Poster Session I

Conference: 2021: 71st ACA Annual Meeting
07/31/2021: 4:00 PM - 5:00 PM
Poster Session 


A glimpse at the Balch-Olmstead fullerene legacy

The technique of cocrystallization developed in the Balch-Olmstead laboratory provided a unique solution to problems relating to fullerene disorder. It caused the field of fullerene structure determination to explode, enabling the characterization of historic endohedral fullerenes that defied the once-sacred Isolated Pentagon Rule and challenging our understanding of encapsulation chemistry. A timeline of the Balch-Olmstead legacy's greatest highlights will be presented. 

View Abstract 876

Poster Author

Mrittika Roy, University of California, Davis Davis, CA 

Additional Author

Alan Balch, University of California, Davis Davis, CA 

A structural investigation into an alpha-mannosidase found in Bacteroides thetaiotaomicron

Glycoside hydrolase family 38 (GH38) enzymes contain a myriad of alpha-mannosidases ranging in substrate specificity. All of the enzymes within this family share a common catalytic retaining mechanism with a conserved catalytic residue of aspartic acid. Despite structural studies across the kingdoms - notably from Drosophila melanogaster to Streptococcus pyrogenes - these enzymes vary strongly in kinetic preferences and structure. Recently, we've expressed a predicted alpha-mannosidase originating from Bacteroides thetaiotaomicron. This enzyme is predicted to contain a metal-binding region within the structure and may contain similar stabilizing disulfide-bridges similar to those in the Drosophila melanogaster alpha-mannosidase. The recombinant enzyme has been structurally modelled within ITASSER1,2,3 and current work focuses on crystallization of the enzyme for model validation.

1. Yang, J., Yan, R., Roy, A., Xu, D., Poisson, J., & Zhang, Y. (2015). The I-TASSER Suite: Protein structure and function prediction. Nature Methods, 12, 7-8. doi:10.1038/nmeth.3213.
2. Roy, A., Kucukural, A., & Zhang, Y. (2010). I-TASSER: a unified platform for automated protein structure and function prediction. Nature Protocols, 5, 725-738. doi: 10.1038/nprot.2010.5.
3. Zhang, Y.. (2008) I-TASSER server for protein 3D structure prediction. BMC Bioinformatics, 9(40). doi: 10.1186/1471-2105-9-40. 

View Abstract 839

Poster Author

Nicole Fraser, University of Waterloo Waterloo, ON 

Additional Author

David Rose, Dept of Biology, Univ of Waterloo Waterloo

Avoiding Sample Collisions with Puck Visualization for MX

We have created a Convolutional Neural Network (CNN) meant to prevent dangerous collisions within the dewar located at the National Synchrotron Light Source II (NSLS-II) AMX beamline. Our data consists of 1,041 images of B5 SPINE style base housed in Uni-Pucks under liquid nitrogen. These images are separated into three categories, straight, tilted, or empty, depending on the condition of the cap.

An image is taken using the area detector through the Experimental Physics and Industrial Control System (EPICS). After getting the image, each cap is cropped to its own image at its own position. This image is sent through the CNN to predict if the cap is safe for the autonomous robotic gripper. The CNN is composed of three hidden convolutional layers with max pooling between each layer for feature extraction. A flatten and dense layer are used to condense the matrix into a usable vector before being input into the final dense layer for classification.

Using the samples collected, the CNN model described above was able to achieve over ninety percent accuracy in predicting the cap condition. Although the model can predict three important cap conditions, to make a more robust program, we plan on adding obstruction categories as we continue data collection. Future categories, such as ALS caps and loose pins within the puck, will add further protection for the autonomous robotic gripper and allow for safer data collection. 

View Abstract 557

Poster Author

Samuel Clark, Hofstra University Bellport, NY 

Additional Author(s)

Herbert Bernstein, Ronin Institute for Independent Scholarship, c/o NSLS-II, Brookhaven National Lab, Bldg 745 New York, NY 
Dale Kreitler, National Synchrotron Light Source II Port Jefferson, NY 
jean jakoncic, BNL Upton, NY 
Alexei Soares, Brookhaven National Laboratory Shirley, NY 
Robert Sweet, Brookhaven Nat'l Lab
Edwin Lazo, Brookhaven National Laboratory, National Synchrotron Light Source II Upton, NY 

Biochemical analysis and review of the active site evolution of SARS-CoV-2 and other coronaviruses.

Coronaviruses have been a source of significant risk to global health. Almost four million lives have been lost to SARS-CoV-2 (COVID-19). Major efforts are ongoing to mitigate the current pandemic and future outbreaks by designing broad-spectrum drugs to target coronaviruses across species. We identified the positions of residues that participate in binding by using the 3D visualization tool Mol* ( to view known inhibitors interacting with the active site of SARS-CoV-2 proteases, two essential enzymes to virus maturation. Experimental structures from the Protein Data Bank (PDB) were used where available and additional models were generated using Robetta ( Sequences for additional coronaviridae proteases were obtained from NCBI ( A sequence-based comparison was performed using Clustal ( and a structure-based comparison was performed using Dali (, both using SARS-CoV-2 as the template. The preliminary results show some positions with mutations that remain within the same amino acid classification, groupings by structural, chemical, and functional similarity. Other mutations result in a different classification in that position that may result in a significant impact to the active site structure and binding. We will continue to analyze these changes to identify patterns in the mutations and to identify which mutations have the most impact and are most relevant to potential viral evasion of broad-spectrum drugs. 

