I03-Macromolecular Crystallography
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Andrius
Jasilionis
,
Magdalena
Plotka
,
Lei
Wang
,
Sebastian
Dorawa
,
Joanna
Lange
,
Hildegard
Watzlawick
,
Tom
Van Den Bergh
,
Bas
Vroling
,
Josef
Altenbuchner
,
Anna-Karina
Kaczorowska
,
Ehmke
Pohl
,
Tadeusz
Kaczorowski
,
Eva
Nordberg Karlsson
,
Stefanie
Freitag-Pohl
Diamond Proposal Number(s):
[18598]
Abstract: Bacteriophages encode a wide variety of cell wall disrupting enzymes that aid the viral escape in the final stages of infection. These lytic enzymes have accumulated notable interest due to their potential as novel antibacterials for infection treatment caused by multiple-drug resistant bacteria. Here, the detailed functional and structural characterization of Thermus parvatiensis prophage peptidoglycan lytic amidase AmiP, a globular Amidase_3 type lytic enzyme adapted to high temperatures is presented. The sequence and structure comparison with homologous lytic amidases reveals the key adaptation traits that ensure the activity and stability of AmiP at high temperatures. The crystal structure determined at a resolution of 1.8 Å displays a compact α/β-fold with multiple secondary structure elements omitted or shortened compared to protein structures of similar proteins. The functional characterisation of AmiP demonstrates high efficiency of catalytic activity and broad substrate specificity towards thermophilic and mesophilic bacteria strains containing Orn-type or DAP-type peptidoglycan. The here presented AmiP constitutes the most thermoactive and ultrathermostable Amidase_3 type lytic enzyme biochemically characterised with a temperature optimum at 85 °C. The extraordinary high melting temperature Tm 102.6 °C confirms fold stability up to approximately 100 °C. Furthermore, AmiP is shown to be more active over the alkaline pH range with pH optimum at pH 8.5 and tolerates NaCl up to 300 mM with the activity optimum at 25 mM NaCl. This set of beneficial characteristics suggests that AmiP can be further exploited in biotechnology.
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Jan 2023
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Sudarshan
Murthy
,
Maria Giulia
Nizi
,
Mirko M.
Maksimainen
,
Serena
Massari
,
Juho
Alaviuhkola
,
Barbara E.
Lippok
,
Chiara
Vagaggini
,
Sven T.
Sowa
,
Albert
Galera-Prat
,
Yashwanth
Ashok
,
Harikanth
Venkannagari
,
Renata
Prunskaite-Hyyryläinen
,
Elena
Dreassi
,
Bernhard
Lüscher
,
Patricia
Korn
,
Oriana
Tabarrini
,
Lari
Lehtio
Diamond Proposal Number(s):
[23346, 26794, 19951]
Open Access
Abstract: We report [1,2,4]triazolo[3,4-b]benzothiazole (TBT) as a new inhibitor scaffold, which competes with nicotinamide in the binding pocket of human poly- and mono-ADP-ribosylating enzymes. The binding mode was studied through analogues and cocrystal structures with TNKS2, PARP2, PARP14, and PARP15. Based on the substitution pattern, we were able to identify 3-amino derivatives 21 (OUL243) and 27 (OUL232) as inhibitors of mono-ARTs PARP7, PARP10, PARP11, PARP12, PARP14, and PARP15 at nM potencies, with 27 being the most potent PARP10 inhibitor described to date (IC50 of 7.8 nM) and the first PARP12 inhibitor ever reported. On the contrary, hydroxy derivative 16 (OUL245) inhibits poly-ARTs with a selectivity toward PARP2. The scaffold does not possess inherent cell toxicity, and the inhibitors can enter cells and engage with the target protein. This, together with favorable ADME properties, demonstrates the potential of TBT scaffold for future drug development efforts toward selective inhibitors against specific enzymes.
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Jan 2023
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Detectors
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J.
Correa
,
M.
Mehrjoo
,
R.
Battistelli
,
F.
Lehmkühler
,
A.
Marras
,
C. B.
Wunderer
,
T.
Hirono
,
V.
Felk
,
F.
Krivan
,
S.
Lange
,
I.
Shevyakov
,
V.
Vardanyan
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M.
Zimmer
,
M.
Hoesch
,
K.
Bagschik
,
N.
Guerrini
,
B.
Marsh
,
I.
Sedgwick
,
G.
Cautero
,
L.
Stebel
,
D.
Giuressi
,
R. H.
Menk
,
A.
