| 1. |
- Aggarwal, Swati, et al.
(author)
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A protocol for production of perdeuterated OmpF porin for neutron crystallography
- 2021
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In: Protein Expression and Purification. - : Elsevier BV. - 1046-5928. ; 188
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Journal article (peer-reviewed)abstract
- Hydrogen atoms are at the limit of visibility in X-ray structures even at high resolution. Neutron macromolecular crystallography (NMX) is an unambiguous method to locate hydrogens and study the significance of hydrogen bonding interactions in biological systems. Since NMX requires very large crystals, very few neutron structures of proteins have been determined yet. In addition, the most common hydrogen isotope 1H gives rise to significant background due to its large incoherent scattering cross-section. Therefore, it is advantageous to substitute as many hydrogens as possible with the heavier isotope 2H (deuterium) to reduce the sample volume requirement. While the solvent exchangeable hydrogens can be substituted by dissolving the protein in heavy water, complete deuterium labelling – perdeuteration – requires the protein to be expressed in heavy water with a deuterated carbon source. In this work, we developed an optimized method for large scale production of deuterium-labelled bacterial outer membrane protein F (OmpF) for NMX. OmpF was produced using deuterated media with different carbon sources. Mass spectrometry verified the integrity and level of deuteration of purified OmpF. Perdeuterated OmpF crystals diffracted X-rays to a resolution of 1.9 Å. This work lays the foundation for structural studies of membrane protein by neutron diffraction in future.
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| 2. |
- Budayova-Spano, Monika, et al.
(author)
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Large crystal growth for neutron protein crystallography
- 2020
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In: Neutron Crystallography in Structural Biology. - : Elsevier. - 1557-7988 .- 0076-6879. ; 634, s. 21-46
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Book chapter (peer-reviewed)abstract
- The use of neutron protein crystallography (NPX) is expanding rapidly, with most structures determined in the last decade. This growth is stimulated by a number of developments, spanning from the building of new NPX beamlines to the availability of improved software for structure refinement. The main bottleneck preventing structural biologists from adding NPX to the suite of methods commonly used is the large volume of the individual crystals required for a successful experiment. A survey of deposited NPX structures in the Protein Data Bank shows that about two-thirds came from crystals prepared using vapor diffusion, while batch and dialysis-based methods all-together contribute to most of the remaining one-third. This chapter explains the underlying principles of these protein crystallization methods and provides practical examples that may help others to successfully prepare large crystals for NPX.
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| 3. |
- Clabbers, Max T. B., et al.
(author)
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Visualizing drug binding interactions using microcrystal electron diffraction
- 2020
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In: Communications biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Journal article (peer-reviewed)abstract
- Visualizing ligand binding interactions is important for structure-based drug design and fragment-based screening methods. Rapid and uniform soaking with potentially reduced lattice defects make small macromolecular crystals attractive targets for studying drug binding using microcrystal electron diffraction (MicroED). However, so far no drug binding interactions could unambiguously be resolved by electron diffraction alone. Here, we use MicroED to study the binding of a sulfonamide inhibitor to human carbonic anhydrase isoform II (HCA II). We show that MicroED data can efficiently be collected on a conventional transmission electron microscope from thin hydrated microcrystals soaked with the clinical drug acetazolamide (AZM). The data are of high enough quality to unequivocally fit and resolve the bound inhibitor. We anticipate MicroED can play an important role in facilitating in-house fragment screening for drug discovery, complementing existing methods in structural biology such as X-ray and neutron diffraction. Clabbers et al. utilize MicroED to present the structure of both apo and inhibitor-bound human carbonic anhydrase II at a high resolution to clearly identify the interaction of the inhibitor, acetazolamide. This method eases the difficulty of both crystallizing the protein and soaking the inhibitor in a smaller protein crystal.
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| 4. |
- Donahue, Marie L., et al.