View Abstract 860

Poster Author

Mickayla Bacorn

Bioinformatics and 3D Structural Analysis of the Coronavirus Main Protease Active Site

Coronaviruses (Coronaviridae) such as SARS-CoV-2 (severe acute respiratory syndrome coronavirus) and MERS-CoV (Middle East respiratory syndrome coronavirus) have been the source of recent outbreaks and global health concerns. While vaccines have been essential for controlling the SARS-CoV-2 (COVID-19) pandemic, it is uncertain whether they will be effective against future coronavirus strains. Therefore, identification or design of a broad-spectrum drug
that targets highly conserved regions of the main protease of multiple coronavirus strains is essential in the long term. As part of a virtual summer research experience with the RCSB PDB, bioinformatics tools were employed to predict and construct 3D models of the coronavirus main protease (MPro) using SARS-CoV-2 as the template, with a focus on mutational trends and active sites. This study focused on the active sites of MPro, a cysteine protease essential for viral assembly and replication. Sequence alignments and
structure modeling of MPro structures has identified conserved regions across multiple coronavirus strains. Inhibition of MPro halts coronavirus replication, making it an ideal drug target, and studies of MPro may foster and accelerate the discovery of high affinity broad-spectrum drugs. This work was supported by an NSF REU to RCSB PDB. RCSB PDB is funded by the National Science Foundation (DBI-1832184), the US Department of Energy (DE-SC0019749), and the National Cancer Institute, National Institute of Allergy and Infectious Diseases, and National Institute of General Medical Sciences of the National Institutes of Health under grant R01GM133198 

View Abstract 861

Poster Author

Amy Wu Wu, University of Puerto Rico, Mayagüez Campus

Additional Author(s)

Mickayla Bacorn
MaryAgnes Balogun, Morgan State University
Cassandra Olivas, California State University, Stanislaus Ripon, CA 
Christine Zardecki, RCSB Protein Data Bank Piscataway, NJ 
Sagar Khare, Institute for Quantitative Biomedicine
Stephen Burley, RCSB Protein Data Bank, Rutgers University Piscataway, NJ 
Joseph Lubin

Extraordinary Structures of Orphan Methyltransferases with Their Substrate DNA

"Recognition of DNA by proteins, both sequence and structure specific, is important in the functioning of the cell, such as in the processes of replication, transcription, and DNA repair. Twenty-five years after base flipping, a phenomenon whereby a base in normal B-DNA is swung completely out of the helix into an extrahelical position, was first observed in HhaI methyltransferase, we are still learning from and surprised by structures of protein-DNA complexes. The novel structures of the bacterium Caulobacter crescentus cell cycle-regulated DNA adenine methyltransferase (CcrM), as well as the newly discovered CamA enzyme (named for Clostridioides difficile adenine methyltransferase A) in complexes with double-stranded DNA containing their recognition sequence, will be discussed. Each of these enzymes affect their DNA substrate in a unique number of ways that are critical for their level of discrimination of their recognition DNA sequence.

CcrM in C.crescentus is responsible for maintenance methylation immediately after replication and methylates the adenine of hemimethylated GANTC. CcrM contains an N-terminal methyltransferase domain and a C-terminal nonspecific DNA-binding domain. CcrM is a dimer, with each monomer contacting primarily one DNA strand: the methyltransferase domain of one molecule binds the target strand, recognizes the target sequence, and catalyzes methyl transfer, while the C-terminal domain of the second molecule binds the non-target strand. The DNA contacts at the five base pair recognition site results in dramatic DNA distortions including bending, unwinding and base flipping. The two DNA strands are pulled apart, creating a bubble comprising four recognized base pairs. The five bases of the target strand are recognized meticulously by stacking contacts, van der Waals interactions and specific Watson–Crick polar hydrogen bonds to ensure high enzymatic specificity.

In the developed world, C. difficile is one of the leading causes of hospital-acquired infections. CamA-mediated methylation of the last adenine in CAAAAA is required for normal sporulation and biofilm production by this bacterium, a key step in disease transmission. Thus, selective inhibition of CamA has great therapeutic potential. CamA contains an N-terminal methyltransferase domain as well as a C-terminal DNA recognition domain. Major and minor groove DNA contacts in the recognition site involve base-specific hydrogen bonds, van der Waals contacts and the Watson-Crick pairing of a rearranged A:T base pair. These interactions provide sufficient sequence discrimination to ensure high specificity. In addition, this DNA methyltransferase has unusual features that may aide in discovery of a new selective antibiotic to combat C. difficile infection.

Knowledge acquired from these structures may also relate to other projects in our laboratory relating to mammalian epigenetics." 

