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23 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I04-Macromolecular Crystallography	Structural insights into the complex of oncogenic KRas4B G12V and Rgl2, a RalA/B activator Mishal Tariq , Teppei Ikeya , Naoyuki Togashi , Louise Fairall , Shun Kamei , Sannojah Mayooramurugan , Lauren R, Abbott , Anab Hasan , Carlos Bueno-Alejo , Sakura Sukegawa , Beatriz Romartinez-Alonso , Miguel Angel Muro Campillo , Andrew J. Hudson , Yutaka Ito , John W. R. Schwabe , Cyril Dominguez , Kayoko Tanaka Life Science Alliance 7 DOI: 10.26508/lsa.202302080 Diamond Proposal Number(s): [19880] Open Access Show Abstract ▼ Abstract: About a quarter of total human cancers carry mutations in Ras isoforms. Accumulating evidence suggests that small GTPases, RalA, and RalB, and their activators, Ral guanine nucleotide exchange factors (RalGEFs), play an essential role in oncogenic Ras-induced signalling. We studied the interaction between human KRas4B and the Ras association (RA) domain of Rgl2 (Rgl2RA), one of the RA-containing RalGEFs. We show that the G12V oncogenic KRas4B mutation changes the interaction kinetics with Rgl2RA. The crystal structure of the KRas4BG12V: Rgl2RA complex shows a 2:2 heterotetramer where the switch I and switch II regions of each KRasG12V interact with both Rgl2RA molecules. This structural arrangement is highly similar to the HRasE31K:RALGDSRA crystal structure and is distinct from the well-characterised Ras:Raf complex. Interestingly, the G12V mutation was found at the dimer interface of KRas4BG12V with its partner. Our study reveals a potentially distinct mode of Ras:effector complex formation by RalGEFs and offers a possible mechanistic explanation for how the oncogenic KRas4BG12V hyperactivates the RalA/B pathway.	Oct 2023
I22-Small angle scattering & Diffraction	A cross-sectional study of bone nanomechanics in hip fracture and aging Richard Stavri , Tabitha Tay , Crispin C. Wiles , Erica Di Federico , Oliver Boughton , Shaocheng Ma , Angelo Karunaratne , John H. Churchwell , Rajarshi Bhattacharya , Nicholas J. Terrill , Justin P. Cobb , Ulrich Hansen , Richard L. Abel Life 13 DOI: 10.3390/life13061378 Diamond Proposal Number(s): [17664, 13337] Open Access Show Abstract ▼ Abstract: Bone mechanics is well understood at every length scale except the nano-level. We aimed to investigate the relationship between bone nanoscale and tissue-level mechanics experimentally. We tested two hypotheses: (1) nanoscale strains were lower in hip fracture patients versus controls, and (2) nanoscale mineral and fibril strains were inversely correlated with aging and fracture. A cross-sectional sample of trabecular bone sections was prepared from the proximal femora of two human donor groups (aged 44–94 years): an aging non-fracture control group (n = 17) and a hip-fracture group (n = 20). Tissue, fibril, and mineral strain were measured simultaneously using synchrotron X-ray diffraction during tensile load to failure, then compared between groups using unpaired t-tests and correlated with age using Pearson’s correlation. Controls exhibited significantly greater peak tissue, mineral, and fibril strains than the hip fracture (all p < 0.05). Age was associated with a decrease in peak tissue (p = 0.099) and mineral (p = 0.004) strain, but not fibril strain (p = 0.260). Overall, hip fracture and aging were associated with changes in the nanoscale strain that are reflected at the tissue level. Data must be interpreted within the limitations of the observational cross-sectional study design, so we propose two new hypotheses on the importance of nanomechanics. (1) Hip fracture risk is increased by low tissue strain, which can be caused by low collagen or mineral strain. (2) Age-related loss of tissue strain is dependent on the loss of mineral but not fibril strain. Novel insights into bone nano- and tissue-level mechanics could provide a platform for the development of bone health diagnostics and interventions based on failure mechanisms from the nanoscale up.	Jun 2023
