonance. HOIP-NZF1 binds to NEMO and ubiquitin with a low binding affinity KD of 86.7 μM and 135.2 μM respectively, suggesting that the binding is preferential to the ubiquitinated NEMO. HOIP overall does not initiate the ubiquitination process, but catalyzes the chain elongation on the mono ubiquitinated substrate. HOIL-1L could be responsible for priming of the first ubiquitin on the substrate, but the exact mechanism is yet to be explored 

View Abstract 1289

Poster Author

Mamta Iyer, Chapman University Irvine, CA 

Additional Author

Simin Rahighi, Chapman University School of Pharmacy Irvine, CA 

Structural insights into combinations of histone H4 modifications recognized by the ATAD2B bromodomain

The ATPase family, AAA domain-containing protein 2 (ATAD2, or ANCCA) is an AAA nuclear co-regulator protein containing two ATPase domains and a bromodomain. It's closely related paralogue, ATAD2B (KIAA1240), also contains two ATPase domains and a bromodomain. The AAA ATPase domains are broadly associated with ATP driven molecular remodeling reactions, while bromodomains are evolutionarily conserved chromatin 'reader' domains, known to regulate gene expression through recognition of histone post-translational modifications (PTMs). Extensive studies on ATAD2 discuss its upregulation and correlation with poor prognosis in various cancers, particularly breast cancer, and have successfully mapped the different histone interactions needed for targeting ATAD2 to the chromatin. Despite sharing high structural and sequence similarity with ATAD2, little is known about the unique role of ATAD2B. ATAD2B is expressed during neuronal differentiation and in tumor progression. However, limited information is available on how ATAD2B is involved, or upregulated in various tumors, especially in breast cancer. Our group has recently published a broad range of histone modifications recognized by the ATAD2B bromodomain. These modifications mostly include acetyllysine moieties on histone H4.
Here, we present the structural and functional insights into the factors driving the recognition of various histone post-translational modifications and how their cross-talk can modulate the chromatin 'reader' activity of the ATAD2B bromodomain. Our ITC data confirms that the ATAD2B bromodomain can interact with multiply modified histone H4 ligands. We observe that some combinations of histone PTMs are permissible, while others diminish the binding activity. We also present novel crystal structures of the ATAD2B bromodomain in complex with two acetylated histone ligands. These high resolution structures highlight the polar contacts and hydrophobic interactions that form the molecular basis for the histone H4 acetyllysine recognition. Furthermore, immunoblot experiments confirm that the ATAD2B bromodomain can bind modified H4 histones extracted from breast cancer model-MCF7 cells.
Taken together, our data provide crucial information on how cross-talk between multiple histone modifications modulate the bromodomain activity of ATAD2B and how the histone code dynamics may alter its role as a regulator of gene expression.

Funding Sources:
NIGMS R01GM129338, NCI P01CA240685 

View Abstract 921

Poster Author

Margaret Phillips, Univeristy of Vermont Burlington, VT 

Structural Studies of TDE0362: A Virulence Factor in Treponema denticola

Structural Studies of TDE0362: A Virulence Factor in Treponema denticola.

Clark, N.D.1, Kurniyati, K.2, Li, C.2, and Malkowski, M.G1.

1Department of Structural Biology, Jacob's School of Medicine & Biomedical Science, State University of New York: University at Buffalo, Buffalo, NY, USA; 2Department of Oral and Craniofacial Molecular Biology, Philips Institute of Oral Health Research, Virginia Commonwealth University, Richmond, VA, USA.

Treponema denticola is a key pathogen of chronic periodontitis. During infection, Treponema denticola thrives within subgingival plaques formed in the periodontal pocket, despite direct interactions with the host immune system. To facilitate host immune evasion, Treponema denticola produces a number of virulence factors, including TDE0362, which cleave host immune proteins. TDE0362 encodes a two-domain protein with two conserved bacterial Ig-like domains at the N-terminus and a C-terminal cysteine protease domain (C362). C362 shows sequence and structural homology to the papain superfamily of cysteine proteases, with highest homology to IdeS, an IgG-specific protease from Streptococcus pyogenes. The survival of Streptococcus pyogenes is dependent on the ability to avoid the innate and adaptive immune responses. IdeS specifically cleaves the hinge region of IgG, dissecting the Fab and Fc domains, providing a defense against Fc-mediated phagocytic killing. Treponema denticola is also resistant to phagocytosis. However, despite the similarity to IdeS, C362 does not cleave IgG, suggesting a different mechanism for this resistance. We utilized Se-methionine incorporated protein in conjunction with SAD phasing methods and synchrotron radiation to elucidate the crystal structure of C362 to 2.19 Å. The structure contains 4 monomers in the asymmetric unit in space group P21. The observed structural architecture is conserved in each monomer and is similar to that seen in the IdeS crystal structure. Each monomer consists of 10 β-strands and 10 α-helices, which are organized into two distinct lobes. In its unliganded state, the putative active site residues are highly mobile, suggesting that substrate binding may be required for stabilization.

This research was supported by NIH NIGMS 5R01DE023080 and NIH R25GM095459. 

