B21-High Throughput SAXS
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Diamond Proposal Number(s):
[15868]
Open Access
Abstract: Phosphoenolpyruvate carboxylase (PEPc) is an essential enzyme in plants. A photosynthetic form is
present both as dimer and tetramer in C4 and CAM metabolism. Additionally, non-photosynthetic
PEPcs are also present. The single, non-photosynthetic PEPc of the unicellular cyanobacterium
Synechococcus PCC 7002 (Synechococcus), involved in the TC A cycle, was examined. Using size
exclusion chromatography (SEC) and small angle X-ray scattering (SAXS), we observed that PEPc in
Synechococcus exists as both a dimer and a tetramer. This is the first demonstration of two different
oligomerization states of a non-photosynthetic PEPc. High concentration of Mg2+, the substrate
PEP and a combination of low concentration of Mg2+ and HCO3
− induced the tetramer form of the
carboxylase. Using SEC-SAXS analysis, we showed that the oligomerization state of the carboxylase
is concentration dependent and that, among the available crystal structures of PEPc, the scattering
profile of PEPc of Synechococcus agrees best with the structure of PEPc from Escherichia coli. In
addition, the kinetics of the tetramer purified in presence of Mg2+ using SEC, and of the mixed
population purified in presence of Mg2+ using a Strep-tagged column were examined. Moreover,
the enzyme showed interesting allosteric regulation, being activated by succinate and inhibited by
glutamine, and not affected by either malate, 2-oxoglutarate, aspartic acid or citric acid.
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Feb 2020
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I13-1-Coherence
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Peter G.
Martin
,
Christopher P.
Jones
,
Silvia
Cipiccia
,
Darren
Batey
,
Keith R.
Hallam
,
Yukihiko
Satou
,
Ian
Griffiths
,
Christoph
Rau
,
David A.
Richards
,
Keisuke
Sueki
,
Tatsuya
Ishii
,
Thomas B.
Scott
Diamond Proposal Number(s):
[16702]
Open Access
Abstract: Both the three-dimensional internal structure and elemental distribution of near-field radioactive fallout particulate material released during the March 2011 accident at the Fukushima Daiichi Nuclear Power Plant is analysed using combined high-resolution laboratory and synchrotron radiation x-ray techniques. Results from this study allow for the proposition of the likely formation mechanism of the particles, as well as the potential risks associated with their existence in the environment, and the likely implications for future planned reactor decommissioning. A suite of particles is analyzed from a locality 2 km from the north-western perimeter of the site – north of the primary contaminant plume in an area formerly attributed to being contaminated by fallout from reactor Unit 1. The particles are shown to exhibit significant structural similarities; being amorphous with a textured exterior, and containing inclusions of contrasting compositions, as well as an extensive internal void volume – bimodal in its size distribution. A heterogeneous distribution of the various elemental constituents is observed inside a representative particle, which also exhibited a Fukushima-derived radiocesium (134Cs, 135Cs and 137Cs) signature with negligible natural Cs. We consider the structure and composition of the particle to suggest it formed from materials associated with the reactor Unit 1 building explosion, with debris fragments embedded into the particles surface. Such a high void ratio, comparable to geological pumice, suggests such material formed during a rapid depressurisation and is potentially susceptible to fragmentation through attrition.
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Jan 2020
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Open Access
Abstract: Bone morphogenetic proteins (BMPs) are secreted ligands of the transforming growth factor-β (TGF-β) family that control embryonic patterning, as well as tissue development and homeostasis. Loss of function mutations in the type II BMP receptor BMPR2 are the leading cause of pulmonary arterial hypertension (PAH), a rare disease of vascular occlusion that leads to high blood pressure in the pulmonary arteries. To understand the structural consequences of these mutations, we determined the crystal structure of the human wild-type BMPR2 kinase domain at 2.35 Å resolution. The structure revealed an active conformation of the catalytic domain that formed canonical interactions with the bound ligand Mg-ADP. Disease-associated missense mutations were mapped throughout the protein structure, but clustered predominantly in the larger kinase C-lobe. Modelling revealed that the mutations will destabilize the protein structure by varying extents consistent with their previously reported functional heterogeneity. The most severe mutations introduced steric clashes in the hydrophobic protein core, whereas those found on the protein surface were less destabilizing and potentially most favorable for therapeutic rescue strategies currently under clinical investigation.
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Dec 2019
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I09-Surface and Interface Structural Analysis
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Zachary W.
Lebens-higgins
,
David M.
Halat
,
Nicholas V.
Faenza
,
Matthew J.
Wahila
,
Manfred
Mascheck
,
Tomas
Wiell
,
Susanna K.
Eriksson
,
Paul
Palmgren
,
Jose
Rodriguez
,
Fadwa
Badway
,
Nathalie
Pereira
,
Glenn G.
Amatucci
,
Tien-lin
Lee
,
Clare P.