B21-High Throughput SAXS I04-Macromolecular Crystallography	Structure of SALL4 zinc finger domain reveals link between AT-rich DNA binding and Okihiro syndrome James A. Watson , Raphaël Pantier , Uma Jayachandran , Kashyap Chhatbar , Beatrice Alexander-Howden , Valdeko Kruusvee , Michal Prendecki , Adrian Bird , Atlanta G. Cook Life Science Alliance 6 DOI: 10.26508/lsa.202201588 Open Access Show Abstract ▼ Abstract: Spalt-like 4 (SALL4) maintains vertebrate embryonic stem cell identity and is required for the development of multiple organs, including limbs. Mutations in SALL4 are associated with Okihiro syndrome, and SALL4 is also a known target of thalidomide. SALL4 protein has a distinct preference for AT-rich sequences, recognised by a pair of zinc fingers at the C-terminus. However, unlike many characterised zinc finger proteins, SALL4 shows flexible recognition with many different combinations of AT-rich sequences being targeted. SALL4 interacts with the NuRD corepressor complex which potentially mediates repression of AT-rich genes. We present a crystal structure of SALL4 C-terminal zinc fingers with an AT-rich DNA sequence, which shows that SALL4 uses small hydrophobic and polar side chains to provide flexible recognition in the major groove. Missense mutations reported in patients that lie within the C-terminal zinc fingers reduced overall binding to DNA but not the preference for AT-rich sequences. Furthermore, these mutations altered association of SALL4 with AT-rich genomic sites, providing evidence that these mutations are likely pathogenic.	Jan 2023
I03-Macromolecular Crystallography	H2S biogenesis by cystathionine beta-synthase: mechanism of inhibition by aminooxyacetic acid and unexpected role of serine Maria Petrosino , Karim Zuhra , Jola Kopec , Andrew Hutchin , Csaba Szabo , Tomas Majtan Cellular And Molecular Life Sciences 79 DOI: 10.1007/s00018-022-04479-9 Diamond Proposal Number(s): [29346] Open Access Show Abstract ▼ Abstract: Cystathionine beta-synthase (CBS) is a pivotal enzyme of the transsulfuration pathway responsible for diverting homocysteine to the biosynthesis of cysteine and production of hydrogen sulfide (H2S). Aberrant upregulation of CBS and overproduction of H2S contribute to pathophysiology of several diseases including cancer and Down syndrome. Therefore, pharmacological CBS inhibition has emerged as a prospective therapeutic approach. Here, we characterized binding and inhibitory mechanism of aminooxyacetic acid (AOAA), the most commonly used CBS inhibitor. We found that AOAA binds CBS tighter than its respective substrates and forms a dead-end PLP-bound intermediate featuring an oxime bond. Surprisingly, serine, but not cysteine, replaced AOAA from CBS and formed an aminoacrylate reaction intermediate, which allowed for the continuation of the catalytic cycle. Indeed, serine rescued and essentially normalized the enzymatic activity of AOAA-inhibited CBS. Cellular studies confirmed that AOAA decreased H2S production and bioenergetics, while additional serine rescued CBS activity, H2S production and mitochondrial function. The crystal structure of AOAA-bound human CBS showed a lack of hydrogen bonding with residues G305 and Y308, found in the serine-bound model. Thus, AOAA-inhibited CBS could be reactivated by serine. This difference may be important in a cellular environment in multiple pathophysiological conditions and may modulate the CBS-inhibitory activity of AOAA. In addition, our results demonstrate additional complexities of using AOAA as a CBS-specific inhibitor of H2S biogenesis and point to the urgent need to develop a potent, selective and specific pharmacological CBS inhibitor.	Aug 2022
B21-High Throughput SAXS I04-1-Macromolecular Crystallography (fixed wavelength)	Structure and function of an atypical homodimeric actin capping protein from the malaria parasite Abris Adam Bendes , Petri Kursula , Inari Kursula Cellular And Molecular Life Sciences 79 DOI: 10.1007/s00018-021-04032-0 Open Access Show Abstract ▼ Abstract: Apicomplexan parasites, such as Plasmodium spp., rely on an unusual actomyosin motor, termed glideosome, for motility and host cell invasion. The actin filaments are maintained by a small set of essential regulators, which provide control over actin dynamics in the different stages of the parasite life cycle. Actin filament capping proteins (CPs) are indispensable heterodimeric regulators of actin dynamics. CPs have been extensively characterized in higher eukaryotes, but their role and functional mechanism in Apicomplexa remain enigmatic. Here, we present the first crystal structure of