General Interest 2

Conference: 2021: 71st ACA Annual Meeting
08/01/2021: 12:00 PM - 3:00 PM
Oral Session 


General Interest sessions are the forum for topics of broad interest to the crystallographic community or for presentations that do not fit the specific theme of other sessions. All presentations are selected from submitted abstracts


Visualization of the constellation of protons in the product-inhibited state of human manganese superoxide dismutase

Oxidoreductases are integral to human vitality though most of their mechanisms are unclear due to the difficulty in observing the protons that are used as chemical tools to drive the electron transfers in a process called concerted proton-electron transfer (CPET). Human mitochondrial manganese superoxide dismutase (MnSOD) is a prominently studied oxidoreductase in clinical research due to its central role in the oxidative stress response where it uses CPETs to convert superoxide (O2•-) into either oxygen (O2) or hydrogen peroxide (H2O2). Like many other oxidoreductases, the protonation states and proton transfers (PTs) needed to facilitate the ETs are unclear. Our group's recent work published in Nature Communications on room temperature, wildtype Mn3+SOD, and Mn2+SOD structures revealed portions of the CPET mechanism including an unusual back-and-forth amide deprotonation-and-protonation at Gln143 that is proximal to the Mn ion. Neutron diffraction's ability to resolve proton positions while being inert to the electronic states of metals and other paramagnetic centers (unlike X-rays) is indispensable for filling the gaps of mechanistic knowledge that are present for oxidoreductases.

Another layer of complexity is provided by human MnSOD's unique feature of product-inhibition that limits the output of H2O2. The capacity to become inhibited by its product has shown to be a necessity for the normal functioning of human cells, and if perturbed, leads to dramatic cell cycle effects. MnSOD uses this alternative pathway 50% of the time during steady-state conditions and is thought to be characterized by decoupling ETs and PTs into separate kinetic steps. O2•- binds and abstracts an electron from Mn2+ without occurrence of PTs to yield a complex of (Mn3+–O22-) incapable of performing ETs. To relieve inhibition at the active site, kinetic and mutagenesis studies suggest an alternative array of PTs is used to protonate peroxide anion to hydrogen peroxide. This demonstrates the activity of an oxidoreductase is regulated by a chemical decision to perform electron and proton transfers in concert though the basis of such chemistry has not been experimentally explored.

Comparison of other human MnSOD point mutants and MnSOD from other species suggests that the precise position of Gln143 affects the extent of product inhibition. We obtained neutron diffraction data to 2.3 Å resolution on a perdeuterated Trp161Phe MnSOD crystal cryotrapped with the peroxide ligand. The Trp161Phe slightly perturbs the packing at the active site leading to a shift of the Gln143 position and helped enrich the product-inhibited complex. The position of the peroxide molecule coincides with the X-ray counterpart. From the neutron diffraction data, X-ray absorption spectra, and quantum mechanical calculations, we have derived (1) the chemical significance of the Gln143 position for inhibition, (2) the protonation and electronics conditions leading to product inhibition, and (3) the proton transfer array used to protonate the peroxo-Mn complex and relieve inhibition. The work sets a foundation for investigating CPET mechanisms of oxidoreductases that are largely unknown. 

View Abstract 596


Jahaun Azadmanesh, Univ. Nebraska Medical Center Omaha, NE 

Additional Author(s)

WIlliam Lutz, University of Nebraska Medical Center Omaha, NE 
Leighton Coates, Oak Ridge National Laboratory Oak Ridge, TN 
Kevin Weiss, Oak Ridge National Laboratory Oak Ridge, TN 
Gloria Borgstahl, The Eppley Inst For Cancer Res Omaha, NE 

The TELSAM Protein Polymer significantly Improves the Speed of Crystallization of target proteins

The development of novel crystallization approaches that require less time, effort, and expense can significantly increase the success rate of target proteins crystallization and accelerate structure determination of many biotechnology and disease-relevant proteins. Specifically, improved crystallization methods will accelerate study of the molecular mechanisms of disease. Polymer forming crystallization chaperones (PFCCs) are a potentially better type of crystallization chaperone. One potential PFCC is the sterile alpha motif domain (SAM) of the human Translocation ETS Leukemia protein. In this study, the effectiveness of TELSAM protein polymers to reliably form well-diffracting crystals of Capillary Morphogenesis Gene 2 (CMG2) is investigated. CMG2 is involved in cancer, where its overexpression is associated with increased tumor grade and poor patient survival. The long-term goal of this research is to develop crystallization methods that could lead to diffraction-quality crystals from greater than 70% of targeted proteins of interest. A TELSAM-target protein fusion can crystallize more rapidly than the same target protein alone. In this work the results on CMG2 alone, 1TEL-flex-CMG2, 1TEL-flex-CMG2+PGM, 1TEL-flex-DARPin will be presented. TELSAM accelerates the rate of crystal formation by as much as 27-fold versus the target protein alone, likely by using avidity to stabilize weak crystal contacts made by the target protein. In addition, TELSAM-target protein fusions can form well-ordered, diffracting crystals using flexible TELSAM-target linkers. The TELSAM polymers themselves need not directly touch one another in the crystal lattice to make crystal lattice. We conclude that TELSAM is a powerful crystallization chaperone warranting future investigation. 