(author)
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Using social media to understand drivers of urban park visitation in the Twin Cities, MN
- 2018
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In: Landscape and Urban Planning. - : Elsevier BV. - 0169-2046 .- 1872-6062. ; 175, s. 1-10
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Journal article (peer-reviewed)abstract
- Green space and parks in urban environments provide a range of ecosystem services and public benefits. However, planners and park managers can lack tools and resources to gather local information on how parks are used and what makes them desirable places for recreation and a wide variety of uses. Traditional survey methods to monitor park use and user preferences can be costly, time consuming, and challenging to apply at scale. Here, we overcome this limitation by using geotagged social media data to assess patterns of visitation to urban and peti-urban green space across park systems in the metropolitan area of the Twin Cities, Minnesota, USA. We find that parks with nearby water features, more amenities, greater accessibility from the presence of trails, and that are located within neighborhoods with higher population density, are associated with higher rates of visitation. As cities grow and shifts in demographics occur, more responsive management of public green space will become increasingly important to ensure urban parks provide ecosystem services and meet users' needs. Using social media data to rapidly assess park use at a lower cost than traditional surveys has the potential to inform public green space management with targeted information on user behavior and values of urban residents.
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| 5. |
- Fisher, Zoe, et al.
(author)
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Biological Structures
- 2017
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In: Experimental Methods in the Physical Sciences. - 1079-4042. ; 49, s. 1-75
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Book chapter (peer-reviewed)abstract
- Neutron scattering methods are excellent for probing the detailed structure of biological systems, which rely on the intricate interplay of a large number of molecules from proteins and nucleic acids to lipids, hormones, and metabolites. With recent instrument developments and emergence of both new neutron sources and techniques, many biological systems that are not yet amenable to characterization by neutron scattering will become accessible in the near future, which will allow new experiments to be developed with a range of biologically relevant samples, offering new insights in life science. In this chapter, we will describe neutron methods for biological structure characterization on different length scales from atomic resolution to macromolecular length scales-up to micrometers. The dynamics of biological molecules are described by Seydel in Chapter 2 of this thematic volume.
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| 6. |
- Fisher, Zoe, et al.
(author)
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Neutron Crystallographic Studies Reveal Hydrogen Bond and Water-Mediated Interactions between a Carbohydrate-Binding Module and Its Bound Carbohydrate Ligand.
- 2015
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In: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 54:42, s. 6435-6438
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Journal article (peer-reviewed)abstract
- Carbohydrate-binding modules (CBMs) are key components of many carbohydrate-modifying enzymes. CBMs affect the activity of these enzymes by modulating bonding and catalysis. To further characterize and study CBM-ligand binding interactions, neutron crystallographic studies of an engineered family 4-type CBM in complex with a branched xyloglucan ligand were conducted. The first neutron crystal structure of a CBM-ligand complex reported here shows numerous atomic details of hydrogen bonding and water-mediated interactions and reveals the charged state of key binding cleft amino acid side chains.
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| 7. |
- Hamstead, Zoé A., et al.
(author)
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Geolocated social media as a rapid indicator of park visitation and equitable park access
- 2018
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In: Computers, Environment and Urban Systems. - : Elsevier BV. - 0198-9715 .- 1873-7587. ; 72, s. 38-50
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Journal article (peer-reviewed)abstract
- Understanding why some parks are used more regularly or intensely than others can inform ways in which urban parkland is developed and managed to meet the needs of a rapidly expanding urban population. Although geolocated social media (GSM) indicators have been used to examine park visitation rates, studies applying this approach are generally limited to flagship parks, national parks, or a small subset of urban parks. Here, we use geolocated Flickr and Twitter data to explore variation in use across New York City's 2143 diverse parks and model visitation based on spatially-explicit park characteristics and facilities, neighborhood-level accessibility features and neighborhood-level demographics. Findings indicate that social media activity in parks is positively correlated with proximity to public transportation and bike routes, as well as particular park characteristics such as water bodies, athletic facilities, and impervious surfaces, but negatively associated with green space and increased proportion of minority ethnicity and minority race in neighborhoods in which parks are located. Contrary to previous studies which describe park visitation as a form of nature-based recreation, our findings indicate that the kinds of green spaces present in many parks may not motivate visitation. From a social equity perspective, our findings may imply that parks in high-minority neighborhoods are not as accessible, do not accommodate as many visitors, and/or are of lower quality than those in low-minority neighborhoods. These implications are consistent with previous studies showing that minority populations disproportionately experience barriers to park access. In applying GSM data to questions of park access, we demonstrate a rapid, big data approach for providing information crucial for park management in a way that is less resource-intensive than field surveys.