View Abstract 1250

Poster Author

Nicholas Clark, Department of Structural Biology, State University of New York: University at Buffalo BUFFALO, NY 

Additional Author(s)

Kurni Kurniyati, Department of Oral and Craniofacial Molecular Biology, Phillips Institute for Oral Health Research Richmond, VA 
Chunhao Li, Department of Oral and Craniofacial Molecular Biology, Phillips Institute for Oral Health Research Richmond, VA 
Michael Malkowski, University at Buffalo Buffalo, NY 

Structures of MfnG, an O-methyltranseferase involved in the biosynthese of marformycins, from multiple crystal forms

Marformycins are anti-infective natural products isolated from a deep sea sediment-derived Streptomyces drozdowiczii strain. These cyclodepsipetides contain O-methyl-D-Tyr. Liu et al., (Org. Lett. 2015, 17, 1509–1512) identified a SAM-dependent O-methyltransferase, MfnG, in the marformycins biosynthetic gene cluster and found it capable of methylating the phenoic oxygen of both D-Tyr and L-Tyr in vitro.

To better understand this enzyme's structural recognition and function, we have determined the MfnG structure using X-ray crystallography. Despite adding S-adenosyl-L-methionine (SAM/AdoMet) to the protein during crystallization, we found the spent product, S-Adenosyl-L-homocysteine (SAH/AdoHcy), bound. Since the SAH is unreactive, we were able to soak in L-Tyrosine to obtain a structure with the methyl doner product (SAH) and a methyl acceptor substrate (L-Tyr).

We found MfnG could crystalize from a number of different screening conditions and that these crystals had different unit cell parameters. To date, we have phased 5 forms (2 forms in P212121, 2 forms in P21 and a P1 form), which contain one to four dimers (2-8 protomers) per asymmetric unit. Here we compare the packing arrangement in these different crystal packing forms.


This work was supported in part by the NSF (STC 1231306), NIH/NIGMS (R01-GM115261, R35-GM133706), NIH/NIAID (R01-AI165079), NIH/NCI (R01-CA217255, R21-CA255894), US DOD (W81XWH-21-1-0789), Texas CPRIT (RR170014) and the Robert A. Welch Foundation (C-1970). The content is solely the responsibility of the authors and does not necessarily represent the official views of the Funders. 

View Abstract 1323

Poster Author

Mitchell D. Miller, Rice University Houston, TX 

Additional Author(s)

Kuan-Lin Wu, Rice University Houston, TX 
Weijun Xu, Rice University Houston, TX 
Han Xiao, Rice University Houston, TX 
George N. Phillips, Jr., Rice University Houston, TX 

Study of L-captopril Binding to VIM-20 by X-ray Crystallography Method

Verona Integron-encoded Metallo--lactamase (VIM) is one of the families of B1 subclass of beta-lactamases (BLs) and VIM-20 is one those 74 variants of VIM. We have crystallized the VIM-20 in different crystallization conditions and succeeded in growing crystals large enough for diffraction experiments in MCSG-1 screen. The crystals were soaked into L-captopril and obtained diffraction data at 1.37 Å. The structure of VIM-20/L-captopril complex was solved by using the PHENIX and Coot software packages. The final structure featuring Rwork and Rfree values of 0.185 and 0.218 with 100% ligand occupancy has confirmed that the thiol group of the L-captopril knocks off the catalytic hydroxyl group present in the active site of VIM-20. This finding concluded that the L-captopril can inhibit VIM-20, further supported by the IC50 data.

Key words: MBLs, SBLs, Enzymes, Inhibitors, Activity Assay 

View Abstract 970

Poster Author

Surendra Silwal, Miami University Oxford, OH 

The Role of Glu467 in NIS Synthetase DesD: Structure, Thermodynamics, and Kinetics.

Pathogenetic bacteria are becoming more resistant to current antibiotics with increased exposure. A simple infection that could be easily eliminated previously becomes more difficult to get rid of with the high resistance of MRSA, anthrax, and others. Creating a new class of antibiotics will be critical for our future ability to survive infections. The long term goal of the Hoffmann lab is to utilize structure-based drug design to design a new class of antibiotics. Our target is a unique family of proteins in bacteria called NIS Synthetases, involved in siderophore biosythesis; our model enzyme is Desferrioxamine D (DesD) from Streptomyces coelicolor.

Siderophores are small molecular iron chelators that are produced by microbes and whose most notable function is to sequester iron from the host and provide this essential metal nutrient to microbes. They are synthesized from multiple different pathways, however, the NIS pathways are relatively understudied, and their siderophore products are increasingly associated with bacterial virulence. NIS pathways always have one member of a unique family of enzymes called NIS Synthetases-excellent drug targets because of their associating with virulence, unique structure, and unique chemistry. A model enzyme from this family is DesD from Streptomyces coelicolor bacterium, which catalyzes the last three bonds made in the siderophore dfoE.