View Abstract 605


sara soleimani

Additional Author(s)

Supeshala Sarath Nawarathnage
Parag Gajjar, Brigham Young University Provo, UT 
Braydan Bezzant, Brigham Young University Provo, UT 
Moriah Longhurst, Brigham Young University Provo, UT 
Tobin Smith, Brigham Young University Provo, UT 
Maria Pedroza, Brigham Young University Provo, UT 
Seth Brown, Brigham Young University Provo, UT 
Tzanko Duokov, Macromolecular Crystallography Group, Structural Molecular Biology Resource, SSRL Menlo Park, CA 
James Moody
Diana Ramirez, California State Univesity, Chico Chico, CA 

Structural Science Awakens – with a splash of water to the (inter)face

Water molecules play crucial roles in biology. However, as water energetics are difficult to characterize experimentally, they are often ignored in ligand discovery or computational approaches are used in lieu to approximate their contributions to ligand binding. Here we describe an experimentally-driven approach in which we utilize variable-temperature, high-resolution crystallography and calorimetry to characterize the contributions of individual water molecules to ligand binding. By probing water organization via a protein mutation, we show that water networks can make dominant contributions to ligand binding. Further perturbation of water networks with ligand modifications provides pragmatic insights into relative changes in the free energy of binding. Overall, the work highlights the importance of water molecules in understanding and designing protein-ligand interactions. 

View Abstract 575


Marcus Fischer, St. Jude Children’s Research Hospital Memphis, TN 

Using (3+1)D Space to Investigate a Modulated Superstructure Mystery

Two commensurately modulated structures (PDB entries 4n3e (Sliwiak et al., 2015) and 6sjj (Smietanska et al., 2020)) were solved using translational noncrystallographic symmetry (tNCS). The data required the use of large supercells, sevenfold and ninefold, respectively, to properly index the reflections. Commensurately modulated structures can be challenging to solve. Molecular-replacement software such as Phaser can detect tNCS and either handle it automatically or, for more challenging situations, allow the user to enter a tNCS vector, which the software then uses to place the components. Although this approach has been successful in solving these types of challenging structures, it does not make it easy to understand the underlying modulation in the structure or how these two superstructures are related. The mystery was how could a slight change in crystallization condition have resulted in the observed large change in the size of the superstructure (from 7x to 9x) with no significantly observed large changes in the chains composing the superstructure. A way to investigate this problem is to view the atoms and associated parameters as following periodic atomic modulation functions (AMFs) in higher dimensional space, and what is being observed in these superstructures are the points where these higher dimensional AMFs intersect physical 3D space (Lovelace & Borgstahl, 2021). In this case, although the two 3D structures, with a sevenfold and a ninefold superstructure, seem to be quite different, describing those structures within the higher dimensional superspace approach makes a strong case that they are closely related, as they show very similar AMFs and can be described with one unique (3+1)D structure. With this approach, the addition of the small molecule caused only a small change (q Vector) to the intersection of the (3+1)D structure with 3D space. We find the modulation is easier to understand, especially when represented as an animated gif (Fig. 1).

Lovelace, J. J. & Borgstahl, G. E. O. (2021). Acta Crystallographica Section D 77.
Sliwiak, J., Dauter, Z., Kowiel, M., McCoy, A. J., Read, R. J. & Jaskolski, M. (2015). Acta Crystallogr D Biol Crystallogr 71, 829-843.
Smietanska, J., Sliwiak, J., Gilski, M., Dauter, Z., Strzalka, R., Wolny, J. & Jaskolski, M. (2020). Acta Crystallographica Section D 76, 653-667.