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| 8. |
- Koruza, Katarina, et al.
(author)
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Biophysical Characterization of Cancer-Related Carbonic Anhydrase IX
- 2020
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In: International Journal of Molecular Sciences. - : MDPI AG. - 1422-0067. ; 21:15
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Journal article (peer-reviewed)abstract
- Upregulation of carbonic anhydrase IX (CA IX) is associated with several aggressive forms of cancer and promotes metastasis. CA IX is normally constitutively expressed at low levels in selective tissues associated with the gastrointestinal tract, but is significantly upregulated upon hypoxia in cancer. CA IX is a multi-domain protein, consisting of a cytoplasmic region, a single-spanning transmembrane helix, an extracellular CA catalytic domain, and a proteoglycan-like (PG) domain. Considering the important role of CA IX in cancer progression and the presence of the unique PG domain, little information about the PG domain is known. Here, we report biophysical characterization studies to further our knowledge of CA IX. We report the 1.5 Å resolution crystal structure of the wild-type catalytic domain of CA IX as well as small angle X-ray scattering and mass spectrometry of the entire extracellular region. We used matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to characterize the spontaneous degradation of the CA IX PG domain and confirm that it is only the CA IX catalytic domain that forms crystals. Small angle X-ray scattering analysis of the intact protein indicates that the PG domain is not randomly distributed and adopts a compact distribution of shapes in solution. The observed dynamics of the extracellular domain of CA IX could have physiological relevance, including observed cleavage and shedding of the PG domain.
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| 9. |
- Koruza, Katarina, et al.
(author)
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From initial hit to crystal optimization with microseeding of human carbonic anhydrase IX—A case study for neutron protein crystallography
- 2018
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In: Crystals. - : MDPI AG. - 2073-4352. ; 8:11
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Journal article (peer-reviewed)abstract
- Human carbonic anhydrase IX (CA IX) is a multi-domain membrane protein that is therefore difficult to express or crystalize. To prepare crystals that are suitable for neutron studies, we are using only the catalytic domain of CA IX with six surface mutations, named surface variant (SV). The crystallization of CA IX SV, and also partly deuterated CA IX SV, was enabled by the use of microseed matrix screening (MMS). Only three drops with crystals were obtained after initial sparse matrix screening, and these were used as seeds in subsequent crystallization trials. Application of MMS, commercial screens, and refinement resulted in consistent crystallization and diffraction-quality crystals. The crystallization protocols and strategies that resulted in consistent crystallization are presented. These results demonstrate not only the use of MMS in the growth of large single crystals for neutron studies with defined conditions, but also that MMS enabled re-screening to find new conditions and consistent crystallization success.
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| 10. |
- Koruza, Katarina, et al.
(author)
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Using neutron crystallography to elucidate the basis of selective inhibition of carbonic anhydrase by saccharin and a derivative
- 2019
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In: Journal of Structural Biology. - : Elsevier BV. - 1047-8477. ; 205:2, s. 147-154
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Journal article (peer-reviewed)abstract
- Up-regulation of carbonic anhydrase IX (CA IX) expression is an indicator of metastasis and associated with poor cancer patient prognosis. CA IX has emerged as a cancer drug target but development of isoform-specific inhibitors is challenging due to other highly conserved CA isoforms. In this study, a CA IXmimic construct was used (CA II with seven point mutations introduced, to mimic CA IX active site) while maintaining CA II solubility that make it amenable to crystallography. The structures of CA IXmimic unbound and in complex with saccharin (SAC) and a saccharin-glucose conjugate (SGC) were determined using joint X-ray and neutron protein crystallography. Previously, SAC and SGC have been shown to display CA isoform inhibitor selectivity in assays and X-ray crystal structures failed to reveal the basis of this selectivity. Joint X-ray and neutron crystallographic studies have shown active site residues, solvent, and H-bonding re-organization upon SAC and SGC binding. These observations highlighted the importance of residues 67 (Asn in CA II, Gln in CA IX) and 130 (Asp in CA II, Arg in CA IX) in selective CA inhibitor targeting.
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