I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Declan A.
Gray
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Joshua B. R.
White
,
Abraham O.
Oluwole
,
Parthasarathi
Rath
,
Amy J.
Glenwright
,
Adam
Mazur
,
Michael
Zahn
,
Arnaud
Basle
,
Carl
Morland
,
Sasha L.
Evans
,
Alan
Cartmell
,
Carol V.
Robinson
,
Sebastian
Hiller
,
Neil A.
Ranson
,
David N.
Bolam
,
Bert
Van Den Berg
Diamond Proposal Number(s):
[13587, 18598]
Open Access
Abstract: In Bacteroidetes, one of the dominant phyla of the mammalian gut, active uptake of large nutrients across the outer membrane is mediated by SusCD protein complexes via a “pedal bin” transport mechanism. However, many features of SusCD function in glycan uptake remain unclear, including ligand binding, the role of the SusD lid and the size limit for substrate transport. Here we characterise the β2,6 fructo-oligosaccharide (FOS) importing SusCD from Bacteroides thetaiotaomicron (Bt1762-Bt1763) to shed light on SusCD function. Co-crystal structures reveal residues involved in glycan recognition and suggest that the large binding cavity can accommodate several substrate molecules, each up to ~2.5 kDa in size, a finding supported by native mass spectrometry and isothermal titration calorimetry. Mutational studies in vivo provide functional insights into the key structural features of the SusCD apparatus and cryo-EM of the intact dimeric SusCD complex reveals several distinct states of the transporter, directly visualising the dynamics of the pedal bin transport mechanism.
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Jan 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19800]
Open Access
Abstract: Dye‐decolorizing peroxidases (DyPs) constitute a superfamily of heme‐containing peroxidases that are related neither to animal nor to plant peroxidase families. These are divided into four classes (types A, B, C, and D) based on sequence features. The active site of DyPs contains two highly conserved distal ligands, an aspartate and an arginine, the roles of which are still controversial. These ligands have mainly been studied in class A‐C bacterial DyPs, largely because no effective recombinant expression systems have been developed for the fungal (D‐type) DyPs. In this work, we employ ancestral sequence reconstruction (ASR) to resurrect a D‐type DyP ancestor, AncDyPD‐b1. Expression of AncDyPD‐b1 in Escherichia coli results in large amounts of a heme‐containing soluble protein and allows for the first mutagenesis study on the two distal ligands of a fungal DyP. UV‐Vis and resonance Raman (RR) spectroscopic analyses, in combination with steady‐state kinetics and the crystal structure, reveal fine pH‐dependent details about the heme active site structure and show that both the aspartate (D222) and the arginine (R390) are crucial for hydrogen peroxide reduction. Moreover, the data indicate that these two residues play important but mechanistically different roles on the intraprotein long‐range electron transfer process.
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Jan 2021
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I03-Macromolecular Crystallography
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Samuel L.
Rose
,
Svetlana V.
Antonyuk
,
Daisuke
Sasaki
,
Keitaro
Yamashita
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Kunio
Hirata
,
Go
Ueno
,
Hideo
Ago
,
Robert R.
Eady
,
Takehiko
Tosha
,
Masaki
Yamamoto
,
S. Samar
Hasnain
Diamond Proposal Number(s):
[15991]
Open Access
Abstract: Copper-containing nitrite reductases (CuNiRs), encoded by nirK gene, are found in all kingdoms of life with only 5% of CuNiR denitrifiers having two or more copies of nirK. Recently, we have identified two copies of nirK genes in several α-proteobacteria of the order Rhizobiales including Bradyrhizobium sp. ORS 375, encoding a four-domain heme-CuNiR and the usual two-domain CuNiR (Br2DNiR). Compared with two of the best-studied two-domain CuNiRs represented by the blue (AxNiR) and green (AcNiR) subclasses, Br2DNiR, a blue CuNiR, shows a substantially lower catalytic efficiency despite a sequence identity of ~70%. Advanced synchrotron radiation and x-ray free-electron laser are used to obtain the most accurate (atomic resolution with unrestrained SHELX refinement) and damage-free (free from radiation-induced chemistry) structures, in as-isolated, substrate-bound, and product-bound states. This combination has shed light on the protonation states of essential catalytic residues, additional reaction intermediates, and how catalytic efficiency is modulated.