Grey
,
Louis F. J.
Piper
Diamond Proposal Number(s):
[22250, 22148]
Open Access
Abstract: Aluminum is a common dopant across oxide cathodes for improving the bulk and cathode-electrolyte interface (CEI) stability. Aluminum in the bulk is known to enhance structural and thermal stability, yet the exact influence of aluminum at the CEI remains unclear. To address this, we utilized a combination of X-ray photoelectron and absorption spectroscopy to identify aluminum surface environments and extent of transition metal reduction for Ni-rich LiNi0.8Co0.2−yAlyO2 (0%, 5%, or 20% Al) layered oxide cathodes tested at 4.75 V under thermal stress (60 °C). For these tests, we compared the conventional LiPF6 salt with the more thermally stable LiBF4 salt. The CEI layers are inherently different between these two electrolyte salts, particularly for the highest level of Al-doping (20%) where a thicker (thinner) CEI layer is found for LiPF6 (LiBF4). Focusing on the aluminum environment, we reveal the type of surface aluminum species are dependent on the electrolyte salt, as Al-O-F- and Al-F-like species form when using LiPF6 and LiBF4, respectively. In both cases, we find cathode-electrolyte reactions drive the formation of a protective Al-F-like barrier at the CEI in Al-doped oxide cathodes.
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Dec 2019
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Open Access
Abstract: The effect of hydrostatic pressure up to P = 1.7 GPa on the fluctuation conductivity σ′(T) and pseudogap ∆*(T) in Y0.95Pr0.05Ba2Cu3O7−δ single crystal with critical temperature Тс = 85.2 K (at P = 0) was investigated. The application of pressure leads to the increase in Tc with dTc/dP = +1.82 K∙GPa−1 while the resistance decreases as dlnρ(100 K)/dP = −(10.5 ± 0.2) %∙GPa−1. Regardless of the pressure, in the temperature interval from Tc to T0 (~88 K at P = 0) the behaviour of σ′(T) is well described by the Aslamazov – Larkin (AL – 3D) fluctuation theory, and above the T0 by the Lawrence – Doniach theory (LD). The Maki-Thompson (MT – 2D) fluctuation contribution is not observed. This indicates the presence of structural defects in the sample induced by Pr. Here it is determined for the first time that when the pressure is applied to the Y1−xPrxBa2Cu3O7−δ single crystal, the pseudogap increases as dlnΔ*/dP = 0.17 GPa–1.
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Dec 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[18938]
Open Access
Abstract: Cytochrome P450 reductases (CPRs) are diflavin oxidoreductases that supply electrons to type II cytochrome P450 monooxygenases (CYPs). In addition, it can also reduce other proteins and molecules, including cytochrome c, ferricyanide, and different drugs. Although various CPRs have been functionally and structurally characterized, the overall mechanism and its interaction with different redox acceptors remain elusive. One of the main problems regarding electron transfer between CPRs and CYPs is the so-called “uncoupling”, whereby NAD(P)H derived electrons are lost due to the reduced intermediates’ (FAD and FMN of CPR) interaction with molecular oxygen. Additionally, the decay of the iron-oxygen complex of the CYP can also contribute to loss of reducing equivalents during an unproductive reaction cycle. This phenomenon generates reactive oxygen species (ROS), leading to an inefficient reaction. Here, we present the study of the CPR from Candida tropicalis (CtCPR) lacking the hydrophobic N-terminal part (Δ2–22). The enzyme supports the reduction of cytochrome c and ferricyanide, with an estimated 30% uncoupling during the reactions with cytochrome c. The ROS produced was not influenced by different physicochemical conditions (ionic strength, pH, temperature). The X-ray structures of the enzyme were solved with and without its cofactor, NADPH. Both CtCPR structures exhibited the closed conformation. Comparison with the different solved structures revealed an intricate ionic network responsible for the regulation of the open/closed movement of CtCPR.
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Dec 2019
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B21-High Throughput SAXS
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Sarah C.
Lee
,
Richard
Collins
,
Yu-pin
Lin
,
Mohammed
Jamshad
,
Claire
Broughton
,
Sarah A.
Harris
,
Benjamin S.
Hanson
,
Cecilia
Tognoloni
,
Rosemary A.
Parslow
,
Ann E.
Terry
,
Alison
Rodger
,
Corinne J.
Smith
,
Karen J.
Edler
,
Robert
Ford
,
David I.
Roper
,
Timothy R.