View Abstract 874

Poster Author

John Horton, MD Anderson Cancer Center of The University of Texas Houston, TX 

Additional Author

Clayton Wookcock, The University of Texas-MD Anderson Cancer Center Houston, TX 

Factors of Atomic Electron Scattering (FAES): A resource for Gaussian parameterization of integral ionic, fractionally charged, and neutral electron scattering factors

Electrostatic potential maps derived from cryo–EM can contain a wealth of information about charged states. However, access to such information is obstructed by the absence of appropriately parameterized ionic electron scattering factors. Existing parameterizations remain either incomplete or incompatible with least-squares refinement programs. To rectify this, we introduce FAES (Factors of Atomic Electron Scattering), a web server publicly accessible at . This resource supplies Gaussian parameterizations of elastic electron scattering factors into three forms and calculates fractionally charged scattering factors by computing linearly weighted sums of adjacent integral neighbors. Using atomic scattering amplitudes tabulated in the International Tables for Crystallography, FAES provides numerical fitting coefficients, statistical goodness-of-fit values, and accompanying visual plots for all supported fits. We also derive elastic and estimated inelastic cross-sections from FAES parameterizations at a range of accelerating voltages relevant to transmission electron microscopy. 

View Abstract 539

Poster Author

Ambarneil Saha, University of California, Los Angeles Los Angeles, CA 

Additional Author(s)

Madeline Evans, University of California, Los Angeles Los Angeles, CA 
Thomas Holton, University of California, Los Angeles Los Angeles, CA 
Jose Rodriguez, UCLA Los angeles, CA 

Magnetic behavior of Cu-intercalated MnPSe3

There has been a growing interest in magnetic van der Waals (vdW) compounds owing to their two-dimensional magnetic properties, making them particularly suited for the developing field of spintronics. One particular family of vdW compounds, transition-metal phosphorous trichalcogenides (MPX3, M = Mn, Ni, Fe, Cu, Co, etc. X = S and Se), has shown a great potential in the field of magnonics. We study the magnetic nature of MnPSe3 by doping Cu into the structure and analyzing its impact on the long-range magnetic order. Powders of Mn1-xCuxPSe3 have been synthesized using high-temperature solid-state method. Phase purity was confirmed using synchrotron powder X-Ray Diffraction (XRD), Pair Distribution Function (PDF), and powder neutron diffraction. From our PDF and XRD analysis, we find that Cu is most favorably found to be intercalated in the vdW gap of MnPSe3, inducing a long-range magnetic order transformation as observed in magnetic susceptibility and powder neutron diffraction data. Herein, we report magnetic susceptibility, PDF, XRD, carrier density, neutron diffraction data, and DFT calculations for Mn1-xCuxPSe3. 

View Abstract 758

Poster Author

Mohamed Nawwar, The Ohio State University Columbus, OH 

Additional Author(s)

Sogol Lotfi, The Ohio State University Columbus, OH 
Vicky Doan-Nguyen, The Ohio State University Columbus, OH 

Operando oxidation and reduction neutron scattering studies on pristine and Pt-coated ceria nanorods

Ceria is widely used as a three-way catalyst support (TWC) for the conversion of automotive emissions due to its excellent redox properties and high oxygen storage capacity (OSC). It has been broadly reported that OSC can be drastically improved by increasing the concentration of oxygen defects and that the redox properties of ceria are controlled by the type, size, distribution, and location (surface or bulk) of the oxygen vacancies. Recently, a partially reduced Ce3O5+x defect phase was discovered on the surface of ceria nanorods, which is distinct from the bulk Frenkel-type defect structure. The higher OSC performance of ceria nanorods relative to other morphologies (such as nanocubes) was attributed to the presence of a higher concentration of these surface defects.

Platinum group metals have been shown to strongly interact with ceria under redox conditions, further improving its catalytic properties. In this work, we elucidate the effects of Pt loading on the surface and bulk defects of ceria nanorods, through an in situ neutron scattering study of as prepared vs 1% Pt-coated ceria nanorods, under redox flow. Bragg diffraction results indicate that the bulk structure of both as prepared and 1% Pt-coated ceria nanorods can be indexed to a classic Fluorite structure. Interestingly, pair distribution function analysis (PDF), in accordance with Bragg results, indicate that the Pt-coated rods have a larger lattice parameter and crystallite domain size than the as prepared rods. This suggests that the Pt-coated rods contain a higher concentration of Ce3+ and are more easily reducible than the as prepared rods. Further PDF results reveal that the Pt-coated rods contain a secondary surface defect phase, which is lacking in the as-prepared rods. These structural differences in the as prepared and Pt-coated rods explain the superior catalytic performance of the Pt-coated rods. Unlike many studies in scientific literature, the as prepared and Pt-coated nanorods used in this study more closely resemble real catalytic converters, which are pre-conditioned at high temperatures in humid oxidizing conditions. Thus, the results from this study provide insights into the structure of ceria nanorod based catalysts under real operating conditions, which can aid in the design and optimization of future catalytic materials. 