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Jan 2021
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Nathan David
Wright
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Patrick
Collins
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Lizbe
Koekemoer
,
Tobias
Krojer
,
Romain
Talon
,
Elliot
Nelson
,
Mingda
Ye
,
Radoslaw
Nowak
,
Joseph
Newman
,
Jia Tsing
Ng
,
Nick
Mitrovic
,
Helton
Wiggers
,
Frank
Von Delft
Open Access
Abstract: Despite the tremendous success of X-ray cryo-crystallography in recent decades, the transfer of crystals from the drops in which they are grown to diffractometer sample mounts remains a manual process in almost all laboratories. Here, the Shifter, a motorized, interactive microscope stage that transforms the entire crystal-mounting workflow from a rate-limiting manual activity to a controllable, high-throughput semi-automated process, is described. By combining the visual acuity and fine motor skills of humans with targeted hardware and software automation, it was possible to transform the speed and robustness of crystal mounting. Control software, triggered by the operator, manoeuvres crystallization plates beneath a clear protective cover, allowing the complete removal of film seals and thereby eliminating the tedium of repetitive seal cutting. The software, either upon request or working from an imported list, controls motors to position crystal drops under a hole in the cover for human mounting at a microscope. The software automatically captures experimental annotations for uploading to the user's data repository, removing the need for manual documentation. The Shifter facilitates mounting rates of 100–240 crystals per hour in a more controlled process than manual mounting, which greatly extends the lifetime of the drops and thus allows a dramatic increase in the number of crystals retrievable from any given drop without loss of X-ray diffraction quality. In 2015, the first in a series of three Shifter devices was deployed as part of the XChem fragment-screening facility at Diamond Light Source, where they have since facilitated the mounting of over 120 000 crystals. The Shifter was engineered to have a simple design, providing a device that could be readily commercialized and widely adopted owing to its low cost. The versatile hardware design allows use beyond fragment screening and protein crystallography.
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Jan 2021
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I20-EDE-Energy Dispersive EXAFS (EDE)
I20-Scanning-X-ray spectroscopy (XAS/XES)
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Diamond Proposal Number(s):
[17574]
Open Access
Abstract: Deep eutectic solvents (DES) and their hydrated mixtures are used for solvothermal routes towards greener functional nanomaterials. Here we present the first static structural and in situ studies of the formation of iron oxide (hematite) nanoparticles in a DES of choline chloride[thin space (1/6-em)]:[thin space (1/6-em)]urea where xurea = 0.67 (aka. reline) as an exemplar solvothermal reaction, and observe the effects of water on the reaction. The initial speciation of Fe3+ in DES solutions was measured using extended X-ray absorption fine structure (EXAFS), while the atomistic structure of the mixture was resolved from neutron and X-ray diffraction and empirical potential structure refinement (EPSR) modelling. The reaction was monitored using in situ small-angle neutron scattering (SANS), to determine mesoscale changes, and EXAFS, to determine local rearrangements of order around iron ions. It is shown that iron salts form an octahedral [Fe(L)3(Cl)3] complex where (L) represents various O-containing ligands. Solubilised Fe3+ induced subtle structural rearrangements in the DES due to abstraction of chloride into complexes and distortion of H-bonding around complexes. EXAFS suggests the complex forms [–O–Fe–O–] oligomers by reaction with the products of thermal hydrolysis of urea, and is thus pseudo-zero-order in iron. In the hydrated DES, the reaction, nucleation and growth proceeds rapidly, whereas in the pure DES, the reaction initially proceeds quickly, but suddenly slows after 5000 s. In situ SANS and static small-angle X-ray scattering (SAXS) experiments reveal that nanoparticles spontaneously nucleate after 5000 s of reaction time in the pure DES before slow growth. Contrast effects observed in SANS measurements suggest that hydrated DES preferentially form 1D particle morphologies because of choline selectively capping surface crystal facets to direct growth along certain axes, whereas capping is restricted by the solvent structure in the pure DES.
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Jan 2021
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I05-ARPES
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Wujun
Shi
,
Benjamin J.
Wieder
,
Holger L.
Meyerheim
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Yan
Sun
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Yang
Zhang
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Yiwei
Li
,
Lei
Shen
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Yanpeng
Qi
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Lexian
Yang
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Jagannath
Jena
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Peter
Werner
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Klaus
Koepernik
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Stuart
Parkin
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Yulin
Chen
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Claudia
Felser
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B. Andrei
Bernevig
,
Zhijun
Wang
Abstract: Topological physics and strong electron–electron correlations in quantum materials are typically studied independently. However, there have been rapid recent developments in quantum materials in which topological phase transitions emerge when the single-particle band structure is modified by strong interactions. Here we demonstrate that the room-temperature phase of (TaSe4)2I is a Weyl semimetal with 24 pairs of Weyl nodes. Owing to its quasi-one-dimensional structure, (TaSe4)2I also hosts an established charge-density wave instability just below room temperature. We show that the charge-density wave in (TaSe4)2I couples the bulk Weyl points and opens a bandgap. The correlation-driven topological phase transition in (TaSe4)2I provides a route towards observing condensed-matter realizations of axion electrodynamics in the gapped regime, topological chiral response effects in the semimetallic phase, and represents an avenue for exploring the interplay of correlations and topology in a solid-state material.
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Jan 2021
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E02-JEM ARM 300CF
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Diamond Proposal Number(s):
[19130, 20614]
Abstract: Cellulose is crystallized by plants and other organisms into fibrous nanocrystals. The mechanical properties of these nanofibers and the formation of helical superstructures with energy dissipating and adaptive optical properties depend on the ordering of polysaccharide chains within these nanocrystals, which is typically measured in bulk average. Direct measurement of the local polysaccharide chain arrangement has been elusive. In this study, we use the emerging technique of scanning electron diffraction to probe the packing of polysaccharide chains across cellulose nanofibers and to reveal local ordering of the chains in twisting sections of the nanofibers. We then use atomic force microscopy to shed light on the size dependence of the inherent driving force for cellulose nanofiber twisting. The direct measurement of crystalline twisted regions in cellulose nanofibers has important implications for understanding single-cellulose-fibril properties that influence the interactions between cellulose nanocrystals in dense assemblies. This understanding may enable cellulose extraction and separation processes to be tailored and optimized.