The literature on DesD has indicated a qualitative broad substrate specificity due to its iterative, successive bonds to the same substrate. We will test this hypothesis with quantitative binding data using isothermal titration calorimetry, and predict a significant preference for HSC (small substrate) and dfoG (large substrate) over analogs (dfoB or cadaverine) or products (dfoE). These studies will additionally allow us to describe the differences in binding to small vs. increasingly larger substrates in a quantitative exploration of iterative activity. To ensure that binding dynamics are not upstaged by catalytic turnover, we will use catalytically inactive variant Arg306Q DesD in assay using both cofactor and substrates.

Our previously published structures of DesD agree with the literature in a very important feature: the novel ATP binding site, notable for highly strained phosphate bonds enabling hydrolysis at the beta phosphate rather than the gamma (producing pyrophosphate rather than phosphate.) To explore the critical role for magnesium coordination and phosphate structure, we will present the structure, kinetics, and binding studies of variant Glu467Ala, which has a dual catalytic and magnesium ion coordination role. The combination of data will test the hypothesis that the absence of phosphate strain permits unproductive ATP hydrolysis at high [ATP] concentrations.

This project is key to future studies mapping the binding site and testing the binding of designed inhibitors to siderophore synthesis. The successful creation of an antibiotic targeted against NIS Synthetases would represent a new drug class against some of the most pathological and virulent bacteria posing threats to the human immune system. 

View Abstract 1272

Poster Author

Ashley Dumas, California Lutheran University Ridgecrest, CA 

Additional Author

Katherine Hoffmann, California Lutheran University Thousand Oaks, CA 

Towards understanding the conformational changes behind electron bifurcation in thermophilic metalloenzymes using small-angle X-ray scattering

Electron bifurcation (EB) is a recently discovered avenue of biological energy conservation that enables an unfavorable oxidation-reduction (redox) reaction by coupling it with a favorable one, so that the net reaction is favorable (Peters, et al, Curr Opin Chem Biol, 2016). The multi-subunit electron-transferring flavoprotein-menaquinone oxidoreductase ABCX (EtfABCX) is one enzyme that catalyzes EB through the exergonic reduction of menaquinone (high potential/favorable) coupled to the endergonic reduction of ferredoxin (low potential/unfavorable). Reduced ferredoxin drives many global microbial processes, including production of hydrogen gas (H2), methane (CH4), and nitrogen (N2) fixation. EtfABCX is a membrane-bound dimer (EtfABCX2) found in the thermophilic bacterium, Thermotoga maritima (Adams, FEMS Microbiol Rev, 1994). For EB to occur, the two-electron donor, enzyme cofactors, and electron acceptors must be orchestrated for efficient conformational gating so that the created higher potential electron does not lose its energy to the lower potential electron. The static cryo-EM structure of EtfABCX (Feng, et al, PNAS, 2021) greatly enhances our knowledge, but further investigation is needed to fully understand the EB mechanism as an uncharacterized conformational change is expected to decouple the electrons in the bifurcation from one another, the existence of which will likely occur in solution as observed with small-angle X-ray scattering (SAXS).

A truncation of EtfABCX, EtfAB, has been studied by both High-Throughput-SAXS (HT-SAXS) and Size-Exclusion Chromatography and Multi-Angle Light Scattering-coupled SAXS (SEC-MALS-SAXS) on the SIBYLS beamline at the Advanced Light Source. Data showed domain disassociation of the EtfAB subcomplex upon reduction by NADH, supporting the hypothesis that conformational changes are involved in EB. Results from EM and SEC-MALS-SAXS of EtfABCX indicate the enzyme forms a transient mixture of dimers and tetramers (EtfABCX2-EtfABCX2). The tetramer exists as an in vitro artifact resulting from hydrophobic patches integral to membrane insertion of the dimer (Feng, et al, PNAS, 2021). The data associated with the tetrameric fraction required software-assisted deconvolution of the SEC-MALS-SAXS intensity peaks (Meisburger, et al, JACS, 2016), but the resulting analysis proved inconclusive. To obtain the dimer's SAXS curve, SEC-MALS-SAXS was measured of EtfABCX in the presence of nonionic detergents to eliminate the tetramer population. Now, EtfABCX's conformational changes through varying redox states are being characterized using anaerobic SAXS. The resulting data from these experiments will be analyzed to elucidate allosteric interactions of EtfABCX that enable EB and the subsequent generation of H2, CH4, and N2 fixation. 

View Abstract 1328

Poster Author

Dan Murray, Lawrence Berkeley National Laboratory Berkeley, CA 

Additional Author(s)

Gerrit Schut, Department of Biochemistry and Molecular Biology, University of Georgia Athens, GA 
Xiaoxuan Ge, Department of Biochemistry and Molecular Biology, University of Georgia Athens, GA 
Michal Hammel, LBNL Berkeley, CA 
Michael Adams, Department of Biochemistry and Molecular Biology, University of Georgia Athens, GA 
Greg Hura, Molecular Biophysics and Integrated Bioimaging Division (MBIB), Lawrence Berkeley National Laborator Berkeley, CA 

Understanding the role of pyridoxal reductase (PDXI) in pyridoxal 5’-phosphate (PLP) homeostasis.