View Abstract 833

Poster Author

Sreya Paladugu, The University of Tennessee, Knoxville Knoxville, TN 

Additional Author(s)

Peter Metz, Oak Ridge National Laboratory Oak Ridge, TN 
JUE LIU, Oak Ridge National Laboratory Oak Ridge, TN 
Zili Wu, Oak Ridge National Lab
KATHARINE PAGE, University of Tennessee Oak Ridge, TN 

Optimizing the conditions for GGDPS crystal growth

Several incurable cancers are characterized by abnormal protein production and secretion. This includes excessive monoclonal protein (MP) secretion in multiple myeloma (MM) and excessive glycosylated mucin production in pancreatic ductal adenocarcinoma (PDAC). Aberrant production and secretion of these cancer-related proteins lead to increased disease progression indicated by enhanced metastasis, tumor growth, and drug resistance. As both cancers are incurable and have a high chance of developing drug resistance as the diseases progress, new treatments are highly desirable. Research has shown that inhibition of geranylgeranyl diphosphate synthase (GGDPS) disrupts the function of the intracellular trafficking Rab family of proteins. GGDPS is responsible for synthesizing the 20-carbon isoprenoid group (geranylgeranyl diphosphate or GGDP) that is added to the carboxy terminus of Rab. This addition is essential for Rab's function in intracellular trafficking. Disrupting Rab geranylgeranylation leads to disruption of monoclonal proteins and mucin trafficking resulting in the activation of the unfolded protein response and apoptosis.

Our collaborators have synthesized new potent inhibitors with high specificity for GGDPS. The goal of this study is to obtain the crystal structure of these inhibitors bound to GGDPS to understand the mechanisms behind GGDPS binding and to better rationalize the design of future GGDPS inhibitors. Currently, we have obtained initial GGDPS protein crystals that require further optimization. These crystals were seen a month after the initial screening and exhibited either a needle or a feather-like structure. We are working on growing diffraction quality GGDPS protein crystals for drug development. 

View Abstract 603

Poster Author

Andrew Pham LaVista, NE 

Additional Author(s)

Lucas Struble, The Eppley Inst For Cancer Res
Sarah Holstein, Department of Internal Medicine
Gloria Borgstahl, The Eppley Inst For Cancer Res Omaha, NE 

Porting Crystal Structure Data to Virtual Reality using CAD2VR

Three-dimensional crystal structure data is most often displayed on two-dimensional surfaces such as printed images or computer screens. These displays create visualization issues for extended structures like minerals or metal organic frameworks (MOFs) where packing the structure causes many atoms to overlap. With virtual reality hardware becoming more accessible, the next natural step for crystallography is to visualize 3D structures in 3D space.

CAD2VR software was developed at Sandia National Laboratories to visualize Computer Aided Drafting (CAD) Models in virtual reality (VR). It offers a variety of interaction tools that, when applied to the atomic scale, would be useful for crystallographers. A plugin is being developed that can import crystal structure models into VR. Features include measuring atomic distances, bond lengths, and bond angles; displaying the unit cell; and drawing polyhedron volumes to measure void spaces.

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 753

Poster Author

Nichole Valdez, Sandia National Laboratories Albuquerque, NM 

Additional Author(s)

Mark Rodriguez, Sandia National Laboratories
John Krukar, Sandia National Laboratories
Matthew Gallegos, Sandia National Laboratories
James Harris, Sandia National Laboratories

Structural basis of inhibition of the NUDIX family ORF141

NUDIX hydrolases exist in a wide variety of species, ranging from humans to viruses. Initially, these enzymes were considered "housecleaning" enzymes based upon their ability to remove mutagenic nucleotides such as 8-oxo-dGTP. Since then, many more functions of NUDIX enzymes have been discovered and characterized such as: mRNA degradation, regulation of coenzyme A, and hydrolysis of nucleoside diphosphate sugars. Named after their shared ability, this superfamily catalyzes the hydrolysis of nucleoside diphosphate linked to a moiety X, hence the name "NUDIX". This superfamily is characterized by the presence of a sequence signature motif of 23 amino acids, G1N[5X]E7N[7X]R15NE16NXXE19NE20NXG22NU, known as the NUDIX box. This motif has been identified in over 20,000 species, all with differing genome sizes and number of NUDIX genes. The NUDIX signature sequence has three glutamates (E16NXX19NE20N) that are at the center of the β-strand-loop-helix-loop motif. Moreover, they bind the catalytic divalent cations that bridge the protein and the pyrophosphate of the substrate. Although the sequence signature motif allows for the identification of these enzymes into the superfamily, the classification of these enzymes into families requires information regarding the reaction they catalyze, preferred substrate, quaternary structure, and phenotype to formulate rules.
Previously, the NUDIX family represented by ORF141 was identified to be nucleoside triphosphatases with a preference for pyrimidine deoxy-nucleoside triphosphates, such as deoxyguanosine triphosphate (dGTP). Recent studies have discovered ORF141 to be an atypical NUDIX that prefers geranyl pyrophosphate, which lacks the typical nucleoside base of the archetypical NUDIX substrates.
To establish the structural determinants of substrate specificity for ORF141, we have determined the structure of apo ORF141 to 1.54 Å by x-ray crystallography. Interestingly, ORF141's NUDIX fold displays a hairpin insertion when compared to the minimal NUDIX fold observed in the NUDIX enzyme family with a preference for dGTP. We determined that bisphosphonates inhibit ORF141. To define the structural basis of inhibition, we determined the structure of ORF141 with three bisphosphonates: risedronate, [2-(n-pentylamino)ethane-1,1-diyl]bisphosphonic acid (BR6), and 2-(n-hexylamino) ethane-1,1-diyl]bisphosphonic acid (BR18). Each of the complex structures show that Mg2+ atoms mediate their binding to the glutamates of the NUDIX signature sequence. Interestingly, when bound to the bisphosphonates, ORF141's hairpin insertion displays a conformational change of up to 11 Å away from the active site and proportional to the length of the bisphosphonate's R derivative. For example, the 3-pyrindinylethyl group of the risedronate exerts less of a displacement in comparison to the longer hexylamine group of BR6. 

