Treatment of the trichlorotin-capped trinuclear nickel cluster, Ni
3
(dppm)
3
(μ
3
-Cl)(μ
3
-SnCl
3
),
1
, with 4 eq. NaHB(Et)
3
yields a μ
3
-SnH capped trinuclear nickel cluster, Ni
3
(dppm)
3
(μ
3
...-H)(μ
3
-SnH),
2
dppm = bis(diphenylphosphino)methane. Single-crystal X-ray diffraction, nuclear magnetic resonance (NMR) spectroscopy, and computational studies together support that cluster
2
is a divalent tin hydride. Complex
2
displays a wide range of reactivity including oxidative addition of bromoethane across the Sn center. Addition of 1 eq. iodoethane to complex
2
releases H
2
(g) and generates an ethyltin-capped nickel cluster with a μ
3
-iodide, Ni
3
(dppm)
3
(μ
3
-I)(μ
3
-Sn(CH
2
CH
3
)),
4
. Notably, insertion of alkynes into the Sn-H bond of
2
can be achieved
via
addition of 1 eq.
1
-hexyne to generate the 1-hexen-2-yl-tin-capped nickel cluster, Ni
3
(dppm)
3
(μ
3
H)(μ
3
-Sn(C
6
H
11
)),
5
. Addition of H
2
(g) to
5
regenerates the starting material,
2
, and hexane. The formally 44-electron cluster
2
also displays significant redox chemistry with two reversible one-electron oxidations (
E
= −1.3 V, −0.8 V
vs.
Fc
0/+
) and one-electron reduction process (
E
= −2.7 V
vs.
Fc
0/+
) observed by cyclic voltammetry.
The synthesis, structure, and reactivity of a μ
3
-SnH capped trinuclear nickel cluster, Ni
3
(dppm)
3
(μ
3
-H)(μ
3
-SnH), is reported. This complex undergoes oxidative addition chemistry, alkyne insertion, and subsequent hydrogenation.
Treatment of the trichlorotin-capped trinuclear nickel cluster, Ni3(dppm)3(μ3-Cl)(μ3-SnCl3), 1, with 4 eq. NaHB(Et)3 yields a μ3-SnH capped trinuclear nickel cluster, Ni3(dppm)3(μ3-H)(μ3-SnH), 2 dppm ...= bis(diphenylphosphino)methane. Single-crystal X-ray diffraction, nuclear magnetic resonance (NMR) spectroscopy, and computational studies together support that cluster 2 is a divalent tin hydride. Complex 2 displays a wide range of reactivity including oxidative addition of bromoethane across the Sn center. Addition of 1 eq. iodoethane to complex 2 releases H2 (g) and generates an ethyltin-capped nickel cluster with a μ3-iodide, Ni3(dppm)3(μ3-I)(μ3-Sn(CH2CH3)), 4. Notably, insertion of alkynes into the Sn–H bond of 2 can be achieved via addition of 1 eq. 1-hexyne to generate the 1-hexen-2-yl-tin-capped nickel cluster, Ni3(dppm)3(μ3H)(μ3-Sn(C6H11)), 5. Addition of H2 (g) to 5 regenerates the starting material, 2, and hexane. The formally 44-electron cluster 2 also displays significant redox chemistry with two reversible one-electron oxidations (E = −1.3 V, −0.8 V vs. Fc0/+) and one-electron reduction process (E = −2.7 V vs. Fc0/+) observed by cyclic voltammetry.
The type II secretion system (T2SS) is a cell envelope-spanning macromolecular complex that is prevalent in Gram-negative bacterial species. It serves as the predominant virulence mechanism of many ...bacteria including those of the emerging human pathogens Vibrio vulnificus and Aeromonas hydrophila. The system is composed of a core set of highly conserved proteins that assemble an inner membrane platform, a periplasmic pseudopilus and an outer membrane complex termed the secretin. Localization and assembly of secretins in the outer membrane requires recognition of secretin monomers by two different partner systems: an inner membrane accessory complex or a highly sequence-diverse outer membrane lipoprotein, termed the pilotin. In this study, we addressed the question of differential secretin assembly mechanisms by using cryo-electron microscopy to determine the structures of the secretins from A. hydrophila (pilotin-independent ExeD) and V. vulnificus (pilotin-dependent EpsD). These structures, at approximately 3.5 Å resolution, reveal pentadecameric stoichiometries and C-terminal regions that carry a signature motif in the case of a pilotin-dependent assembly mechanism. We solved the crystal structure of the V. vulnificus EpsS pilotin and confirmed the importance of the signature motif for pilotin-dependent secretin assembly by performing modelling with the C-terminus of EpsD. We also show that secretin assembly is essential for membrane integrity and toxin secretion in V. vulnificus and establish that EpsD requires the coordinated activity of both the accessory complex EpsAB and the pilotin EpsS for full assembly and T2SS function. In contrast, mutation of the region of the S-domain that is normally the site of pilotin interactions has little effect on assembly or function of the ExeD secretin. Since secretins are essential outer membrane channels present in a variety of secretion systems, these results provide a structural and functional basis for understanding the key assembly steps for different members of this vast pore-forming family of proteins.