Purpose: Pyridoxal 5'-phosphate (PLP), the biologically active form of vitamin B6 is a cofactor for over 180 B6 enzymes (PLP-dependent enzymes) that are involved in critical biochemical reactions, e.g. amino acid, heme, and neurotransmitter biosynthesis. In prokaryotes, yeasts and plants, PLP is obtained from de novo and salvage pathways. The salvage pathway involves the enzymes pyridoxal kinase (PL kinase) and pyridoxine 5'-phosphate oxidase (PNPO) that use the B6 vitamers, pyridoxine (PN), pyridoxal (PL) and pyridoxamine (PM) to produce PLP. Humans can only synthesize PLP through the salvage pathway. Deficiency of PLP in the cell leads to several diseases. PLP is a highly reactive molecule and toxic, therefore its concentration in the cell is regulated by phosphatases that dephosphorylate it to PL and/or feedback inhibition of PL kinase and PNPO.
A third salvage enzyme, PDXI, with a reductase activity was recently discovered in bacteria and plants, catalyzing PL in the presence of NADPH to form PN and NADP+. Despite, its importance as a PLP homeostasis protein for efficient salvage of PL, only limited information on its structure and function is known. This study is aimed at characterizing E. coli PDXI with respect to its catalytic conversion of PL to PN, substrate binding specificity, regulation, and atomic structure.
Method: E. coli cells containing the cloned pdxI were grown in 6 liters of LB media and after reaching an O.D.600nm of greater than 0.6, were induced with 0.5mM IPTG. Cells were grown for an additional 4 hours at 37 C and harvested by centrifugation. The expressed PDXI was purified using a nickel column via fast protein liquid chromatography (FPLC). Protein fractions with single band at 33 kDa as checked by SDS-PAGE were combined and dialyzed overnight in a buffer containing 50mM NaH2PO4, 150mM NaCl, pH 7.5. The pure apo-protein (>90%) as judged by SDS-PAGE was used for crystallization with the Crystal Gryphon robot using a wide range of crystallization conditions. The condition 0.1 M MgCl, 0.1 M MES:NaOH, pH 6.5, and 30% PEG 400 gave the best crystal, which was used to collect X-ray diffraction data. The crystal structure of PDXI has been solved by a molecular replacement with the Phenix program, and the model subsequently refined using the Phenix and COOT programs. Further crystallographic studies of PDXI in complex with its substrates PL and/or NADPH or its products PN and/or NADP+ are ongoing. Kinetic studies are also ongoing using UV/vis spectroscopic method by measuring a decrease of NADPH at 340 nm. The mechanism and kinetic constants (km and Kcat) for PDXI will be determined. ITC and/or MST measurements will also be used to analyze the binding (Kd) of B6 vitamers to apo-PDXI to study substrate specificity.
Results: The crystal structure of apo-PDXI has been determined to 2.2 Å. This is the first such structure of a PDXI protein. PDXI folds as a TIM barrel and consists of 11 α-helices and 8 β-strands. Specific interactions of PL or NADPH with the protein await further crystallographic study. The kinetic and binding studies are also ongoing.
Conclusion: It is expected that the study will provide detailed molecular level information that will be useful in understanding PDXI cellular function and provide insight into PLP homeostasis. 

View Abstract 1051

Poster Author

Akua Donkor, Virginia Commonwealth University Richmond, VA 

Additional Author(s)

Mohini Ghatge, Virginia Commonwealth University Richmond, VA 
Faik Musayev, Virginia Commonwealth University Richmond, VA 
Roberto Contestabile, Sapienza University of Rome
Martino Di Salvo, Sapienza University of Rome
Martin Safo, Virginia commonwealth university RICHMOND, VA 

Viral RNA Dependent RNA Polymerase forms Amyloids Like Fibrils via Liquid-Liquid Phase Separation

Noroviruses (NoV), responsible for severe gastroenteritis, are members of the Caliciviridae family of positive-sense RNA viruses. Currently, human norovirus infection is responsible for ~200,000 deaths annually worldwide, yet there are no effective vaccines/antivirals currently available. RNA-dependent RNA polymerase (RdRP) is considered a promising drug target because of its critical role in genome replication and the synthesis/amplification of sub-genomic RNA. Using light scattering measurements and confocal microscopy, we show that RdRP of human GII.4 NoV forms liquid-liquid phase droplets. With time, these liquid-liquid phase condensates undergo liquid to solid transition resulting in the formation of higher-order oligomers/fibrils. The formation of the higher-order oligomers with increasing temperatures was also confirmed using size exclusion chromatography and analytical ultra-centrifugation. Furthermore, using amyloid-specific dye-based assays such as fluorescence-based Thioflavin-T and Congo red binding in addition to transmission electron microscopy (TEM) analysis, we discovered that RdRP forms amyloid-like fibrils at physiological conditions in vitro. Circular Dichroism (CD) spectroscopy of RdRP with increasing temperatures revealed an increase in the β-sheet content and loss of α-helical content as typically observed in amyloid-forming proteins. Bioinformatics analysis of the RdRP sequence using three independent web-based servers suggests that RdRP has multiple hot spots spread across the sequence that may help in the formation of amyloid-like fibrils corroborating our experimental data. Overexpression of RdRP in Escherichia coli and HEK293T cells also showed the formation of distinct puncta indicating amyloid inclusions. These results set the stage for further investigations to assess the functional role of amyloid-like fibrils in the viral life cycle, and test the hypothesis that liquid-liquid phase condensates formed by RdRP provide a platform for sequestering other non-structural proteins and viral RNA to form replication factories during norovirus infection. 