View Abstract 826

Poster Author

Kim Phan

Additional Author(s)

Maurice Bessman, Johns Hopkins University Baltimore, MD 
Juan Rodríguez, Universidad de Buenos Aires
Sandra Gabelli, Johns Hopkins University Baltimore, MD 

Structural Comparison of Faecalibacterium prausnitzii α-glycosidases and Sucrase-Isomaltase

The human gastrointestinal system is home to a very diverse microbiome that is made up of approximately 100 trillion microorganisms. This microbiome has a significant impact on human health, as it can influence the immune system, the central nervous system and the body's metabolism. Faecalibacterium prausnitzii is one of the most abundant microorganisms found in a healthy gut, where it makes up about 5% of the microbiome. This gastrointestinal microorganism produces 2 enzymes belonging to the glycoside hydrolase family 31, which will be referred to as Fp-αG1 and Fp-αG2. These α-glycosidases have notable structural similarities to the N-terminal subunit of sucrase-isomaltase. Sucrase-Isomaltase (SI) is a gastrointestinal enzyme found in humans, and is responsible for hydrolyzing carbohydrates with α-1,6, α-1,4 and α-1,2 glycosidic bonds. The objective of this project is to compare the enzymatic activity of F. prausnitzii α-glycosidases to the N-terminal subunit of SI, in order to investigate the structural similarities between the proteins. Real time kinetic assays and computational models will be used to identify structural features contributing to the differences in substrate affinity. These investigations into protein structure and enzymatic activity of the α-glucosidase proteins will provide a clearer insight on the structure and function of the Fp-αG1, Fp-αG2, and SI. 

View Abstract 836

Poster Author

Anna Jewczynko, University of Waterloo Waterloo, ON 

Additional Author

David Rose, Dept of Biology, Univ of Waterloo Waterloo

Structural Insight into Replication Fidelity and Primer Shuttling in Human Mitochondrial DNA Polymerase Gamma

DNA polymerase gamma (PolG) is the sole DNA replicase in human mitochondria. Nucleoside Reverse Transcriptase Inhibitors (NRTIs) act as chain terminators once incorporated by the HIV reverse transcriptase (RT), effectively inhibiting viral replication. However, PolG is an off target of NRTIs, leading to mitochondrial DNA deletions and mutations that clinically manifest as neurological and cardiovascular diseases. Thus, it is important to understand the mechanism behind incorporation and excision of NRTIs to reduce drug toxicity. Using X-ray crystallography, we have previously provided structural mechanism behind pre-incorporation drug discrimination in the polymerase (pol) site, shared by both enzymes. However, the mechanisms behind post-incorporation drug recognition and excision by the exonuclease (exo) site are still unknown. To this end, we first set out to study PolG under normal replication circumstances. We've determined cryo-EM structures of four different PolG-DNA complexes at near-atomic resolution. Here, we reveal both the error detection mechanism in the pol site post-incorporation and error excision mechanism in the exo site. Global subunit movement together with displacement of the primer by the "ejection loop" facilitates the shuttling of the primer from pol to exo sites. In addition, we report previously unobserved transition conformation, which gives deeper insight into initiation of primer shuttling and communication between pol and exo sites. Global and local structural changes associated with error recognition and excision show dynamic and well-coordinated communication between pol and exo sites required for high fidelity DNA replication. This information can be exploited to improve current NRTI designs to reduce PolG-mediated drug toxicity. 

