I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[34223]
Open Access
Abstract: DNA polymerase β (Pol β) is an important polymerase that functions in DNA repair within the Base Excision Repair and Non-Homologous End-Joining pathways. It is estimated to function in the repair of up to 50,000 DNA lesions per cell per day, within the base excision repair pathway (BER). Given the significant role Pol β plays in repairing DNA, genetic variants of Pol β have the potential to perturb repair, resulting in mutation accumulation which can potentiate cancer formation. Here we identify an unstudied human germline variant of Pol β, the S180R variant (rs1585898410), which introduces a significant amino acid alteration within the dNTP binding pocket of the enzyme. We demonstrate that S180R is a low fidelity variant of Pol β due to its loss of the ability to discriminate correct nucleotides from incorrect nucleotides. We also show that this variant exhibits a much slower rate of nucleotide incorporation, which could further disrupt repair capacity in vivo. Structural data reveal that this variant not only has structural changes that may disrupt dNTP binding but also a loss of primer terminus positioning and dynamic flexibility of the fingers domain in the binary state, which likely are driving the low fidelity of S180R Pol β. This study highlights the importance of binary positioning and nucleotide coordinating residues for maintaining nucleotide selectivity, polymerase function, and fidelity. It also emphasizes the importance of further study of this human germline Pol β variant in vivo.
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Jan 2026
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9948]
Abstract: Copper is an essential micronutrient for bacteria, needed for important copper enzymes such as terminal respiratory oxidases. However, in excess, copper is toxic to bacteria. This toxicity is caused by its ability to bind tightly to proteins through the formation of Cu-Cys and Cu-His bonds. To control toxicity, bacteria have evolved homeostatic systems to safely handle the copper they need while efficiently sequestering and effluxing excess copper ions. We previously found that GapA, the abundant glycolytic glyceraldehyde-3-phosphate dehydrogenase enzyme in the Staphylococcus aureus cytosol, becomes associated with copper within cells cultured in medium containing excess copper. We found that this association of GapA with copper resulted in inhibition of its enzyme activity. Here, we have characterised this binding of copper ions to S. aureus GapA in vitro to determine the mechanism of copper inhibition of GAPDH. We found that purified recombinant GapA binds a single Cu(I) ion with high affinity. Crystallographic structural determination showed association of this copper ion with two active site residues, Cys151 and His178, known to be important for catalysis. This observation was confirmed by characterisation of mutated variants lacking these residues, which showed reduced ability to bind Cu(I) ions. Finally, we demonstrated that the cytosolic copper metallochaperone, CopZ, exhibits a tighter affinity for Cu(I) and can remove copper from GapA in vitro. Together, our data demonstrate the mechanism by which excess copper binds to the S. aureus GapA enzyme and irreversibly inhibit its activity and how the cellular homeostasis system is capable of resolving this inhibition.
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Dec 2025
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[32728]
Open Access
Abstract: Certain members of the bacterial cytochrome P450 152 family (CYP152) are peroxygenases that catalyse the decarboxylation of fatty acids into terminal olefins making them attractive biocatalysts for biofuel production. To date, the characterisation of decarboxylating CYP152s has mainly focused on their reaction with saturated fatty acid substrates. CYP152s are often co-purified with a bound substrate, which is generally removed before further experiments are conducted. In the present work we identified that heterologous over-expressed CYP152 from Staphylococcus aureus (OleTSa) is co-purified with the trans-monounsaturated C18:1 fatty acid, elaidic acid. We report the spectral, thermodynamic and kinetic characteristics of OleTSa bound to both elaidic acid and its saturated counterpart, stearic acid. Despite differing spectral profiles, metabolic and kinetic studies reveal that OleTSa is capable of decarboxylating elaidic acid, converting it to heptadeca-1,8-diene following addition of hydrogen peroxide, at the same rate and chemoselectivity as the conversion of stearic acid to 1-heptadecane. The X-ray crystal structure of the as purified OleTSa in complex with elaidic acid is also presented, allowing for several key residues to be identified for site-directed mutagenesis studies. The influence of the site-directed variants on C18:0 and C18:1 product formation, binding thermodynamics and kinetics have been investigated, showing that while spectral differences occur as a likely result of perturbing the binding pocket, this does not alter the chemoselectivity of the enzyme. Our work provides important insights into the mechanism of decarboxylation of an unsaturated fatty acid substrate by OleTSa potentially expanding the sustainable substrate space available for CYP152s.
Graphical abstract
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Oct 2025
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I03-Macromolecular Crystallography
I23-Long wavelength MX
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Open Access
Abstract: Glycine N-acyltransferase (GLYAT; EC 2.3.1.13, Accession ID: AAI12537) is a key enzyme in mammalian homeostasis that has been linked to several pathologies in humans, including cancer. Here we report the first crystal structure of a member of the GLYAT family, both in the apo form as well as bound to benzoyl-CoA. Binding of glycine could be inferred from an acetate molecule from the crystallization solution. A detailed analysis of its structure and the effects of mutations of key residues helped elucidate the catalytic mechanism, showing a general base-catalyzed reaction driven by a potential low-barrier hydrogen bond (LBHB) formed between the catalytic Glu-His dyad. This work will aid further studies of GLYAT and other members of the family.