Abstract
Serial crystallography at X-ray free-electron lasers (XFELs) permits the determination of radiation-damage free static as well as time-resolved protein structures at room temperature. ...Efficient sample delivery is a key factor for such experiments. Here, we describe a multi-reservoir, high viscosity extruder as a step towards automation of sample delivery at XFELs. Compared to a standard single extruder, sample exchange time was halved and the workload of users was greatly reduced. In-built temperature control of samples facilitated optimal extrusion and supported sample stability. After commissioning the device with lysozyme crystals, we collected time-resolved data using crystals of a membrane-bound, light-driven sodium pump. Static data were also collected from the soluble protein tubulin that was soaked with a series of small molecule drugs. Using these data, we identify low occupancy (as little as 30%) ligands using a minimal amount of data from a serial crystallography experiment, a result that could be exploited for structure-based drug design.
Intensive care unit professionals have experience in critical care and its proportionality, collegial decision-making, withholding or withdrawal of treatment deemed futile, and communication with ...patients’ relatives. These elements rely on ethical values from which we must not deviate in a pandemic situation. The recommendations made by the Ethics Commission of the French Intensive Care Society reflect an approach of responsibility and solidarity towards our citizens regarding the potential impact of a pandemic on critical care resources in France, with the fundamental requirement of respect for human dignity and equal access to health care for all.
Serial and time-resolved macromolecular crystallography are on the rise. However, beam time at X-ray free-electron lasers is limited and most third-generation synchrotron-based macromolecular ...crystallography beamlines do not offer the necessary infrastructure yet. Here, a new setup is demonstrated, based on the JUNGFRAU detector and Jungfraujoch data-acquisition system, that enables collection of kilohertz serial crystallography data at fourth-generation synchrotrons. More importantly, it is shown that this setup is capable of collecting multiple-time-point time-resolved protein dynamics at kilohertz rates, allowing the probing of microsecond to second dynamics at synchrotrons in a fraction of the time needed previously. A high-quality complete X-ray dataset was obtained within 1 min from lysozyme microcrystals, and the dynamics of the light-driven sodium-pump membrane protein KR2 with a time resolution of 1 ms could be demonstrated. To make the setup more accessible for researchers, downstream data handling and analysis will be automated to allow on-the-fly spot finding and indexing, as well as data processing.
We consider the Eden model on the d-dimensional hypercubical unoriented lattice, for large d. Initially, every lattice point is healthy, except the origin which is infected. Then, each infected ...lattice point contaminates any of its neighbours with rate 1. The Eden model is equivalent to first passage percolation, with exponential passage times on edges. The Eden conjecture states that the limit shape of the Eden model is a Euclidean ball. By pushing the computations of Dhar 5 a little further with modern computers and efficient implementation we obtain improved bounds for the speed of infection. This shows that the Eden conjecture does not hold in dimension superior to 22 (the lowest known dimension was 35).
•We show that the Eden conjecture does not hold in dimension superior to 22.•Dhar's computations (Dhar, 1988) are pushed a little further with modern computers.•Bounds for the speed of infection along the axis are improved.•This result is combined with a bound for the speed of infection along the diagonal.
We consider the Eden model on the d-dimensional hypercubical unoriented lattice , for large d. Initially, every lattice point is healthy, except the origin which is infected. Then, each infected ...lattice point contaminates any of its neighbours with rate 1. The Eden model is equivalent to first passage percolation, with exponential passage times on edges. The Eden conjecture states that the limit shape of the Eden model is a Euclidean ball. By putting the computations of Dhar Dha88 a little further with modern computers and efficient implementation we obtain improved bounds for the speed of infection. This shows that the Eden conjecture does not hold in dimension superior to 22 (the lower known dimension was 35).
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•-Lactate dehydrogenase from Selenomonas ruminantium displays an allosteric capacity without large conformational reorganizations.•-The enzymatic properties differ from those ...encountered with canonical lactate dehydrogenases.•-This enzyme could be seen as the relics of an ancestral step prior the complete acquisition of allosteric regulation in lactate dehydrogenases.
In this work, we investigated the lactate dehydrogenase (LDH) from Selenomonas ruminantium (S. rum), an enzyme that differs at key amino acid positions from canonical allosteric LDHs. The wild type (Wt) of this enzyme recognises pyuvate as all LDHs. However, introducing a single point mutation in the active site loop (I85R) allows S. Rum LDH to recognize the oxaloacetate substrate as a typical malate dehydrogenase (MalDH), whilst maintaining homotropic activation as an LDH. We report the tertiary structure of the Wt and I85RLDH mutant.
The Wt S. rum enzyme structure binds NADH and malonate, whilst also resembling the typical compact R-active state of canonical LDHs. The structure of the mutant with I85R was solved in the Apo State (without ligand), and shows no large conformational reorganization such as that observed with canonical allosteric LDHs in Apo state. This is due to a local structural feature typical of S. rum LDH that prevents large-scale conformational reorganization. The S. rum LDH was also studied using Molecular Dynamics simulations, probing specific local deformations of the active site that allow the S. rum LDH to sample the T-inactive state. We propose that, with respect to the LDH/MalDH superfamily, the S. rum enzyme possesses a specificstructural and dynamical way to ensure homotropic activation.
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