View Abstract 609


Jeffrey Lovelace, Univ. of Nebraska Medical Center Omaha, NE 

Additional Author

Gloria Borgstahl, The Eppley Inst For Cancer Res Omaha, NE 

Coffee Break

Peptidoglycan binding by a pocket on the accessory NTF2-domain of Pgp2 directs helical cell shape of Campylobacter jejuni

Every year, over 600 million people worldwide contract campylobacteriosis, a bacterial food-borne gastroenteritis primarily caused by Campylobacter jejuni. The helical cell shape of C. jejuni, a key colonization factor, is determined by the structure of the peptidoglycan (PG) layer. The helical structure of PG is determined by Pgp2, a LD-carboxypeptidase that cleaves the terminal D-Ala residue from both monomeric and cross-linked PG tetrapeptides. The interaction interface between Pgp2 and PG to select sites for peptide trimming is unknown. Here, we report a 1.6 Å resolution crystal structure that contains a conserved LD-carboxypeptidase domain and a previously uncharacterized domain with an NTF2-like fold (NTF2). We identified a pocket in the NTF2 domain formed by conserved residues that is located approximately 40 Å from the LD-carboxypeptidase active site. Site-directed mutagenesis combined with NMR-monitored titration studies were used to define the interaction interfaces of Pgp2 with several PG fragments, which bound to the active site within the LD-carboxypeptidase domain and the pocket of the NTF2 domain. We propose a model for Pgp2 binding to PG strands involving both the LD-carboxypeptidase and the NTF2 domains to guide catalytic activity to induce a helical cell shape. 

View Abstract 702


Chang Sheng-Huei Lin Vancouver

Additional Author(s)

Anson Chan, University of British Columbia
Jenny Vermeulen, University of British Columbia
Jacob Brockerman, University of British Columbia
Arvind Soni, University of British Columbia
Martin Tanner, University of British Columbia
Erin Gaynor, University of British Columbia
Lawrence McIntosh, University of British Columbia
Jean-Pierre Simorre, University of Grenoble Alpes
Michael Murphy

Structural analysis of fibrillar polymorphs in AD (Alzheimer Disease) brain tissue and Pair distribution function of associated fibrils

X-ray microdiffraction in the SAXS (Small Angle X-ray scattering) and WAXS (Wide Angle X-ray scattering) regimes can be used to probe the structural organization of amyloid and NFT (Neurofibrillary tangle) deposits in thin section of tissue from AD subjects. The Wide-angle X-ray scattering from these deposits is dominated by a layer line at 4.7 Å spacing which provides insight into fibrillar structure. The polymorphism of these structures is being analyzed using data from the SAXS regime that generates information about cross-sectional shape/ size and cylindrical pair distribution function of fibrils involved.

The cylindrical pair distribution function differs from the conventional pair distribution function derived from solution scattering in that it corresponds to a histogram of interatomic distances projected on a plane perpendicular to fiber axis. In this study, the cylindrical pair distribution function is being utilized to determine the maximum diameters of fibrils and fibrillar aggregates within intact tissue. This work is resulting in mapping of structural variations of pathological protein deposits within brain tissue which will contribute to a better understanding of the molecular basis of the progression of AD.

This work is supported by NIH Grant number R21AG068972. 

View Abstract 615


Prakash Nepal, Dept. of Bioengineering Boston, MA 

Additional Author(s)

Abdullah Al Bashit, Northeastern University Medford, MA 
Lee Makowski, Northeastern University Boston, MA 

Solving protein structure using highly realistic diffraction photographs generated from MD trajectory of crystalline lysozyme

We have designed and implemented an MD-based pipeline to emulate the entire process of solving crystallographic structures of biomolecules. As a starting point, we have used the structure of lysozyme solved in-house (tetragonal lattice, resolution 2.1 Å). Using this structure, we constructed a supercell containing 5×5×5=125 crystal unit cells and explicit interstitial solvent (SPC/E water). The supercell was used as a periodic boundary box to record 0.3 μs MD trajectory of crystalline lysozyme at room temperature employing Amber 18 software with ff14SB force field. A series of snapshots from this trajectory were then used to simulate a set of diffraction photographs. Normally, crystallographic diffraction pattern is calculated using FFT-based methods or, alternatively, direct summation methods, producing an array of structure factors rather than diffraction photographs per se. In our study we take a different approach. Specifically, we treat each atom in the system (including bulk water) as a scattering center, applying the fundamental Huygens-Fresnel principle to calculate the complete diffraction image of the crystal (not limited to the diffraction spots). The example of such highly realistic MD-based diffraction photograph is shown in Figure 1.