Poster Author

Joon Park, University of Texas Medical Branch Galveston, TX 

Structural insight into the neosubstrate selectivity of thalidomide metabolite

Thalidomide (Thal) exerts adverse effects such as teratogenicity, however, is used for the therapy of multiple myeloma and other haematologic malignancies as immunomodulatory imide drugs (IMiDs). The molecular mechanism of thalidomide's pharmacological action has been gradually elucidated through the search for multiple target proteins that thalidomide acts on. Celebron (CRBN) is the intracellular receptor for Thal and induces Thal-dependent degradation of target protein (neosubstrate) as a component of a E3-ubiquitin ligase. Although C2H2 zinc finger (ZF) transcription factors, IKZF1 and SALL4, are concerned in immunomodulatory effects and teratogenicity of Thal, respectively, a primary Thal metabolite, 5-hydroxythalidomide (5HT), induces degradation of SALL4 but not IKZF1. Due to the action of the enzyme cytochrome P450 in the body, the administered thalidomide produces 5HT. Here, we focused on the molecular mechanism in which the selectivity of Thal toward C2H2 ZF-type neosubstrates is altered with its metabolism. First, we characterized the enantioselectivity of the formation in the SALL4-CRBN complex. The (S)-enantiomer of Thal and 5-HT showed more effect than the (R)-enantiomer, which is consistent to "Left-hand (S-form) theory of teratogenicity" of Thal. Based on the enantioselectivity, we determined the crystal structures of the ternary complexes of the Thal-binding domain (TBD) of human CRBN and the second ZF domain (ZF2) of human SALL4 induced by (S)-Thal and (S)-5HT. As a result, Thal and 5HT positioned between the interface of SALL4 ZF2 and CRBN TBD to mediate the protein-protein interaction as molecular glues. Although both compounds occupy at the same position in the SALL4-CRBN complex, the 5-hydroxy group of 5HT forms an additional hydrogen bond with CRBN TBD through a water molecule, which enhances the formation of the SALL4-CRBN complex. The 5-hydroxy group is also located near the 2nd and 9th residues of the β-hairpin structure in SALL4 ZF2, and these residues are different from IKZF1. The complex formation and proteasomal degradation experiments using the residue-swap mutants of SALL4 and IKZF1 elucidated the variation in the 2nd residue of β-hairpin structure defines the neosubstrate selectivity of 5HT. Thalidomide's action on its target is altered through its metabolism in the body and if the hydroxylation of thalidomide found in this study is avoided, a new designed drug can be expected to reduce teratogenicity. Furthermore, our findings indicate that the structural differences found in C2H2 ZF-type transcription factors may be exploited to increase the efficiency of action of IMiDs, including thalidomide, on target proteins required for drug efficacy. 

View Abstract 834

Poster Author

Hirotake Furihata, The University of Tokyo Tokyo

Additional Author(s)

Satoshi Yamanaka, Proteo-Science Center, Ehime University Ehime
Toshiaki Honda, Department of Nanopharmaceutical Sciences, Nagoya Institute of Technology Nagoya
Norio Shibata, Department of Nanopharmaceutical Sciences, Nagoya Institute of Technology Nagoya
Masaru Tanokura, Department of Applied Biological Chemistry,The University of Tokyo Toyko
Tatsuya Sawasaki, Proteo-Science Center, Ehime University Ehime
Takuya Miyakawa, Department of Applied Biological Chemistry,The University of Tokyo Toyko

Structural insights into the mechanisms of substrate recognition and catalysis for the N-methyltransferases involved in benzylisoquinoline alkaloid metabolism

Methyltransferases (MTs) are a diverse family of enzymes responsible for the addition of a methyl group from a donor molecule (typically S-adenosyl methionine) to target substrates. Methyltransferases are found widely across nature and are critical for many cellular activities, including central metabolism, DNA and RNA processing, signal transduction, and protein localization. The ubiquity of MTs in multiple contexts highlights the importance of methyl transfer for many biological processes. In addition to their intrinsic importance to biology, the diversity of sequences and 3-dimensional structures of MTs, particularly in the domain responsible for recognizing methyl acceptor substrates, underlines the importance of characterizing the broad range of molecular mechanisms for substrate recognition and catalysis used within this enzyme family.

MTs play a key role in the production of benzylisoquinoline alkaloids (BIAs), a class of specialized metabolites produced in plants such as the opium poppy. High value natural (morphine, codeine) and non-natural (oxycodone, naloxone) BIAs are considered essential medicines. Several novel or rare BIAs are being actively studied for their potent and diverse pharmacological properties, and to explore their potential as lead compounds in drug discovery. However, the promiscuity of many of the natural enzymes involved with BIA biosynthesis, including MTs, is widely believed to limit the yields of targeted, high-value products from engineered metabolic pathways. As a result, there is great interest and urgency to develop a rational molecular basis for controlling the specificity and activity of BIA biosynthetic enzymes, including MTs. A diversity of BIA MTs have been shown to methylate the multiple oxygen atoms (OMT) or central nitrogen atom (NMT) within the BIA substrate. While several functionally distinct BIA OMTs are known, all characterized BIA NMTs can be grouped into 3 functional classes.

Previously, the structures of two functionally distinct BIA NMTs have been reported, showing surprisingly distinct modes of acceptor substrate recognition [1, 2]. Recently, we reported structure-function studies of the third distinct class of NMTs involved with BIA biosynthesis, the tetrahydroprotoberberine NMT from Glaucium flavum (dmin = 1.6-1.8 Å) [3]. Our newest structures highlight the importance of complementarity between the uniquely L-shaped substrate recognition pocket and the (S)-cis configuration of the BIA substrate for stereospecific substrate selection.. This presentation will focus on how our most recent results provide novel insights into the functional roles of conserved residues within and near the enzyme active site, and the roles of the size and shape of the substrate recognition pocket in the selection of the appropriate BIA substrate.