View Abstract 1299

Poster Author

Soni Kaundal, Baylor College of Medicine, Houston Houston, TX 

Additional Author(s)

Anish Thachangattuthodi, Baylor College of Medicine HOUSTON, TX 
Liya Hu, Baylor College of Medicine Houston, TX 
Sue Crawford, Baylor College of Medicine Houston, TX 
Jeroen Pollet, Baylor College of Medicine Houston, TX 
Mary Estes, Baylor College of Medicine Houston, TX 
BVV Prasad, Baylor College of Medicine Houston, TX 

X-ray Crystallographic Studies of pri-miR-21

Micro RNA (miRNA) are noncoding RNAs that are involved in gene silencing and transcriptional regulation. microRNAs are transcribed as long stem-loop structures (pri-miRNA). These molecules undergo double-stranded cleavage by the enzyme drosha in the nucleus to form a shorter stem-loop called pre-miRNA. After export from the nucleus to the cytoplasm, pre-miRNA undergoes another double-stranded cleavage by the enzyme dicer to eventually form mature miRNA of 20-22 nucleotide residues in length. miR-21 is considered an "onco-miR" because it is one of a family of micro RNAs that are upregulated in some cancers. To understand the structure of miR-21, we performed X-ray crystallography utilizing a general toolkit of RNA binding proteins. Here we describe the structure of nearly full-length pri-miR-21 in complex with an RNA-binding Fab solved at 1.8 Å resolution. All 55 nucleotides of the miR-21 double helical region are visible in the structure including the drosha and dicer cleavage sites. A ligand-bound ternary complex was also captured at 2.0 Å resolution using the tool compound neomycin. Neomycin binds near the dicer cleavage site and distorts the conformation of the RNA backbone near the A29 bulge region. 

View Abstract 1276

Poster Author

Doug Davies, UCB Bainbridge Island, WA 

Additional Author(s)

Thomas Edwards, UCB Boston Bedford, MA 
Stephen Mayclin, UCB Seattle Bainbridge Island, WA 
Ellen Wallace, UCB Seattle Bainbridge Island, WA 
Jessica Williamson, UCB Boston Boston, MA 
Madison Weiss, UCB Seattle Bainbridge Island, WA 

Exploring ATAD2 bromodomain structure and function differences in the dynamic epigenetic landscape

The ATPase family, AAA-domain containing protein 2 (ATAD2) is highly overexpressed in unrelated cancers and associated with poor patient outcomes. ATAD2 contains a C-terminal bromodomain that "reads" post-translational modifications (PTM) that occur on histone proteins present in the nucleosome core particle. The bromodomain is a structurally conserved motif consisting of a left-handed alpha helical bundle with a deep binding pocket that is known to recognize acetylated lysine. The epigenetic landscape forms a combinatorial code on histone proteins, which contain multiple modifications at any given time. Yet, it is unknown how the presence of other modifications adjacent to the acetylated lysine residues impact bromodomain protein recognition and function. We hypothesized that the presence of nearby post-translational modifications including methylation and phosphorylation would modulate the ability of the ATAD2 bromodomain to recognize its acetylated lysine binding partners. Previously, we systematically screened for multiple PTM combinations recognized by the ATAD2 bromodomain. In our current study, we characterized the interactions with these histone ligands using isothermal titration calorimetry (ITC), and solved structures of the bromodomain-ligand complexes using X-ray crystallography. Our results indicate that the histone binding activity of the ATAD2 bromodomain is impacted by the presence of nearby PTMs, which is likely due to changes observed in the binding pocket interactions. Our results provide new insights on how the bromodomain functions to target the ATAD2 protein to chromatin, and how this interaction may be fine-tuned via dynamic changes in the epigenetic landscape. 

View Abstract 943

Poster Author

Kiera Malone, University of Vermont Burlington, VT 

Additional Author(s)

Karen Glass, University of Vermont, Pharmacology Department Burlington, VT 
Jay Nix, Molecular Biology Consortium Berkeley, CA 

Features in selected area continuous rotation electron diffraction measurements that may be sensitive to molecular handedness of 3D crystals