Greer
,
T.
Nicholls
,
W.
Nichols
,
U.
Pedersen
,
P.
Shikhaliev
,
N.
Tartoni
,
H. J.
Hyun
,
S. H.
Kim
,
S. Y.
Park
,
K. S.
Kim
,
F.
Orsini
,
F. J.
Iguaz
,
F.
Büttner
,
B.
Pfau
,
E.
Plönjes
,
K.
Kharitonov
,
M.
Ruiz-Lopez
,
R.
Pan
,
S.
Gang
,
B.
Keitel
,
H.
Graafsma
Open Access
Abstract: The PERCIVAL detector is a CMOS imager designed for the soft X-ray regime at photon sources. Although still in its final development phase, it has recently seen its first user experiments: ptychography at a free-electron laser, holographic imaging at a storage ring and preliminary tests on X-ray photon correlation spectroscopy. The detector performed remarkably well in terms of spatial resolution achievable in the sample plane, owing to its small pixel size, large active area and very large dynamic range; but also in terms of its frame rate, which is significantly faster than traditional CCDs. In particular, it is the combination of these features which makes PERCIVAL an attractive option for soft X-ray science.
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Jan 2023
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I04-Macromolecular Crystallography
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Michelle H.
Nelson
,
Sara
Fritzell
,
Robert
Miller
,
Doreen
Werchau
,
Danielle
Van Citters
,
Anneli
Nilsson
,
Lynda
Misher
,
Lill
Ljung
,
Robert
Bader
,
Adnan
Deronic
,
Allison G.
Chunyk
,
Lena
Schultz
,
Laura A.
Varas
,
Nadia
Rose
,
Maria
Håkansson
,
Jane
Gross
,
Christina
Furebring
,
Peter
Pavlik
,
Anette
Sundstedt
,
Niina
Veitonmäki
,
Hilario J.
Ramos
,
Anna
Säll
,
Anna
Dahlman
,
David
Bienvenue
,
Laura
Von Schantz
,
Catherine J.
Mcmahan
,
Maria
Askmyr
,
Gabriela
Hernandez-Hoyos
,
Peter
Ellmark
Diamond Proposal Number(s):
[23282]
Open Access
Abstract: 4–1BB (CD137) is an activation-induced costimulatory receptor that regulates immune responses of activated CD8 T and natural killer cells, by enhancing proliferation, survival, cytolytic activity, and IFNγ production. The ability to induce potent antitumor activity by stimulating 4–1BB on tumor-specific cytotoxic T cells makes 4–1BB an attractive target for designing novel immuno-oncology therapeutics. To minimize systemic immune toxicities and enhance activity at the tumor site, we have developed a novel bispecific antibody that stimulates 4–1BB function when co-engaged with the tumor-associated antigen 5T4. ALG.APV-527 was built on the basis of the ADAPTIR bispecific platform with optimized binding domains to 4–1BB and 5T4 originating from the ALLIGATOR-GOLD human single-chain variable fragment library. The epitope of ALG.APV-527 was determined to be located at domain 1 and 2 on 4–1BB using X-ray crystallography. As shown in reporter and primary cell assays in vitro, ALG.APV-527 triggers dose-dependent 4–1BB activity mediated only by 5T4 crosslinking. In vivo, ALG.APV-527 demonstrates robust antitumor responses, by inhibiting growth of established tumors expressing human 5T4 followed by a long-lasting memory immune response. ALG.APV-527 has an antibody-like half-life in cynomolgus macaques and was well tolerated at 50.5 mg/kg. ALG.APV-527 is uniquely designed for 5T4-conditional 4–1BB–mediated antitumor activity with potential to minimize systemic immune activation and hepatotoxicity while providing efficacious tumor-specific responses in a range of 5T4-expressing tumor indications as shown by robust activity in preclinical in vitro and in vivo models. On the basis of the combined preclinical dataset, ALG.APV-527 has potential as a promising anticancer therapeutic for the treatment of 5T4-expressing tumors.