Having generated an array of 180 diffraction images, we then used the standard suite of crystallography programs, HKL2000/XDS, Coot and Phenix, to process these images, extract structure factors and ultimately calculate protein coordinates using the experimental crystal structure as a molecular replacement model. The procedure has been successful, resulting in Rwork=0.205 and Rfree=0.264. The recovered lysozyme coordinates are within 0.57 Å of the original structure (see Figure 2) and within 0.25 Å of the average MD coordinates. Thus, we have demonstrated that long state-of-the-art MD trajectory of protein crystal can be used to emulate the entire process of crystallographic structure determination at the unparalleled level of realism, leading to an accurate structural model. We envisage that in the future this procedure can be used to interrogate the relationship between protein internal dynamics and crystallographic variables (e.g. B-factors), to improve computational tools used in the field of biomolecular crystallography and to validate and benchmark different MD force fields used for biomolecular simulations.

This study is supported by the joint NSFC-RSF grant to Yi Xue (award 32061133011) and Nikolai Skrynnikov (award 21-44-00033). 

View Abstract 657


Yi Xue, Tsinghua University Beijing

Additional Author(s)

Ning Liu, School of Life Sciences, Tsinghua University Beijing
Nikolai Skrynnikov, St. Petersburg State University, St. Petersburg, Russia; Purdue University, West Lafayette, IN, USA St. Petersburg

Structures of Synthetic Nanobodies in Complex with SARS-CoV-2 Spike or Receptor-Binding Domain Provide Insights for Developing Therapeutics and Vaccines

Structures of Synthetic Nanobodies in Complex with SARS-CoV-2 Spike or Receptor-Binding Domain Provide Insights for Developing Therapeutics and Vaccines

Jiansheng Jiang1, Javeed Ahmad1, Kannan Natarajan1, Lisa F. Boyd1, Rick Huang2, Allison Zeher2, and David H. Margulies1

1Molecular Biology Section, Laboratory of Immune System Biology, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892
2Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892


Rapid structure determination of antibodies or nanobodies complexed with SARS-CoV-2 spike or its receptor-binding domain (RBD) by X-ray crystallography and cryo-EM provide structural insight for developing effective therapies and vaccines to combat the current pandemic and recent circulating variants of the virus. We report crystal structures of synthetic nanobodies (sybody) Sb16, Sb45, and Sb68 bound to the RBD of SARS-CoV-2: two binary complexes of Sb16/RBD and Sb45/RBD; a ternary complex of Sb45+RBD+Sb68 [1,2]; and a cryo-EM structure of Sb45 bound to the trimeric spike protein. Sb16 and Sb45 bind the RBD at the angiotension converting enzyme (ACE)2 interface, positioning their complementarity determining region (CDR) CDR2 and CDR3 loops in opposite orientations diametrically, and recognizing distinct epitopic areas. The buried surface area (BSA) at sybody/RBD interfaces is consistent with neutralization activity. A large shift of the CDR2 loop is identified by comparing Sb16 alone (unliganded) with Sb16/RBD (complex) structures. Structural analysis based on these and more than 100 recent structures of antibodies, nanobodies, and sybodies in complex with RBD or spike reveals the vital role of CDR loops and their dynamic flexibility in recognizing epitopes. Superposition of these crystal structures onto cryo-EM structures of trimeric spike protein indicates that the three sybodies can access both "up" and "down" conformations of the mature spike. Cryo-EM maps of Sb45/spike complexes confirm these models. SARS-CoV-2 variants B.1.1.7 (UK) and B.1.351 (South Africa), which have increased affinity for the cellular receptor ACE2, appear to be less susceptible to human antibodies elicited by current mRNA vaccines (Moderna and Pfizer-BioNTech). Our binding studies indicate that the common RBD escape mutant N501Y is still bound by Sb16 and Sb45, although with lower affinity. The E484K mutation, however, has a much lower affinity for Sb16 and Sb45 due to the loss of hydrogen-bonding networks. Insights provided by structural analysis of these complexes of sybodies with RBD or spike may contribute to developing new therapeutics and to updating the vaccine.

(Supported by the Intramural research program of the NIAID/NIH and NIH IRP Cryo-EM Facility)

[1] Ahmad, Jiang & Margulies et al. 2021, bioRxiv,
[2] Walter & Seeger et al. 2020, bioRxiv, 

View Abstract 480


Jiansheng Jiang, NIAID/NIH Bethesda, MD 

Additional Author(s)

Javeed Dhobi, National Institute of Health Rockville, MD 
Kannan Natarajan, LISB/NIAID/NIH Bethesda, MD 
Lisa F. Boyd, LISB/NIAID/NIH Bethesda, MD 
Rick Huang, Laboratory of Cell Biology/CCR/NCI/NIH Bethesda, MD 
Allison Zeher, Laboratory of Cell Biology/CCR/NCI/NIH Bethesda, MD 
David H. Margulies, LISB/NIAID/NIH Bethesda, MD