Optics
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Open Access
Abstract: As Diamond Light Source embraces the move towards becoming a fourth-generation light source its optics will be required to perform under increasingly demanding conditions. Foremost amongst these conditions will be the increasing power densities the optics are subjected to and the reducing real estate they have to perform in. With these new challenges comes the need for greater understanding of how optics are assembled and how consistently the activity is carried out. In this paper, the effect of bolt pretension during assembly of monochromators on distortion of the optical surface is investigated through numerical simulation. The results reveal skewed convex distortion of the optical surface in the meridional direction when uneven clamping force is applied, highlighting the importance of taking the potential for distortion of the optical surface due to clamping force into consideration.
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May 2022
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B16-Test Beamline
Optics
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Open Access
Abstract: Aberrations introduced during fabrication degrade the performance of X-ray optics and their ability to achieve diffraction limited focusing. Corrective optics can counteract these errors by introducing wavefront perturbations prior to the optic which cancel out the distortions. Here we demonstrate two-dimensional wavefront correction of an aberrated Kirkpatrick-Baez mirror pair using adaptable refractive structures. The resulting two-dimensional wavefront is measured using hard X-ray ptychography to recover the complex probe wavefield with high spatial resolution and model the optical performance under coherent conditions. The optical performance including the beam caustic, focal profile and wavefront error is examined before and after correction with both mirrors found to be diffraction limited after correcting. The results will be applicable to a wide variety of high numerical aperture X-ray optics aiming to achieve diffraction limited focussing using low emittance sources.
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May 2022
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I03-Macromolecular Crystallography
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Tika R.
Malla
,
Lennart
Brewitz
,
Dorian-Gabriel
Muntean
,
Hiba
Aslam
,
C. David
Owen
,
Eidarus
Salah
,
Anthony
Tumber
,
Petra
Lukacik
,
Claire
Strain-Damerell
,
Halina
Mikolajek
,
Martin
Walsh
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[27088]
Open Access
Abstract: The SARS-CoV-2 main protease (Mpro) is a medicinal chemistry target for COVID-19 treatment. Given the clinical efficacy of β-lactams as inhibitors of bacterial nucleophilic enzymes, they are of interest as inhibitors of viral nucleophilic serine and cysteine proteases. We describe the synthesis of penicillin derivatives which are potent Mpro inhibitors and investigate their mechanism of inhibition using mass spectrometric and crystallographic analyses. The results suggest that β-lactams have considerable potential as Mpro inhibitors via a mechanism involving reaction with the nucleophilic cysteine to form a stable acyl–enzyme complex as shown by crystallographic analysis. The results highlight the potential for inhibition of viral proteases employing nucleophilic catalysis by β-lactams and related acylating agents.
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May 2022
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I19-Small Molecule Single Crystal Diffraction
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Diamond Proposal Number(s):
[15768]
Open Access
Abstract: Biological systems employ multimetallic assemblies to achieve a range of functions. Here we demonstrate the preparation of metal–organic cages that contain either homobimetallic or heterobimetallic vertices. These vertices are constructed using 2-formyl-6-diphenylphosphinopyridine, which forms ligands that readily bridge between a pair of metal centers, thus enforcing the formation of bimetallic coordination motifs. Two pseudo-octahedral homometallic MI12L4 cages (MI = CuI or AgI) were prepared, with a head-to-head configuration of their vertices confirmed by X-ray crystallography and multinuclear NMR for AgI. The phosphino-pyridine subcomponent also enabled the formation of a class of octanuclear CdII4CuI4L4 tetrahedral cages, representing an initial example of self-assembled cages containing well-defined heterobimetallic vertices.
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May 2022
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B21-High Throughput SAXS
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14739, 29790, 20229]
Open Access
Abstract: IMP dehydrogenase(IMPDH) is an essential enzyme that catalyzes the rate-limiting step in the guanine nucleotide pathway. In eukaryotic cells, GTP binding to the regulatory domain allosterically controls the activity of IMPDH by a mechanism that is fine-tuned by post-translational modifications and enzyme polymerization. Nonetheless, the mechanisms of regulation of IMPDH in bacterial cells remain unclear. Using biochemical, structural, and evolutionary analyses, we demonstrate that, in most bacterial phyla, (p)ppGpp compete with ATP to allosterically modulate IMPDH activity by binding to a, previously unrecognized, conserved high affinity pocket within the regulatory domain. This pocket was lost during the evolution of Proteobacteria, making their IMPDHs insensitive to these alarmones. Instead, most proteobacterial IMPDHs evolved to be directly modulated by the balance between ATP and GTP that compete for the same allosteric binding site. Altogether, we demonstrate that the activity of bacterial IMPDHs is allosterically modulated by a universally conserved nucleotide-controlled conformational switch that has divergently evolved to adapt to the specific particularities of each organism. These results reconcile the reported data on the crosstalk between (p)ppGpp signaling and the guanine nucleotide biosynthetic pathway and reinforce the essential role of IMPDH allosteric regulation on bacterial GTP homeostasis.