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Jan 2023
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B07-B-Versatile Soft X-ray beamline: High Throughput
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Diamond Proposal Number(s):
[31119]
Open Access
Abstract: Tin-containing layers with different degrees of oxidation are uniformly distributed along the length of silicon nanowires formed by a top-down method by applying metalorganic chemical vapor deposition. The electronic and atomic structure of the obtained layers is investigated by applying nondestructive surface-sensitive X-ray absorption near edge spectroscopy using synchrotron radiation. The results demonstrated, for the first time, a distribution effect of the tin-containing phases in the nanostructured silicon matrix compared to the results obtained for planar structures at the same deposition temperatures. The amount and distribution of tin-containing phases can be effectively varied and controlled by adjusting the geometric parameters (pore diameter and length) of the initial matrix of nanostructured silicon. Due to the occurrence of intense interactions between precursor molecules and decomposition by-products in the nanocapillary, as a consequence of random thermal motion of molecules in the nanocapillary, which leads to additional kinetic energy and formation of reducing agents, resulting in effective reduction of tin-based compounds to a metallic tin state for molecules with the highest penetration depth in the nanostructured silicon matrix. This effect will enable clear control of the phase distributions of functional materials in 3D matrices for a wide range of applications.
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Jan 2023
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Stefan
Gahbauer
,
Galen J.
Correy
,
Marion
Schuller
,
Matteo P.
Ferla
,
Yagmur Umay
Doruk
,
Moira
Rachman
,
Taiasean
Wu
,
Morgan
Diolaiti
,
Siyi
Wang
,
R. Jeffrey
Neitz
,
Daren
Fearon
,
Dmytro S.
Radchenko
,
Yurii S.
Moroz
,
John J.
Irwin
,
Adam R.
Renslo
,
Jenny C.
Taylor
,
Jason E.
Gestwicki
,
Frank
Von Delft
,
Alan
Ashworth
,
Ivan
Ahel
,
Brian K.
Shoichet
,
James S.
Fraser
Open Access
Abstract: The nonstructural protein 3 (NSP3) of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) contains a conserved macrodomain enzyme (Mac1) that is critical for pathogenesis and lethality. While small-molecule inhibitors of Mac1 have great therapeutic potential, at the outset of the COVID-19 pandemic, there were no well-validated inhibitors for this protein nor, indeed, the macrodomain enzyme family, making this target a pharmacological orphan. Here, we report the structure-based discovery and development of several different chemical scaffolds exhibiting low- to sub-micromolar affinity for Mac1 through iterations of computer-aided design, structural characterization by ultra-high-resolution protein crystallography, and binding evaluation. Potent scaffolds were designed with in silico fragment linkage and by ultra-large library docking of over 450 million molecules. Both techniques leverage the computational exploration of tangible chemical space and are applicable to other pharmacological orphans. Overall, 160 ligands in 119 different scaffolds were discovered, and 153 Mac1-ligand complex crystal structures were determined, typically to 1 Å resolution or better. Our analyses discovered selective and cell-permeable molecules, unexpected ligand-mediated conformational changes within the active site, and key inhibitor motifs that will template future drug development against Mac1.
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Jan 2023
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Krios IV-Titan Krios IV at Diamond
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Friederike
Leesch
,
Laura
Lorenzo-Orts
,
Carina
Pribitzer
,
Irina
Grishkovskaya
,
Josef
Roehsner
,
Anastasia
Chugunova
,
Manuel
Matzinger
,
Elisabeth
Roitinger
,
Katarina
Belačić
,
Susanne
Kandolf
,
Tzi-Yang
Lin
,
Karl
Mechtler
,
Anton
Meinhart
,
David
Haselbach
,
Andrea
Pauli
Diamond Proposal Number(s):
[2018127]
Abstract: Ribosomes are produced in large quantities during oogenesis and are stored in the egg. However, the egg and early embryo are translationally repressed1,2,3,4. Here, using mass spectrometry and cryo-electron microscopy analyses of ribosomes isolated from zebrafish (Danio rerio) and Xenopus laevis eggs and embryos, we provide molecular evidence that ribosomes transition from a dormant state to an active state during the first hours of embryogenesis. Dormant ribosomes are associated with four conserved factors that form two modules, consisting of Habp4–eEF2 and death associated protein 1b (Dap1b) or Dap in complex with eIF5a. Both modules occupy functionally important sites and act together to stabilize ribosomes and repress translation. Dap1b (also known as Dapl1 in mammals) is a newly discovered translational inhibitor that stably inserts into the polypeptide exit tunnel. Addition of recombinant zebrafish Dap1b protein is sufficient to block translation and reconstitute the dormant egg ribosome state in a mammalian translation extract in vitro. Thus, a developmentally programmed, conserved ribosome state has a key role in ribosome storage and translational repression in the egg.
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Jan 2023
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I22-Small angle scattering & Diffraction
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Mario
Gonzalez-Jimenez
,
Trent
Barnard
,
Ben A.