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Jan 2021
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Tika R.
Malla
,
Anthony
Tumber
,
Tobias
John
,
Lennart
Brewitz
,
Claire
Strain-damerell
,
C. David
Owen
,
Petra
Lukacik
,
H. T. Henry
Chan
,
Pratheesh
Maheswaran
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Eidarus
Salah
,
Fernanda
Duarte
,
Haitao
Yang
,
Zihe
Rao
,
Martin A.
Walsh
,
Christopher J.
Schofield
Open Access
Abstract: The main viral protease (Mpro) of SARS-CoV-2 is a nucleophilic cysteine hydrolase and a current target for anti-viral chemotherapy. We describe a high-throughput solid phase extraction coupled to mass spectrometry Mpro assay. The results reveal some β-lactams, including penicillin esters, are active site reacting Mpro inhibitors, thus highlighting the potential of acylating agents for Mpro inhibition.
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Jan 2021
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I10-Beamline for Advanced Dichroism
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Diamond Proposal Number(s):
[11784, 12943, 12958]
Open Access
Abstract: From the perspective of surface science, only the topmost atomic layers usually exhibit physical properties that are different to those of the bulk material, whereas the deeper layers are assumed to be bulk-like and remain largely unexplored. Going beyond conventional diffraction and imaging techniques, we have determined the depth dependence of the full 3D spin structure of magnetic skyrmions below the surface of a bulk Cu2OSeO3 sample using the polarization dependence of resonant elastic x-ray scattering (REXS). While the bulk spin configuration showed the anticipated Bloch type structure, it was found that the skyrmion lattice changes to a Néel twisting (i.e., with a different helicity angle) at the surface within a distance of several hundred nm. The exact surface helicity angle and penetration length of this twist have been determined, revealing the detailed internal structure of the skyrmion tube. It was found that the experimental penetration length of the Néel twisting is 7× longer than the theoretical value given by the ratio of J/D. This indicates that apart from the considered spin interactions, i.e., the Heisenberg exchange interaction J and the Dzyaloshinskii-Moriya interaction D, as well as the Zeeman interaction, other effects must play an important role. The findings suggest that the surface reconstruction of the skyrmion lattice is a universal phenomenon, stemming from the breaking of translational symmetry at the interface.
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Jan 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Maria-agustina
Rossi
,
Veronica
Martinez
,
Philip
Hinchliffe
,
Maria F.
Mojica
,
Valerie
Castillo
,
Diego M.
Moreno
,
Ryan
Smith
,
Brad
Spellberg
,
George L.
Drusano
,
Claudia
Banchio
,
Robert A.
Bonomo
,
James
Spencer
,
Alejandro J.
Vila
,
Graciela
Mahler
Diamond Proposal Number(s):
[17212]
Open Access
Abstract: Infections caused by multidrug resistant (MDR) bacteria are a major public health threat. Carbapenems are among the most potent antimicrobial agents that are commercially available to treat MDR bacteria. Bacterial production of carbapenem-hydrolysing metallo-β-lactamases (MBLs) challenges their safety and efficacy, with subclass B1 MBLs hydrolysing almost all β-lactam antibiotics. MBL inhibitors would fulfil an urgent clinical need by prolonging the lifetime of these life-saving drugs. Here we report the synthesis and activity of a series of 2-mercaptomethyl-thiazolidines (MMTZs), designed to replicate MBL interactions with reaction intermediates or hydrolysis products. MMTZs are potent competitive inhibitors of B1 MBLs in vitro (e.g., Ki = 0.44 μM vs. NDM-1). Crystal structures of MMTZ complexes reveal similar binding patterns to the most clinically important B1 MBLs (NDM-1, VIM-2 and IMP-1), contrasting with previously studied thiol-based MBL inhibitors, such as bisthiazolidines (BTZs) or captopril stereoisomers, which exhibit lower, more variable potencies and multiple binding modes. MMTZ binding involves thiol coordination to the Zn(II) site and extensive hydrophobic interactions, burying the inhibitor more deeply within the active site than D/L-captopril. Unexpectedly, MMTZ binding features a thioether–π interaction with a conserved active-site aromatic residue, consistent with their equipotent inhibition and similar binding to multiple MBLs. MMTZs penetrate multiple Enterobacterales, inhibit NDM-1 in situ, and restore carbapenem potency against clinical isolates expressing B1 MBLs. Based on their inhibitory profile and lack of eukaryotic cell toxicity, MMTZs represent a promising scaffold for MBL inhibitor development. These results also suggest sulphur–π interactions can be exploited for general ligand design in medicinal chemistry.
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Jan 2021
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