Considering that biological and synthetic reactivity of small molecule compounds differs between enantiomers, the confident determination of a chiral molecule's absolute configuration is of much importance to pharmaceutical and synthetic chemistry. By virtue of resonant scattering phenomena, which break Friedel symmetry in a diffraction pattern, X-ray crystallography remains the most well-established method for determining the absolute structure of a chiral, enantiopure molecular crystal. As 3D electron diffraction (3D ED) now enables atomic structure determination from crystals on the order of nanometers in size, circumventing the traditional X-ray crystallography bottleneck of growing macroscopic crystals, it is pertinent to study the extent to which information about molecular handedness might be extracted from electron diffraction. In this work, we quantify features in X-ray and electron diffraction intensities that might predict the confident resolution of a chiral crystal's absolute structure. Considering X-ray diffraction as a well-understood model to identify such qualities, we focus both on the magnitudes of intensity differences between Bijvoet pairs for diffraction data collected on a single crystal, and how these measured differences for commonly observed pairs of reflections correlate between diffraction collected on different crystals of either like or opposite hand. Our substrate scope includes crystals with a range of expected anomalous scattering. We assess the degree to which dynamical scattering might impact intensity differences between Bijvoet pairs in measured data at 200 and 300 keV. We also investigate the influence of parameters that may influence the degree of multiple elastic scattering, such as the thickness of the crystal, the scattering mean free path, and the incident wavelength of irradiation, on measured Bijvoet differences. To do so, we collect 3D electron diffraction data on large populations of nanocrystals of chiral, enantiopure substrates and separate data by enantiomer. For each crystal sampled, following diffraction data collection, we also obtain tilt series images to yield intermediate resolution tomograms from which crystal thickness can be estimated, in terms of the number of its elastic mean free path. We are sampling crystalline molecular substrates that vary in atomic composition and scattering cross-section, and collect data at a series of accelerating voltages across different TEMs. Ultimately, we aim to understand the relationship between experimental conditions or substrates and diffraction metrics that might predict accurate absolute structure prediction. If successful, we might understand when scattering differences observed for chiral crystals are meaningful and predictive of crystal chirality. 

View Abstract 1298

Poster Author

Niko Vlahakis, University of California, Los Angeles Los Angeles

Additional Author

Jose Rodriguez, UCLA Los angeles, CA 

Atomic Structure Transformations of C-Doped Ge2Sb2Te5 Using In-Situ X-Ray Scattering

Phase change materials (PCMs) are characterized by their fast transformation speeds from their amorphous to crystalline states, with each state having distinct properties. These unique and distinct properties can be used for data storage and even computation, with their proposed application to enable neuromorphic computing. Ge2Sb2Te5 (GST) is a widely used PCM due to its fast-switching speeds and good data storage capabilities. However, it also has a low crystallization temperature, which makes long-term data storage (>10 years) difficult. One way to improve this is to alloy the material with carbon, which has been found to increase both the transformation temperature and the stability of the cubic phase.
To understand the effects of carbon, we are investigating how atomic structure transformations are modified by carbon doping (from 0% to 12%, molar) via in situ X-ray scattering, including X-ray diffraction (XRD) and pair distribution function (PDF) analysis. These methods provide access across local, mid-range, and average structure scales, and variations thereof through their amorphous-to-crystalline transformation. By identifying this atomic structure over the process, we can establish structure-property relationships and how these vary with composition. This new understanding is necessary to tailor GST, or other PCMs, for current and future applications, such as data storage and computing.

This work was supported by the Laboratory Directed Research and Development program at Sandia National Laboratories, a multimission laboratory managed and operated by National Technology & Engineering Solutions of Sandia, LLC, a wholly owned subsidiary of Honeywell International Inc., for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-NA0003525. This work was performed, in part, at the Center for Integrated Nanotechnologies, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science. The views expressed in the article do not necessarily represent the views of the U.S. DOE or the United States Government. 

View Abstract 1146

Poster Author

John Langhout, University of Florida Gainesville, FL 

Additional Author(s)

Danielle Alverson, University of Florida
Megan Butala, University of Florida Gainesville, FL 

Effect of TELSAM-Target Protein Linkers on Crystal Formation and Quality

Understanding the limitations and characteristics of crystal formation from TELSAM polymers is important in using TELSAM to crystallize proteins that cannot feasibly be crystallized on their own. One variable in TELSAM polymerization that needs to be understood is the effect of the linker between TELSAM and the target protein. To better understand this factor, we attempted to crystallize TELSAM and CMG2 connected with 4 different linkers. We observed the effect of the linker on the conditions under which the proteins crystallized as well as the resolution of the diffraction data from each crystal. Threonine-threonine linkers did not form any crystals. Of the alanine-alanine linkers, only 1 crystal diffracted; it had decent resolution (2.6Å). Threonine-valine linkers produced crystals with the best resolution upon diffraction (as low as 2.3Å typically around 3.5Å) followed closely by alanine-valine(as low as 2.6Å, but typically around 3.5Å). This is consistent with the theory that a threonine-threonine linker would be most rigid and an alanine-alanine linker would be most flexible with the alanine-valine and threonine-valine linkers having intermediate flexibility; and that the best linkers for TELSAM have some flexibility to shift as the polymer forms, but are stiff enough to hold their position and stay in a crystal formation. The diffraction data is currently being refined, and in the future, we intend to test different lengths of linkers as well as different target proteins. 