Russell
,
Nikita V.
Tukachev
,
Uroš
Javornik
,
Laure-Anne
Hayes
,
Andrew J.
Farrell
,
Sarah
Guinane
,
Hans M.
Senn
,
Andrew J.
Smith
,
Martin
Wilding
,
Gregor
Mali
,
Motohiro
Nakano
,
Yuji
Miyazaki
,
Paul
Mcmillan
,
Gabriele C.
Sosso
,
Klaas
Wynne
Diamond Proposal Number(s):
[28529]
Open Access
Abstract: A common feature of glasses is the “boson peak”, observed as an excess in the heat capacity over the crystal or as an additional peak in the terahertz vibrational spectrum. The microscopic origins of this peak are not well understood; the emergence of locally ordered structures has been put forward as a possible candidate. Here, we show that depolarised Raman scattering in liquids consisting of highly symmetric molecules can be used to isolate the boson peak, allowing its detailed observation from the liquid into the glass. The boson peak in the vibrational spectrum matches the excess heat capacity. As the boson peak intensifies on cooling, wide-angle x-ray scattering shows the simultaneous appearance of a pre-peak due to molecular clusters consisting of circa 20 molecules. Atomistic molecular dynamics simulations indicate that these are caused by over-coordinated molecules. These findings represent an essential step toward our understanding of the physics of vitrification.
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Jan 2023
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I12-JEEP: Joint Engineering, Environmental and Processing
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Diamond Proposal Number(s):
[28087, 26608]
Abstract: Ti40Zr10Cu32Pd14Ga4 and Ti40Zr10Cu32Pd14Sn4 (in at.%) bulk metallic glasses (BMGs) with different geometries (wedges, rods, ribbons and discs) were prepared by suction casting, melt spinning and splat quenching, respectively. For comparison, the reference Ti40Zr10Cu36Pd14 BMG was cast as a rod with 2 mm diameter and in wedge-shaped form. High-energy X-ray diffraction measurements yielded a critical casting thickness of 2.4, 2.1 and at least 4 mm for the reference, Ga-containing, and Sn-containing BMGs, respectively. The extension of the supercooled liquid region of about 50 K, measured for the glassy rods and ribbons by differential scanning calorimetry, is larger than that of only 20 K found for the splat-quenched discs. As to the alloys’ mechanical properties, the Ti40Zr10Cu36Pd14 glassy rods deform plastically in compression up to a strain of 3.8% and possess a Young’s modulus of 78 GPa. The Sn- and Ga- containing BMG rods reach respectively a plastic strain of 6.1% and 4.7%, and a Young’s modulus of 72 and 63 GPa. Corrosion tests were performed by electrochemical experiments, and the highest pitting resistance was observed for Ti40Zr10Cu32Pd14Sn4 (pitting overpotential ηpit = 446 mV) compared to Ti40Zr10Cu32Pd14Ga4 (379 mV) and Ti40Zr10Cu36Pd14 (183 mV). The results of live/dead assay and cell viability revealed excellent biocompatibility for the Ga-containing BMGs.
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Jan 2023
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I15-Extreme Conditions
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Diamond Proposal Number(s):
[24144]
Open Access
Abstract: BiFeO3-BaTiO3 (BF-BT) solid solutions exhibit great promise as the basis for high temperature piezoelectric transducers and energy storage dielectrics, but the fundamental mechanisms governing their functional properties require further clarification. In the present study, both pure and niobium-doped 0.7BF-0.3BT ceramics are synthesized by solid state reaction and their structure-property relationships are systematically investigated. It is shown that substituting a low concentration of Ti with Nb at a level of 0.5 at% increases the resistivity of BF-BT ceramics and facilitates ferroelectric switching at high electric field levels. Stable planar piezoelectric coupling factor values are achieved with a variation from 0.35 to 0.45 over the temperature range from 100 to 430 °C. In addition to the ferroelectric-paraelectric phase transformation at the Curie point (~ 430 °C), a frequency-dependent relaxation of the dielectric permittivity and associated loss peak are observed over the temperature range from -50 to +150 °C. These effects are correlated with anomalous enhancement of the remanent polarization and structural (rhombohedral) distortion with increasing temperature, indicating the occurrence of a re-entrant relaxor ferroelectric transformation on cooling. The results of the study provide new insight into the thermal evolution of structure and the corresponding functional properties in BF-BT and related solid solutions.
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Jan 2023
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