Dafforn
Diamond Proposal Number(s):
[9727]
Open Access
Abstract: The E. coli membrane protein ZipA, binds to the tubulin homologue FtsZ, in the early stage of cell division. We isolated ZipA in a Styrene Maleic Acid lipid particle (SMALP) preserving its position and integrity with native E. coli membrane lipids. Direct binding of ZipA to FtsZ is demonstrated, including FtsZ fibre bundles decorated with ZipA. Using Cryo-Electron Microscopy, small-angle X-ray and neutron scattering, we determine the encapsulated-ZipA structure in isolation, and in complex with FtsZ to a resolution of 1.6 nm. Three regions can be identified from the structure which correspond to, SMALP encapsulated membrane and ZipA transmembrane helix, a separate short compact tether, and ZipA globular head which binds FtsZ. The complex extends 12 nm from the membrane in a compact structure, supported by mesoscale modelling techniques, measuring the movement and stiffness of the regions within ZipA provides molecular scale analysis and visualisation of the early divisome.
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Dec 2019
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[16943, 15997]
Open Access
Abstract: Some molecular chaperones are involved not only in assisting the folding of proteins but also, given appropriate conditions, in their degradation. This is the case for Hsp70 and Hsp90 which, in concert with the cochaperone CHIP, direct their bound substrate to degradation through ubiquitination. We generated complexes between the chaperones (Hsp70 or Hsp90), the cochaperone CHIP and, as substrate, a p53 variant containing the GST protein (p53-TMGST). Both ternary complexes (Hsp70:p53-TMGST:CHIP and Hsp90:p53-TMGST:CHIP) ubiquitinated the substrate at a higher efficiency than in the absence of the chaperones. The 3D structures of the two complexes, obtained using a combination of cryoelectron microscopy and crosslinking mass spectrometry, showed the substrate located between the chaperone and the cochaperone, suggesting a ubiquitination mechanism in which the chaperone-bound substrate is presented to CHIP. These complexes are inherently flexible, which is important for the ubiquitination process.
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Dec 2019
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I24-Microfocus Macromolecular Crystallography
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Gerson S.
Profeta
,
Caio V.
Dos Reis
,
André Da S.
Santiago
,
Paulo H. C.
Godoi
,
Angela M.
Fala
,
Carrow I.
Wells
,
Roger
Sartori
,
Anita P. T.
Salmazo
,
Priscila Z.
Ramos
,
Katlin B.
Massirer
,
Jonathan M.
Elkins
,
David H.
Drewry
,
Opher
Gileadi
,
Rafael M.
Counago
Diamond Proposal Number(s):
[16171]
Open Access
Abstract: Calcium/Calmodulin-dependent Protein Kinase Kinase 2 (CAMKK2) acts as a signaling hub, receiving signals from various regulatory pathways and decoding them via phosphorylation of downstream protein kinases - such as AMPK (AMP-activated protein kinase) and CAMK types I and IV. CAMKK2 relevance is highlighted by its constitutive activity being implicated in several human pathologies. However, at present, there are no selective small-molecule inhibitors available for this protein kinase. Moreover, CAMKK2 and its closest human homolog, CAMKK1, are thought to have overlapping biological roles. Here we present six new co-structures of potent ligands bound to CAMKK2 identified from a library of commercially-available kinase inhibitors. Enzyme assays confirmed that most of these compounds are equipotent inhibitors of both human CAMKKs and isothermal titration calorimetry (ITC) revealed that binding to some of these molecules to CAMKK2 is enthalpy driven. We expect our results to advance current efforts to discover small molecule kinase inhibitors selective to each human CAMKK.
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Nov 2019
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Pooja
Sharma
,
Robert
Mahen
,
Maxim
Rossmann
,
Jamie E.
Stokes
,
Bryn
Hardwick
,
David J.
Huggins
,
Amy
Emery
,
Dominique L.
Kunciw
,
Marko
Hyvonen
,
David R.
Spring
,
Grahame J.
Mckenzie
,
Ashok R.
Venkitaraman
Diamond Proposal Number(s):
[7141, 9537]
Open Access
Abstract: The human polo-like kinase PLK1 coordinates mitotic chromosome segregation by phosphorylating multiple chromatin- and kinetochore-binding proteins. How PLK1 activity is directed to specific substrates via phosphopeptide recognition by its carboxyl-terminal polo-box domain (PBD) is poorly understood. Here, we combine molecular, structural and chemical biology to identify a determinant for PLK1 substrate recognition that is essential for proper chromosome segregation. We show that mutations ablating an evolutionarily conserved, Tyr-lined pocket in human PLK1 PBD trigger cellular anomalies in mitotic progression and timing. Tyr pocket mutations selectively impair PLK1 binding to the kinetochore phosphoprotein substrate PBIP1, but not to the centrosomal substrate NEDD1. Through a structure-guided approach, we develop a small-molecule inhibitor, Polotyrin, which occupies the Tyr pocket. Polotyrin recapitulates the mitotic defects caused by mutations in the Tyr pocket, further evidencing its essential function, and exemplifying a new approach for selective PLK1 inhibition. Thus, our findings support a model wherein substrate discrimination via the Tyr pocket in the human PLK1 PBD regulates mitotic chromosome segregation to preserve genome integrity.
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Nov 2019
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