[1] Torres, M. A., Hoffarth, E., Eugenio, L., Savtchouk, J., Chen, X., Morris, J. S., Facchini, P. J., & Ng, K. K. S. (2016) J. Biol. Chem. 291, 23403-23415.
[2] Bennett, M. R., Thompson, M. L., Shepherd, S. A., Dunstan, M. S., Herbert, A. J. Smith, D. R. M., Cronin, V. A., Menon, B. R. K., Levy, C., Micklefield, I. (2018). Angew Chem 57, 10600-10604.
[3] Lang, D. E., Morris, J. S., Rowley, M., Torres, M. A., Maksimovich, V. A., Facchini, P. K., Ng, K. K. S. (2019) J. Biol. Chem. 294, 14482-14498. 

View Abstract 850

Poster Author

Dean Lang

Additional Author(s)

Jeremy Morris, University of Calgary Calgary, Alberta 
Peter Facchini, University of Calgary
Kenneth Ng, University of Windsor

Structural Studies of the Conjugative Entry Exclusion Protein from the F and R100 Plasmids, TraG

Conjugative type IV secretion systems (T4SS) transmit mobile DNA elements in bacteria and are a significant contributor to the evolution of antibiotic resistance. Of the proteins produced from the F and R100 plasmids of Escherichia coli; the representative conjugative plasmids of gram-negative bacteria, TraG is among the largest and consists of a membrane-bound N-terminal domain and a periplasmic C-terminal domain denoted TraG*. Each domain has its own function, the membrane bound N-terminal domain is involved in pilus assembly while TraG* is bifunctional. In the donor cell, it interacts with TraN within the outer membrane to facilitate mating pair stabilisation. However, TraG* is also essential in preventing redundant DNA transfer through its interaction with a cognate TraS in the inner membrane of the recipient cell when the recipient carries the same plasmid. Thermofluor experiments showed N-terminal truncation mutants of TraG* displayed higher stability relative to full-length TraG*, and SEC MALS provides evidence of higher levels of aggregation in the full-length protein relative to the N-terminal truncation mutants. SEC-MALS-SAXS was performed to obtain low-resolution structural models to visualize the conformational changes resulting from the truncation, and crystal trials of TraG* mutants provide evidence of a higher propensity for the crystallisation of N-terminal truncations of TraG*. The 45 N-terminal residues of TraG* are predicted to be highly dynamic, possibly serving as a flexible linker between two independently functioning domains. 

View Abstract 816

Poster Author

Nicholas Bragagnolo, York University Bolton, ON 

Additional Author

Gerald Audette, Dept of Chemistry, York Univ Toronto, ON 

The role of BAM in mediating Fusobacterium nucleatum infection and pathogenesis

Fusobacterium nucleatum is a Gram-negative oral pathogen implicated in periodontal infections and correlated with pre-term births and colorectal cancer. Additionally, this organism plays a vital role in the oral microbiome, adhering to other microbiome components through outer membrane proteins (OMPs) called adhesins. Multiple F. nucleatum adhesin structures have been identified as type Va autotransporters or porins, which are presented as β-barrel OMPs. The β-barrel assembly machinery (BAM) complex is an essential outer membrane protein complex found in all Gram-negative bacteria, functioning in the biogenesis of β-barrel OMPs. Components of this complex have been solved in organisms such as Escherichia coli, Haemophilus ducreyi, Neisseria gonorrhea, and Pseudomonas aeruginosa; however, the composition of the BAM complex in F. nucleatum remains unknown. In E. coli, the BAM complex is composed of an integral membrane protein, BamA, and four periplasmic lipoproteins, BamB-E. In F. nucleatum, only BamA appears to be present in the genome based on our bioinformatics analysis, despite the necessity of both BamA and BamD for organism viability in other Gram-negative bacteria. Since no BamD ortholog was found in F. nucleatum, we hypothesize that the BAM complex in F. nucleatum may use a different mechanism compared to E. coli. Thus, the goals of the project are first, to structurally characterize BamA in F. nucleatum using X-ray crystallography and/or cryo-EM, and second, to determine the composition of the BAM complex in F. nucleatum by isolating and identifying BamA-interacting proteins. 

View Abstract 845

Poster Author

Claire Overly, Purdue University West Lafayette, IN 

Additional Author

Nicholas Noinaj, Purdue University

Towards the structural analysis of an F plasmid protein, TraW

Bacterial conjugation is a form of lateral gene transfer used in bacterial systems to allow for the transfer of genetic material by a contact dependent mechanism. This mechanism has been considered to greatly contribute to the increase of virulence factors among different pathogens. This transmission of conjugative plasmids can be achieved by the type IV secretion system (T4SS). The T4SS is a multiprotein complex spanning the inner and outer membrane of gram-negative bacteria, producing a pilus to allow contact with neighbouring cells. The T4SS is able to produce many different types of pili, the focus of this study is on the F pilus. The F pilus is thin, flexible and capable of extension and retraction. The F pilus is composed of TraA pilin monomers, and the intricate process of assembling these monomers is achieved by many different proteins including TraW. TraW is a protein that is specific to the F-type T4SS and is present in the periplasmic space. TraW is essential for the assembly and extension of the pilus as well as bacterial conjugation. Mutations in TraW stop the pilus from extending. While the interaction between the N-terminal domain of TraW and the C-terminal domain of TrbC is essential for conjugation to move forward. Although the general function of this protein is understood the intricate process required to achieve them is not, understanding this protein at a structural level will provide better insight into the role this protein plays within the T4SS. 