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May 2022
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I04-Macromolecular Crystallography
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Pamela
Sweeney
,
Ashleigh
Galliford
,
Abhishek
Kumar
,
Dinesh
Raju
,
Naveen B.
Krishna
,
Emmajay
Sutherland
,
Caitlin J.
Leo
,
Gemma
Fisher
,
Roopa
Lalitha
,
Likith
Muthuraj
,
Gladstone
Sigamani
,
Verena
Oehler
,
Silvia
Synowsky
,
Sally L.
Shirran
,
Tracey M.
Gloster
,
Clarissa M.
Czekster
,
Pravin
Kumar
,
Rafael G.
Da Silva
Diamond Proposal Number(s):
[14980]
Open Access
Abstract: The enzyme m1A22-tRNA methyltransferase (TrmK) catalyses the transfer of a methyl group to the N1 of adenine 22 in bacterial tRNAs. TrmK is essential for Staphylococcus aureus survival during infection, but has no homologue in mammals, making it a promising target for antibiotic development. Here we characterize the structure and function of S. aureus TrmK using X-ray crystallography, binding assays, and molecular dynamics simulations. We report crystal structures for the S. aureus TrmK apoenzyme as well as in complexes with methyl donor SAM and co-product product SAH. Isothermal titration calorimetry showed that SAM binds to the enzyme with favourable but modest enthalpic and entropic contributions, whereas SAH binding leads to an entropic penalty compensated for by a large favourable enthalpic contribution. Molecular dynamics simulations point to specific motions of the C-terminal domain being altered by SAM binding, which might have implications for tRNA recruitment. In addition, activity assays for S. aureus TrmK-catalysed methylation of A22 mutants of tRNALeu demonstrate that the adenine at position 22 is absolutely essential. In-silico screening of compounds suggested the multi-functional organic toxin plumbagin as a potential inhibitor of TrmK, which was confirmed by activity measurements. Furthermore, LC-MS data indicated the protein was covalently modified by one equivalent of the inhibitor, and proteolytic digestion coupled with LC-MS identified Cys92 in the vicinity of the SAM-binding site as the sole residue modified. These results identify a cryptic binding pocket of S. aureus TrmK, laying a foundation for future structure-based drug discovery.
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May 2022
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I03-Macromolecular Crystallography
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Nikolaos
Georgakopoulos
,
Sandeep
Talapatra
,
Dina
Dikovskaya
,
Sharadha
Dayalan Naidu
,
Maureen
Higgins
,
Jemma
Gatliff
,
Aysel
Ayhan
,
Roxani
Nikoloudaki
,
Marjolein
Schaap
,
Klara
Valko
,
Farideh
Javid
,
Albena T.
Dinkova-Kostova
,
Frank
Kozielski
,
Geoffrey
Wells
Diamond Proposal Number(s):
[12305]
Open Access
Abstract: Inhibitors of Kelch-like ECH-associated protein 1 (Keap1) increase the activity of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) by stalling its ubiquitination and degradation. This enhances the expression of genes encoding proteins involved in drug detoxification, redox homeostasis, and mitochondrial function. Nrf2 activation offers a potential therapeutic approach for conditions including Alzheimer’s and Parkinson’s diseases, vascular inflammation, and chronic obstructive airway disease. Non-electrophilic Keap1-Nrf2 protein–protein interaction (PPI) inhibitors may have improved toxicity profiles and different pharmacological properties to cysteine-reactive electrophilic inhibitors. Here, we describe and characterize a series of phenyl bis-sulfonamide PPI inhibitors that bind to Keap1 at submicromolar concentrations. Structural studies reveal that the compounds bind to Keap1 in a distinct “peptidomimetic” conformation that resembles the Keap1-Nrf2 ETGE peptide complex. This is different to other small molecule Keap1-Nrf2 PPI inhibitors, including bicyclic aryl bis-sulfonamides, offering a starting point for new design approaches to Keap1 inhibitors.
