I03-Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
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Diamond Proposal Number(s):
[29990, 36446]
Open Access
Abstract: The red fluorescent protein mCherry is one of the most widely used fluorescent proteins in biology. Here, we have changed the chromophore chemistry by converting the thioether group of M66 to a thiol group through mutation to cysteine. The new variant, termed mCoral (due to its orange fluorescence hue), has similar brightness to mCherry but improved resistance to hydrogen peroxide. The variant is also responsive to pH with low and high pKa forms that have distinct spectral properties, which DFT analysis suggests is due to protonation state changes in the newly introduced thiol group, as well as the phenol group. The structure of mCoral reveals that the M66C mutation creates a space within the β-barrel structure that is filled by a water molecule, which makes new polar interactions, including the backbone carbonyl group of F65. Molecular dynamics simulations suggest that this additional water molecule, together with local solvation around the chromophore, could play a role in promoting planarity of the full conjugated system comprising the chromophore. The mCoral chromophore makes slightly more H-bonds with water than mCherry. The main water exit point for mCherry is also narrower in mCoral, providing a potential explanation for increased resistance to hydrogen peroxide. Overall, a small structural change to mCherry has resulted in a new fluorescent protein with potentially useful characteristics and an insight into the role of dynamics and water in defining the structure–function relationship in red fluorescent proteins.
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Dec 2025
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I24-Microfocus Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
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Open Access
Abstract: Multi-crystal processing of X-ray diffraction data has become highly automated to keep pace with the current high-throughput capabilities afforded by beamlines. A significant challenge, however, is the automated clustering of such data based on subtle differences such as ligand binding or conformational shifts. Intensity-based hierarchical clustering has been shown to be a viable method of identifying such subtle structural differences, but the interpretation of the resulting dendrograms is difficult to automate. Using isomorphous crystals of bovine, porcine and human insulin, the existing clustering methods in the multi-crystal processing software xia2.multiplex were validated and their limits were tested. It was determined that weighting the pairwise correlation coefficient calculations with the intensity uncertainties was required for accurate calculation of the pairwise correlation coefficient matrix (correlation clustering) and dimension optimization was required when expressing this matrix as a set of coordinates representing data sets (cosine-angle clustering). Finally, the introduction of the OPTICS spatial density-based clustering algorithm into DIALS allowed the automatic output of species-pure clusters of bovine, porcine and human insulin data sets.
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Jun 2025
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Abstract: α-actinin (ACTN) is a pivotal member of the actin-binding protein family, crucial for the anchoring and organization of actin filaments within the cytoskeleton. Four isoforms of α-actinin exist: two non-muscle isoforms (ACTN1 and ACTN4) primarily associated with actin stress fibers and focal adhesions, and two muscle-specific isoforms (ACTN2 and ACTN3) localized to the Z-disk of the striated muscle. Although these isoforms share structural similarities, they exhibit distinct functional characteristics that reflect their specialized roles in various tissues. Genetic variants in α-actinin isoforms have been implicated in a range of pathologies, including cardiomyopathies, thrombocytopenia, and non-cardiovascular diseases, such as nephropathy. However, the precise impact of these genetic variants on the α-actinin structure and their contribution to disease pathogenesis remains poorly understood. This review provides a comprehensive overview of the structural and functional attributes of the four α-actinin isoforms, emphasizing their roles in actin crosslinking and sarcomere stabilization. Furthermore, we present detailed structural modeling of select ACTN1 and ACTN2 variants to elucidate mechanisms underlying disease pathogenesis, with a particular focus on macrothrombocytopenia and hypertrophic cardiomyopathy. By advancing our understanding of α-actinin’s role in both normal cellular function and disease states, this review lays the groundwork for future research and the development of targeted therapeutic interventions.
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Mar 2025
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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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I04-Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
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Diamond Proposal Number(s):
[19946, 23570]
Open Access
Abstract: The third complementary-determining regions of the heavy-chain (CDR3H) variable regions (VH) of some cattle antibodies are highly extended, consisting of 48 or more residues. These `ultralong' CDR3Hs form β-ribbon stalks that protrude from the surface of the antibody with a disulfide cross-linked knob region at their apex that dominates antigen interactions over the other CDR loops. The structure of the Fab fragment of a naturally paired bovine ultralong antibody (D08), identified by single B-cell sequencing, has been determined to 1.6 Å resolution. By swapping the D08 native light chain with that of an unrelated antigen-unknown ultralong antibody, it is shown that interactions between the CDR3s of the variable domains potentially affect the fine positioning of the ultralong CDR3H; however, comparison with other crystallographic structures shows that crystalline packing is also a major contributor. It is concluded that, on balance, the exact positioning of ultralong CDR3H loops is most likely to be due to the constraints of crystal packing.
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Jul 2024
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Katy
Cornish
,
Jiandong
Huo
,
Luke
Jones
,
Parul
Sharma
,
Joseph W.
Thrush
,
Sahar
Abdelkarim
,
Anja
Kipar
,
Siva
Ramadurai
,
Miriam
Weckener
,
Halina
Mikolajek
,
Sai
Liu
,
Imogen
Buckle
,
Eleanor
Bentley
,
Adam
Kirby
,
Ximeng
Han
,
Stephen M.