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Sep 2025
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[15292]
Abstract: Bradyrhizobium japonicum USDA110 is a widely used microorganism in the formulation of bioinoculants for soybean crops, harboring a copper-containing nitrite reductase with low enzymatic activity. The activity of BjNirK at pH 6.5 was higher compared to that at pH 8.0, regardless of the presence of either physiological or artificial electron donors. Thermal shift assays reveal that the enzyme is more stable at pH 6.5 than at pH 8.0. X-ray structural data reveals that the funnel for substrate entry shows a wider cavity when compared to other class I NirK structures. Furthermore, the presence of an additional channel for proton provision is observed, in addition to the primary and secondary proton channels. The T2Cu active site can accommodate one or two water molecules, resulting in a tetra- or pentacoordinated metal site, respectively. The structural data correlates well with both optical visible and resonance Raman spectroscopies, denoting a strong blue character of the T1Cu site in both solid and solution states. Furthermore, EPR-monitored redox titration reveals that the catalytic rate is not constrained by T1Cu−T2Cu intraprotein electron transfer reaction at either pH 6.5 or pH 8.0. Additionally, bioinformatics studies indicate that the interaction between the enzyme and the electron donor is not pH dependent. These two observations suggest that the low activity of BjNirK is not caused by inefficient donor−enzyme interaction or impaired electron transfer. The present results suggest that the structural architecture and enzyme properties in rhizobia are designed to ensure low activity, a trait that is particularly advantageous for symbiosis.
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May 2025
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[2373]
Open Access
Abstract: The de novo design of α-helical coiled-coil peptides is advanced. Using established sequence-to-structure relationships, it is possible to generate various coiled-coil assemblies with predictable numbers and orientations of helices. Here, we target new assemblies, namely, A3B3 heterohexamer α-helical barrels. These designs are based on pairs of sequences with three heptad repeats (abcdefg), programmed with a = Leu, d = Ile, e = Ala, and g = Ser, and b = c = Glu to make the acidic (A) chains and b = c = Lys in the basic (B) chains. These design rules ensure that the desired oligomeric state and stoichiometry are readily achieved. However, controlling the orientation of neighboring helices (parallel or antiparallel) is less straightforward. Surprisingly, we find that assembly and helix orientation are sensitive to the length of the overhang between helices. To study this, cyclically permutated peptide sequences with three heptad repeats (the register) in the peptide sequences were analyzed. Peptides starting at g (g-register) form a parallel 6-helix barrel in solution and in an X-ray crystal structure, whereas the b- and c-register peptides form an antiparallel complex. In lieu of experimental X-ray structures for b- and c-register peptides, AlphaFold-Multimer is used to predict atomistic models. However, considerably more sampling than the default value is required to match the models and the experimental data, as many confidently predicted and plausible models are generated with incorrect helix orientations. This work reveals the previously unknown influence of the heptad register on helical overhang and the orientation of α-helical coiled-coil peptides and provides insights for the modeling of oligopeptide coiled-coil complexes with AlphaFold.
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Apr 2025
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[260673, 29121]
Abstract: The cytochrome P450 family of enzymes are key players in the metabolism of foreign substances in the body, including pharmaceutical compounds, and therefore important to take into consideration during drug development. The main human isoform is CYP3A4, a highly flexible protein that can act on a diverse set of substances and that is inhibited by compounds varying greatly in size. To accompany the different ligands, substantial conformational changes occur that transform the active-site binding pocket between a collapsed form and various open states. A large body of biophysical data including high-resolution structures are available but there is still a lack of understanding of the dynamic properties of CYP3A4. Here, we present the first room-temperature structure of CYP3A4 solved by serial crystallography. The structure is overall very similar to structures solved at cryo-temperature of the un-bound form of the enzyme including the conformation of the active-site lid. We observe that loops are better defined at room-temperature despite the lower resolution of this structure. Based on an internal distance matrix analysis of a large set of CYP3A4 structures, we conclude that the crystal form rather than temperature is determining for how the structures cluster. Finally, a workflow for generating microcrystals suitable for fixed-target serial crystallography data collection is described. This work lays the foundation for future studies of ligand-induced dynamics and structural transitions during the catalytic reaction of CYP3A4.