View Abstract 1278

Poster Author

Celeste Litchfield, Brigham Young University Salt Lake City, UT 

Additional Author(s)

Parag Gajjar, Brigham Young University Provo, UT 
Miles Callahan, Brigham Young University Provo, UT 
Nathan Redd, Brigham Young University Provo, UT 
James Moody

Synthesis and Characterization of Sr12Al14O33

Ca12Al14O33 has many interesting and potentially exploitable properties that arise from its crystal structure. The compound is known for being a room-temperature inorganic electride, and depending on the processing conditions, it can have near-metallic electrical conductivity. The unit cell is cubic, space group I4 ̅3d (no. 220), with a lattice parameter of ∼12 Å and contains two formula units. The Ca, Al, and majority of the O create a framework of twelve cages per unit cell, within which the remaining two O are occluded. Isostructural compounds with different cation combinations may result in larger cages and enhanced properties. Using wet chemical methods and initially dissolving Al(NO3)3 • 9H2O and SrCl2 • 6H2O in deionized H2O, Sr12Al14O33 has been synthesized, but attempts to eliminate secondary phases have not succeeded. Room temperature and high temperature X-ray diffraction data have been used to track the phase evolution. One consideration based on the synthesis is that Cl-, with a significantly larger ionic radius than O2-, is being occluded; the replacement of the divalent O with monovalent Cl would allow for more cages to be filled. However, the cage occupant is difficult to interrogate due to the low occupancy. 

View Abstract 980

Poster Author

Claudia Rawn, University of Tennessee, Knoxville Knoxville TN, TN 

Additional Author(s)

Katherine (Katie) Loughlin, University of Tennessee, Knoxville Knoxville, TN 
Michael Koehler, University of Tennessee, Knoxville Knoxville, TN 

Leveraging Quantum-Chemical In Silico Techniques To Determine Guest Binding Energies for the Crystalline Sponge Method

The crystalline sponge method presents a means to perform single-crystal X-ray diffraction (SC-XRD) analysis on non-crystalline samples [1,2]. The design of new crystalline matrices is necessary to expand the scope and generality of the technique. Computational analysis of crystalline sponge systems provides a way to gain a quantifiable and more detailed understanding of host-guest interactions than through in crystallo analysis alone. Gas-phase geometry optimization and single point energy calculations were performed on existing host-guest metal-organic framework (MOF) complexes based on {[(ZnX2)3(tpt)2]•x(solvent)}n (tpt = tris(4-pyridyl)-1,3,5-triazine, X = I, Br, Cl) to determine guest binding energies [3]. The geometries of the computed gas-phase structures closely matched experimentally-obtained structures. Calculated binding energies were related with guest B-factors to further analyze host-guest interactions. These insights may provide an impetus for further computational studies that will benefit crystal sponge design and selection via virtual screening.

Reference:
[1] Inokuma, Y., Yoshioka, S., Ariyoshi, J., Arai, T., Hitora, Y., Takada, K., Matsunaga, S., Rissanen, K. & Fujita, M. (2013). Nature, 495, 461–466.
[2] Cardenal, A. D. & Ramadhar, T. R. (2021) ACS Cent. Sci., 7, 406–414.
[3] Cardenal, A. D. & Ramadhar, T. R. (2021) CrystEngComm, 23, 7570–7575. 

View Abstract 1273

Poster Author

Timothy Ramadhar, Howard University Washington, DC 

Additional Author

Ashley Cardenal, Howard University Washington, DC 

Progress towards Increasing Automation and Sample Capacity at the GM/CA Beamlines for the upgraded APS (APS-U)

The Advanced Photon Source (APS) is rapidly approaching its yearlong shutdown for the upgrade to the APS-U starting in April of 2023. The GM/CA beamlines at Sector 23 have begun to prepare for the higher brightness of the new source. Installation of new optical focusing elements is underway.

One of the effects of the APS-U is the anticipated increase in the speed of data collection from crystal samples. Furthermore, users will certainly screen more samples at a greater rate. In response to this expectation, we are re-evaluating the capabilities of our current automounters. The possibilities include transitioning to a robotic arm, purchasing a commercial automounter, or upgrading the current automounter. This presentation will focus on upgrading our current automounters and cover other developments that will smooth the transition to the new source.

Our current automounters are a locally modified Berkeley Automounter (BAM). We converted the original model from limited discrete pneumatic positioning to continuous motor positioning for all motions except for the gripper on/off. The advantages of this conversion are:
1) Ability to stop at any position in the horizontal, vertical and gripper rotation
direction.
2) Flexibility to load samples onto either a vertically or horizontally mounted
goniometer.
3) In-house design, construction, maintenance and motion-control of the automounter.
4) Approximately 5 years of operational experience.
5) Tripling of the Dewar capacity from 6 to 18 ALS/Unipucks, for a total of 288 samples.
These improvements will bring us to the APS shutdown, however we expect the APS-U to accentuate the need to increase the robot capacity further. In order to accommodate this increase, a proposal under consideration is the addition of a second 18-puck Dewar, located adjacent to the existing Dewar, perpendicular to the direction of the incident X-rays.

The automounter is one component in the GM/CA automation pathway. A new GUI, PyBluice, will soon be released. It will combine existing and improved capabilities, i.e. loop-centering, screening, raster, and vector, which are necessary for data collection. It will also include the integrated automated data reduction and structure solution packages that are available in JBluIce. The new PyBluIce will balance the need to provide controls for teaching new users and the flexibility for expert users together with the demand for increasing automation.