Laidlaw
,
Michelle
Hill
,
Lauren
Eyssen
,
Chelsea
Norman
,
Audrey
Le Bas
,
John
Clarke
,
William
James
,
James P.
Stewart
,
Miles
Carroll
,
James
Naismith
,
Raymond J.
Owens
Diamond Proposal Number(s):
[27031]
Open Access
Abstract: The Omicron strains of SARS-CoV-2 pose a significant challenge to the development of effective antibody-based treatments as immune evasion has compromised most available immune therapeutics. Therefore, in the ‘arms race’ with the virus, there is a continuing need to identify new biologics for the prevention or treatment of SARS-CoV-2 infections. Here, we report the isolation of nanobodies that bind to the Omicron BA.1 spike protein by screening nanobody phage display libraries previously generated from llamas immunized with either the Wuhan or Beta spike proteins. The structure and binding properties of three of these nanobodies (A8, H6 and B5-5) have been characterized in detail providing insight into their binding epitopes on the Omicron spike protein. Trimeric versions of H6 and B5-5 neutralized the SARS-CoV-2 variant of concern BA.5 both in vitro and in the hamster model of COVID-19 following nasal administration. Thus, either alone or in combination could serve as starting points for the development of new anti-viral immunotherapeutics.
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Jun 2024
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I04-Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
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Abstract: Cytochromes c'-α are nitric oxide (NO)-binding heme proteins derived from bacteria that can thrive in a wide range of temperature environments. Studies of mesophilic Alcaligenes xylosoxidans cytochrome c'-α (AxCP-α) have revealed an unusual NO-binding mechanism involving both heme faces, in which NO first binds to form a distal hexa-coordinate Fe(II)-NO (6cNO) intermediate and then displaces the proximal His to form a proximal penta-coordinate Fe(II)-NO (5cNO) final product. Here we characterize a thermally stable cytochrome c'-α from thermophilic Hydrogenophilus thermoluteolus (PhCP-α) to understand how protein thermal stability affects NO binding. Electron paramagnetic and resonance Raman spectroscopies reveal the formation of a PhCP-α 5cNO product, with time-resolved (stopped-flow) UV-visible absorbance indicating the involvement of a 6cNO intermediate. Relative to AxCP-α, the rates of 6cNO and 5cNO formation in PhCP-α are ∼11-fold and ∼13-fold lower, respectively. Notably, X-ray crystal structures of PhCP-α in the presence and absence of NO suggest that the sluggish formation of the proximal 5cNO product results from conformational rigidity: the Arg-132 residue (adjacent to the proximal His ligand) is held in place by a salt bridge between Arg-75 and Glu-135 (an interaction not present in AxCP-α or a psychrophilic counterpart). Overall, our data provide fresh insights into structural factors controlling NO binding in heme proteins, including 5cNO complexes relevant to eukaryotic NO sensors.
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Jun 2024
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VMXi-Versatile Macromolecular Crystallography in situ
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Open Access
Abstract: A considerable bottleneck in serial crystallography at XFEL and synchrotron sources is the efficient production of large quantities of homogenous, well diffracting microcrystals. Efficient high-throughput screening of batch-grown microcrystals and the determination of ground-state structures from different conditions is thus of considerable value in the early stages of a project. Here, a highly sample-efficient methodology to measure serial crystallography data from microcrystals by raster scanning within standard in situ 96-well crystallization plates is described. Structures were determined from very small quantities of microcrystal suspension and the results were compared with those from other sample-delivery methods. The analysis of a two-dimensional batch crystallization screen using this method is also described as a useful guide for further optimization and the selection of appropriate conditions for scaling up microcrystallization.
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Apr 2024
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VMXi-Versatile Macromolecular Crystallography in situ
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Open Access
Abstract: We present a protocol for the crystallization of proteins using the crystallization facility in the Research Complex at Harwell and subsequent in situ X-ray crystallographic data collection from crystals within the plates at Diamond's Versatile Macromolecular Crystallography in situ (VMXi) beamline. We describe sample requirements, crystallization protocols, and data collection guidelines.
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Mar 2024
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I04-Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
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Halina
Mikolajek
,
Juan
Sanchez-Weatherby
,
James
Sandy
,
Richard J.
Gildea
,
Ivan
Campeotto
,
Harish
Cheruvara
,
John D.
Clarke
,
Toshana
Foster
,
Sotaro
Fujii
,
Ian T.
Paulsen
,
Bhumika S.
Shah
,
Michael A.
Hough
Open Access
Abstract: The utility of X-ray crystal structures determined under ambient-temperature conditions is becoming increasingly recognized. Such experiments can allow protein dynamics to be characterized and are particularly well suited to challenging protein targets that may form fragile crystals that are difficult to cryo-cool. Room-temperature data collection also enables time-resolved experiments. In contrast to the high-throughput highly automated pipelines for determination of structures at cryogenic temperatures widely available at synchrotron beamlines, room-temperature methodology is less mature. Here, the current status of the fully automated ambient-temperature beamline VMXi at Diamond Light Source is described, and a highly efficient pipeline from protein sample to final multi-crystal data analysis and structure determination is shown. The capability of the pipeline is illustrated using a range of user case studies representing different challenges, and from high and lower symmetry space groups and varied crystal sizes. It is also demonstrated that very rapid structure determination from crystals in situ within crystallization plates is now routine with minimal user intervention.
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Jul 2023
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