a homodimeric CP from the malaria parasite and compare the homo- and heterodimeric CP structures in detail. Despite retaining several characteristics of a canonical CP, the homodimeric Plasmodium berghei (Pb)CP exhibits crucial differences to the canonical heterodimers. Both homo- and heterodimeric PbCPs regulate actin dynamics in an atypical manner, facilitating rapid turnover of parasite actin, without affecting its critical concentration. Homo- and heterodimeric PbCPs show partially redundant activities, possibly to rescue actin filament capping in life cycle stages where the β-subunit is downregulated. Our data suggest that the homodimeric PbCP also influences actin kinetics by recruiting lateral actin dimers. This unusual function could arise from the absence of a β-subunit, as the asymmetric PbCP homodimer lacks structural elements essential for canonical barbed end interactions suggesting a novel CP binding mode. These findings will facilitate further studies aimed at elucidating the precise actin filament capping mechanism in Plasmodium.	Feb 2022
I03-Macromolecular Crystallography	PSTPIP1-LYP phosphatase interaction: structural basis and implications for autoinflammatory disorders Jose Antonio Manso , Tamara Marcos , Virginia Ruiz-Martín , Javier Casas , Pablo Alcon , Mariano Sánchez Crespo , Yolanda Bayón , Jose M. De Pereda , Andrés Alonso Cellular And Molecular Life Sciences 79 DOI: 10.1007/s00018-022-04173-w Diamond Proposal Number(s): [10121] Open Access Show Abstract ▼ Abstract: Mutations in the adaptor protein PSTPIP1 cause a spectrum of autoinflammatory diseases, including PAPA and PAMI; however, the mechanism underlying these diseases remains unknown. Most of these mutations lie in PSTPIP1 F-BAR domain, which binds to LYP, a protein tyrosine phosphatase associated with arthritis and lupus. To shed light on the mechanism by which these mutations generate autoinflammatory disorders, we solved the structure of the F-BAR domain of PSTPIP1 alone and bound to the C-terminal homology segment of LYP, revealing a novel mechanism of recognition of Pro-rich motifs by proteins in which a single LYP molecule binds to the PSTPIP1 F-BAR dimer. The residues R228, D246, E250, and E257 of PSTPIP1 that are mutated in immunological diseases directly interact with LYP. These findings link the disruption of the PSTPIP1/LYP interaction to these diseases, and support a critical role for LYP phosphatase in their pathogenesis.	Feb 2022
	The high-throughput production of membrane proteins James Birch , Andrew Quigley Emerging Topics In Life Sciences 6 DOI: 10.1042/ETLS20210196 Open Access Show Abstract ▼ Abstract: Membrane proteins, found at the junctions between the outside world and the inner workings of the cell, play important roles in human disease and are used as biosensors. More than half of all therapeutics directly affect membrane protein function while nanopores enable DNA sequencing. The structural and functional characterisation of membrane proteins is therefore crucial. However, low levels of naturally abundant protein and the hydrophobic nature of membrane proteins makes production difficult. To maximise success, high-throughput strategies were developed that rely upon simple screens to identify successful constructs and rapidly exclude those unlikely to work. Parameters that affect production such as expression host, membrane protein origin, expression vector, fusion-tags, encapsulation reagent and solvent composition are screened in parallel. In this way, constructs with divergent requirements can be produced for a variety of structural applications. As structural techniques advance, sample requirements will change. Single-particle cryo-electron microscopy requires less protein than crystallography and as cryo-electron tomography and time-resolved serial crystallography are developed new sample production requirements will evolve. Here we discuss different methods used for the high-throughput production of membrane proteins for structural biology.	Oct 2021