Acknowledgments: GM/CA@APS is funded by the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006, P30GM138396). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. 

View Abstract 1252

Poster Author

Craig Ogata, Argonne National Lab Argonne, IL 

Additional Author(s)

Qingping Xu, Aronne National Laboratory Lemont, IL 
Mark Hilgart, Aronne National Laboratory Lemont, IL 
Oleg Makarov, Aronne National Laboratory Lemont, IL 
Shenglan Xu, Aronne National Laboratory Lemont, IL 
DAVID KISSICK, Argonne national laboratory Lemont, IL 
Michael Becker, GM/CA@APS, Argonne National Lab
Nagarajan Venugopalan, GM/CA@APS, Argonne National Laboratory Lemont, IL 
Stephen Corcoran, GMCA@APS Lemont, IL 
Dale Ferguson, Aronne National Laboratory Lemont, IL 
Sergey Stepanov, Aronne National Laboratory Lemont, IL 
Janet Smith, Life Sciences Institute, University Of Michigan
Robert Fischetti Argonne, IL 

On the electrolytic method of preparation of nano alumina from aluminum scrap

Aluminum scraps collected from local workshop, were used as electrode to electrolyze distilled water at different pH. A mixture of gibbsite, boehmite, bayerite and a little diaspore were precipitated . Subsequent heating of this mixture of compounds, results in the formation of different phases of aluminum oxide, namely α (corundum), δ, γ and θ, depending - on pH and temperature. If the pH is about 9, a mixture of predominantly nano δ and θ phase result at 900⁰ C . As the temperature is further raised, predominantly θ-phase is produced at 1100⁰ C. On the other hand, if the pH is around 4, micro-meter sized α- phase predominates. 

View Abstract 1021

Poster Author

PRABAL DASGUPTA Konnagar

Additional Author(s)

Praveen Balakrishnan, Jimma University Jimma, Oromiya 
Tsegaye Markos, Jimma University Jimma, Oromiya 

On the use of pseudo-Voigt function in the variance method of size-strain analysis

Although pseudo-Voigt(p-V) function is widely used to describe an experimentally observed powder XRD line, mainly due to ease with which its mathematical calculations could be tackled, Dasgupta[1,2] has shown that under certain conditions fitting of an observed profile with a p-V function, may lead to erroneous results, in case of Warren-Averbach[3] analysis. Pseudo-Voigt functions may be fitted with experimentally observed X-ray powder diffraction profiles and subsequently be subjected to variance method of size-strain analysis to extract particle size and root mean square strain of a crystallite. This work mathematically shows that for spherical crystallite and also for h00 reflections of a cubic crystallite and hho reflections of an octahedron shaped crystals (for which taper parameter L=0), Cauchy content η must be less than 0.328, otherwise no meaningful results in terms of particle size and root-mean-square (r.m.s) strain could be extracted from such profiles. We have made use of variance equation for pseudo-voigt profile deduced by Sanchez-Bajo and Cumbrera[4] and expression for intercept of a sample related variance, W0 given by Klug & Alexander[5] .Taper parameter values were taken from Wilson's paper[6].
References:
1. P. Dasgupta,. Fizika A (Zagreb) 9, 61 (2000)
2..P.Dasgupta,.J..Appl.Cryst 35,267(2002) https://doi.org/10.1107/S0021889801021203
3. B.E. Warren and B.L.Averbach, J.Appl.Phys. (1952) 23, 497https://doi.org/10.1063/1.170223
4. F.Sanchez-Bajo, & F.L Cumbrera,. J.Appl.Cryst.30,427.(1997)
https://doi.org/10.1107/S0021889801021203
5. H.P. Klug H.P & Alexander L.E In: X-ray diffraction procedure for polycrystalline and amorphous materials, (John Wiley, New York) .(1974)
6. A.J.C. Wilson, Proc.Roy.Soc. 80, 286 (1962) 

View Abstract 1018

Poster Author

PRABAL DASGUPTA Konnagar

Additional Author

Endale Abeba Gudeta, Jimma University Jimma, Oromiya 

Unnecessarily Complicated Research

The development of software for new applications requires extensive testing.
Several methods for determining likely Bravais lattice types are already available.
In creating a new one, we realized that their publications seldom indicate
that extensive testing was done. Usually, a few examples of unit cells from
the literature are displayed. We decided that Sella, a new method we are developing,
should have the kind of testing that software engineering requires. Starting from
an example unit cell, in the space S6 a cloud of points was generated. The points
are designed to fall at a prescribed small distance from the example cell. The
perturbed unit cells are then reduced; the resulting reduced cloud should
show continuity and reasonable distances from their corresponding reduced cell. 

View Abstract 1015

Poster Author

Larry Andrews, Ronin Institute Kirkland, WA 

Additional Author

Herbert Bernstein, Ronin Institute for Independent Scholarship, c/o NSLS-II, Brookhaven National Lab, Bldg 745 New York, NY