View Abstract 626

Poster Author

Christina Rodriguez, York University Waterloo, ON 

Additional Author

Gerald Audette, Dept of Chemistry, York Univ Toronto, ON 

Understanding the active site of the SARS-CoV-2 papain-like protease (PLPro)

Coronaviruses are a threat to the health of the global community. Prior to the COVID-19 global pandemic caused by SARS-CoV-2, severe acute respiratory syndrome (SARS-CoV) and Middle East respiratory syndrome (MERS-CoV) were coronaviruses that made the species jump to infect humans. In SARS-CoV-2, papain-like proteases (PLpro) participate in cleaving the polyproteins and liberating non-structural proteins. The non-structural proteins attach to each other and create a replication transcription complex which is used for viral replication. Targeting and disrupting coronavirus proteins with drugs is one strategy for stopping future outbreaks. Studying active site residues that are highly conserved between many variants will help in developing broadly effective inhibitors. We want to be able to identify or create a drug that will work against as many coronaviruses as possible, particularly for variants that differ in the chemical character of their active sites. In order to analyze the active site of SARS-CoV-2 papain-like proteases (PLPro) we performed sequence-based comparisons of the active sites of the SARS-CoV-2 PLPro with other coronavirus PLPro enzymes using sequences obtained from NCBI, and generated computational models with Robetta. A structure-based sequence alignment was performed using the Dali server ( Visualization of the molecular structures of these enzymes was performed using Mol* ( Preliminary results show differences in the chemical character of the active site of the SARS-CoV-2 PLPro enzyme and the active sites of other experimental coronavirus PLPro enzymes. Our studies suggest that a drug effective against one PLPro enzyme may not bind to the PLPro enzyme of a different coronavirus. 

View Abstract 864

Poster Author

MaryAgnes Balogun, Morgan State University

Additional Author(s)

Amy Wu Wu, University of Puerto Rico, Mayagüez Campus
Mickayla Bacorn
Cassandra Olivas, California State University, Stanislaus Ripon, CA 
Christine Zardecki, RCSB Protein Data Bank Piscataway, NJ 
Joseph Lubin
Sagar Khare, Institute for Quantitative Biomedicine
Stephen Burley, RCSB Protein Data Bank, Rutgers University Piscataway, NJ 

What is a single cryo-EM image worth? A survey of high-resolution cryo-EM model system datasets

Significant advancements in both cryo-EM hardware and software have, remarkably, achieved true atomic resolution (~1 Å) cryo-EM reconstructions. Given that this was achieved using reasonably sized particle datasets, this suggests that the improved resolution arises from increased signal contained within individual cryo-EM particle images. Here, we ask the question: what structural features can be reliably identified from small sets of aligned cryo-EM images using various microscope setups? Remarkably, 10 pre-aligned particle images of a high-symmetry structure (i.e apoferritin) from the cold-FEG (CFEG) particle data-set was sufficient to obtain a ~10 A reconstruction, suggesting that substantial structural information may be present at the fold-level within small sets of cryo-EM images. We next compared small pre-aligned sets of particle images (ranging from 1 to 100 particles per set) against the experimental reconstruction (obtained using the full set of images) using established metrics such as the Fourier Ring Correlation (FRC). Unsurprisingly, we discovered that individual particles were too noisy to reliably assess information content using FRC, even when using improved metrics (such as the masked Wiener filter) to assess signal-to-noise ratios (SSNR). However, analysis of 100 pre-aligned particle images was sufficient to identify distinct signatures in the FRC profiles corresponding to secondary structure elements, such as α-helices. We compared FRC profiles from CFEG datasets and conventional XFEG datasets and discovered differences in the FRC profiles that suggest that more high-resolution spectral signal is contained within CFEG compared to XFEG images. We next compared the FRC profiles obtained from experimental reconstructions and atomic models and found that atomic models are mostly insensitive to simulation details. However, alterations to the atomic model result in sharp drops in FRC profiles, indicating that such metrics are highly sensitive to the atomic model being used to compare to experimental projection images. These computational analyses constitute our first attempts towards analyzing small sets of particle projection images. Ultimately, such techniques may be used to build thermodynamic landscapes describing protein structure and function without relying on massive datasets or extensive averaging to extract structural features. 

View Abstract 847

Poster Author

Vinh Truong San Jose, CA 

Additional Author(s)

Tiffany Chen, Kellogg Lab Ithaca, NY 
Elizabeth Kellogg