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Apr 2025
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I14-Hard X-ray Nanoprobe
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Diamond Proposal Number(s):
[27292]
Open Access
Abstract: Soil aggregation is a dynamic process influenced by physical, chemical and biological factors; however, their individual and combined effect on the formation and turnover of aggregates is not well understood. The aim of this study was to examine incorporation of fresh litter inputs of different physicochemical properties including their carbon-to-nitrogen (C/N) ratio – maize (C/N = 12) and straw (C/N = 103) - into aggregates, de novo formed from mineral soil with or without the presence of microbiota. Using rare-earth element oxides, we labelled structures formed during a four-week incubation with a single litter type and traced their incorporation into newly formed aggregates after mixing them together and incubating for a subsequent seven-week period. To visualize them, we used synchrotron-based X-ray fluorescence microspectroscopy, which allowed us to demonstrate that presence of the plant-derived particulate organic matter was the key factor for the aggregate formation. Within the timescale of the experiment, neither microbial abundance nor the community composition had any significant effect. However, the relative increase in straw-associated soil in aggregates larger than 250 μm provided support for our hypothesis regarding impact of carbon-rich organic matter on macroaggregation, likely via promotion of fungal growth and hyphal enmeshing. Phospholipid fatty acid analysis further confirmed relatively higher abundance of fungi in macroaggregates in straw-containing soil. All in all, our study provides insights into the initial stages of aggregate formation following litter additions and development of associated microbial community. The spatial analysis enabled by the X-ray fluorescence microspectroscopy enabled visualization of internal aggregate structures, shedding light on the processes involved, which is not possible with bulk analysis alone.
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Dec 2024
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[26835]
Abstract: During Staphylococcus aureus infections, reactive oxygen species cause DNA damage, including nucleotide base modification. After removal of the defective base, excision repair requires an endonuclease IV (Nfo), which hydrolyzes the phosphodiester bond 5′ to the abasic nucleotide. This class of enzymes, typified by the enzyme from Escherichia coli, contains a catalytic site with three metal ions, previously reported to be all Zn2+. The 1.05 Å structure of Nfo from the Gram-positive organism S. aureus (SaNfo) revealed two inner Fe2+ ions and one Zn2+ as confirmed by dispersive anomalous difference maps. SaNfo has a previously undescribed water molecule liganded to Fe1 forming an octahedral coordination geometry and hydrogen bonded to Tyr33, an active site residue conserved in many Gram-positive bacteria, but which is Phe in Gram-negative species that coordinate Zn2+ at the corresponding site. The 1.9 Å structure of E. coli Nfo (EcNfo), purified without added metals, revealed that metal 2 is Fe2+ and not Zn2+. Octahedral coordination for the sites occupied by Fe2+ suggests a stereoselective mechanism for differentiating between Fe2+ and Zn2+ in this enzyme class. Kinetics and an inhibitor competition assay of SaNfo reveal product inhibition (or slow product release), especially at low ionic strength, caused in part by a Lys-rich DNA binding loop present in SaNfo and Gram-positive species but not in EcNfo. Biological significance of the slow product release is discussed. Catalytic activity in vitro is optimal at 300 mM NaCl, which is consistent with the halotolerant phenotype of S. aureus.
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Dec 2024
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
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Abstract: Multifunctionality, processivity, and thermostability are critical for the cost-effective enzymatic saccharification of non-food plant biomass polymers such as β-glucans, celluloses, and xylans to generate biofuels and other valuable products. We present molecular insights into a processive multifunctional endo-1,3-1,4-β-D-glucanase (Tt_End5A) from the hyperthermophilic bacterium Thermogutta terrifontis. Tt_End5A demonstrated activities against a broad spectrum of β-polysaccharides, including barley glucan, lichenan, carboxymethyl cellulose, regenerated amorphous cellulose (RAC), Avicel, xylan, laminarin, mannan, curdlan, xanthan, and various chromogenic substrates at pH 7 and temperatures ranging from 70-80°C. The enzyme exhibited a high level of processivity on RAC and retained over 90% activity at 80 °C for an extended period, indicating exceptional thermal stability. The 1.20 Å crystal structure of the Tt_End5A catalytic domain revealed an archetypal glycoside hydrolase family 5 (GH5) catalytic TIM-(β/α)8-barrel, supplemented with additional β-strands, elongated α-helices, and a rare cis-non-Pro (His481-cis-Ala482) peptide. A large central cleft was observed in the 3D structure, which is likely related to the enzyme's multifunctionality and processivity. The catalytic domain is preceded by a novel N-terminal multivalent carbohydrate-binding module (CBM) that enhances the enzymatic degradation of insoluble polysaccharides. Mutagenesis studies, ligand interaction analyses, and the structurally conserved positions of E329 and E448 in Tt_End5A suggest that these residues function as the proton donor and nucleophile in the catalytic mechanism. Owing to its multifunctionality and processivity, Tt_End5A can reduce the need for multiple saccharification enzymes to generate fermentable sugars from plant biomass for bioethanol production. Additionally, it holds promise for applications in the pharmaceutical, feed, and food industries.
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Dec 2024
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