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May 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Jingxu
Guo
,
Bin
Liu
,
Midory
Thorikay
,
Minmin
Yu
,
Xiaoyan
Li
,
Zhen
Tong
,
Richard M.
Salmon
,
Randy
Read
,
Peter
Ten Dijke
,
Nicholas W.
Morrell
,
Wei
Li
Diamond Proposal Number(s):
[21426]
Open Access
Abstract: Heterozygous mutations in BMPR2 (bone morphogenetic protein (BMP) receptor type II) cause pulmonary arterial hypertension. BMPRII is a receptor for over 15 BMP ligands, but why BMPR2 mutations cause lung-specific pathology is unknown. To elucidate the molecular basis of BMP:BMPRII interactions, we report crystal structures of binary and ternary BMPRII receptor complexes with BMP10, which contain an ensemble of seven different BMP10:BMPRII 1:1 complexes. BMPRII binds BMP10 at the knuckle epitope, with the A-loop and β4 strand making BMPRII-specific interactions. The BMPRII binding surface on BMP10 is dynamic, and the affinity is weaker in the ternary complex than in the binary complex. Hydrophobic core and A-loop interactions are important in BMPRII-mediated signalling. Our data reveal how BMPRII is a low affinity receptor, implying that forming a signalling complex requires high concentrations of BMPRII, hence mutations will impact on tissues with highest BMPR2 expression such as the lung vasculature.
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May 2022
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Krios II-Titan Krios II at Diamond
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Diamond Proposal Number(s):
[23268]
Open Access
Abstract: Most eukaryotic messenger RNAs (mRNAs) are processed at their 3′ end by the cleavage and polyadenylation specificity factor (CPF/CPSF). CPF mediates the endonucleolytic cleavage of the pre-mRNA and addition of a polyadenosine (poly(A)) tail, which together define the 3′ end of the mature transcript. The activation of CPF is highly regulated to maintain the fidelity of RNA processing. Here, using cryo-EM of yeast CPF, we show that the Mpe1 subunit directly contacts the polyadenylation signal sequence in nascent pre-mRNA. The region of Mpe1 that contacts RNA also promotes the activation of CPF endonuclease activity and controls polyadenylation. The Cft2 subunit of CPF antagonizes the RNA-stabilized configuration of Mpe1. In vivo, the depletion or mutation of Mpe1 leads to widespread defects in transcription termination by RNA polymerase II, resulting in transcription interference on neighboring genes. Together, our data suggest that Mpe1 plays a major role in accurate 3′ end processing, activating CPF, and ensuring timely transcription termination.
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May 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Nicky J.
Willis
,
William
Mahy
,
James
Sipthorp
,
Yuguang
Zhao
,
Hannah L.
Woodward
,
Benjamin N.
Atkinson
,
Elliott D.
Bayle
,
Fredrik
Svensson
,
Sarah
Frew
,
Fiona
Jeganathan
,
Amy
Monaghan
,
Stefano
Benvegnù
,
Sarah
Jolly
,
Luca
Vecchia
,
Reinis R.
Ruza
,
Svend
Kjær
,
Steven
Howell
,
Ambrosius P.
Snijders
,
Magda
Bictash
,
Patricia C.
Salinas
,
Jean-Paul
Vincent
,
E. Yvonne
Jones
,
Paul
Whiting
,
Paul V.
Fish
Diamond Proposal Number(s):
[16814, 19446]
Open Access
Abstract: Notum is a carboxylesterase that suppresses Wnt signaling through deacylation of an essential palmitoleate group on Wnt proteins. There is a growing understanding of the role Notum plays in human diseases such as colorectal cancer and Alzheimer’s disease, supporting the need to discover improved inhibitors, especially for use in models of neurodegeneration. Here, we have described the discovery and profile of 8l (ARUK3001185) as a potent, selective, and brain-penetrant inhibitor of Notum activity suitable for oral dosing in rodent models of disease. Crystallographic fragment screening of the Diamond-SGC Poised Library for binding to Notum, supported by a biochemical enzyme assay to rank inhibition activity, identified 6a and 6b as a pair of outstanding hits. Fragment development of 6 delivered 8l that restored Wnt signaling in the presence of Notum in a cell-based reporter assay. Assessment in pharmacology screens showed 8l to be selective against serine hydrolases, kinases, and drug targets.
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May 2022
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