B24-Cryo Soft X-ray Tomography	Single cell cryo-soft X-ray tomography shows that each Chlamydia trachomatis inclusion is a unique community of bacteria Patrick Phillips , James M. Parkhurst , Ilias Kounatidis , Chidinma Okolo , Thomas M. Fish , James H. Naismith , Martin A. Walsh , Maria Harkiolaki , Maud Dumoux Life 11 DOI: 10.3390/life11080842 Open Access Show Abstract ▼ Abstract: Chlamydiae are strict intracellular pathogens residing within a specialised membrane-bound compartment called the inclusion. Therefore, each infected cell can, be considered as a single entity where bacteria form a community within the inclusion. It remains unclear as to how the population of bacteria within the inclusion influences individual bacterium. The life cycle of Chlamydia involves transitioning between the invasive elementary bodies (EBs) and replicative reticulate bodies (RBs). We have used cryo-soft X-ray tomography to observe individual inclusions, an approach that combines 40 nm spatial resolution and large volume imaging (up to 16 µm). Using semi-automated segmentation pipeline, we considered each inclusion as an individual bacterial niche. Within each inclusion, we identifyed and classified different forms of the bacteria and confirmed the recent finding that RBs have a variety of volumes (small, large and abnormal). We demonstrate that the proportions of these different RB forms depend on the bacterial concentration in the inclusion. We conclude that each inclusion operates as an autonomous community that influences the characteristics of individual bacteria within the inclusion.	Aug 2021
B21-High Throughput SAXS	The structure and flexibility analysis of the Arabidopsis synaptotagmin 1 reveal the basis of its regulation at membrane contact sites Juan L Benavente , Dritan Siliqi , Lourdes Infantes , Laura Lagartera , Alberto Mills , Federico Gago , Noemí Ruiz-López , Miguel A Botella , Maria J. Sanchez-Barrena , Armando Albert Life Science Alliance 4 DOI: 10.26508/lsa.202101152 Diamond Proposal Number(s): [21741] Open Access Show Abstract ▼ Abstract: Non-vesicular lipid transfer at ER and plasma membrane (PM) contact sites (CS) is crucial for the maintenance of membrane lipid homeostasis. Extended synaptotagmins (E-Syts) play a central role in this process as they act as molecular tethers of ER and PM and as lipid transfer proteins between these organelles. E-Syts are proteins constitutively anchored to the ER through an N-terminal hydrophobic segment and bind the PM via a variable number of C-terminal C2 domains. Synaptotagmins (SYTs) are the plant orthologous of E-Syts and regulate the ER–PM communica- tion in response to abiotic stress. Combining different structural and biochemical techniques, we demonstrate that the binding of SYT1 to lipids occurs through a Ca2+-dependent lipid-binding site and by a site for phosphorylated forms of phosphatidylinositol, thus integrating two different molecular signals in response to stress. In addition, we show that SYT1 displays three highly flexible hinge points that provide conformational freedom to facilitate lipid extraction, protein loading, and subsequent transfer between PM and ER.	Aug 2021
I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Structural comparison of GLUT1 to GLUT3 reveal transport regulation mechanism in sugar porter family Tania Filipa Custodio , Peter Aasted Paulsen , Kelly May Frain , Bjorn Panyella Pedersen Life Science Alliance 4 DOI: 10.26508/lsa.202000858 Diamond Proposal Number(s): [23316, 17844, 13062] Show Abstract ▼ Abstract: The human glucose transporters GLUT1 and GLUT3 have a central role in glucose uptake as canonical members of the Sugar Porter (SP) family. GLUT1 and GLUT3 share a fully conserved substrate-binding site with identical substrate coordination, but differ significantly in transport affinity in line with their physiological function. Here, we present a 2.4 Å crystal structure of GLUT1 in an inward open conformation and compare it with GLUT3 using both structural and functional data. Our work shows that interactions between a cytosolic “SP motif” and a conserved “A motif” stabilize the outward conformational state and increases substrate apparent affinity. Furthermore, we identify a previously undescribed Cl− ion site in GLUT1 and an endofacial lipid/glucose binding site which modulate GLUT kinetics. The results provide a possible explanation for the difference between GLUT1 and GLUT3 glucose affinity, imply a general model for the kinetic regulation in GLUTs and suggest a physiological function for the defining SP sequence motif in the SP family.	Feb 2021

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