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Instruments / Technical Area	Publication Details	Published	Actions
Krios III-Titan Krios III at Diamond	The active form of quinol-dependent nitric oxide reductase from Neisseria meningitidis is a dimer M. Arif M. Jamali , Chai C. Gopalasingam , Rachel M. Johnson , Takehiko Tosha , Kazumasa Muramoto , Stephen P. Muench , Svetlana V. Antonyuk , Yoshitsugu Shiro , Samar Hasnain Iucrj 7, 404 - 415 DOI: 10.1107/S2052252520003656 Diamond Proposal Number(s): [19832] Open Access Show Abstract ▼ Abstract: Neisseria meningitidis is carried by nearly a billion humans, causing developmental impairment and over 100 000 deaths a year. A quinol-dependent nitric oxide reductase (qNOR) plays a critical role in the survival of the bacterium in the human host. X-ray crystallographic analyses of qNOR, including that from N. meningitidis (NmqNOR) reported here at 3.15 Å resolution, show monomeric assemblies, despite the more active dimeric sample being used for crystallization. Cryo-electron microscopic analysis of the same chromatographic fraction of NmqNOR, however, revealed a dimeric assembly at 3.06 Å resolution. It is shown that zinc (which is used in crystallization) binding near the dimer-stabilizing TMII region contributes to the disruption of the dimer. A similar destabilization is observed in the monomeric (∼85 kDa) cryo-EM structure of a mutant (Glu494Ala) qNOR from the opportunistic pathogen Alcaligenes (Achromobacter) xylosoxidans, which primarily migrates as a monomer. The monomer–dimer transition of qNORs seen in the cryo-EM and crystallographic structures has wider implications for structural studies of multimeric membrane proteins. X-ray crystallographic and cryo-EM structural analyses have been performed on the same chromatographic fraction of NmqNOR to high resolution. This represents one of the first examples in which the two approaches have been used to reveal a monomeric assembly in crystallo and a dimeric assembly in vitrified cryo-EM grids. A number of factors have been identified that may trigger the destabilization of helices that are necessary to preserve the integrity of the dimer. These include zinc binding near the entry of the putative proton-transfer channel and the preservation of the conformational integrity of the active site. The mutation near the active site results in disruption of the active site, causing an additional destabilization of helices (TMIX and TMX) that flank the proton-transfer channel helices, creating an inert monomeric enzyme.	May 2020
Krios II-Titan Krios II at Diamond	Dimeric structures of quinol-dependent nitric oxide reductases (qNORs) revealed by cryo–electron microscopy Chai C. Gopalasingam , Rachel M. Johnson , George N. Chiduza , Takehiko Tosha , Masaki Yamamoto , Yoshitsugu Shiro , Svetlana V. Antonyuk , Stephen P. Muench , S. Samar Hasnain Science Advances 5 DOI: 10.1126/sciadv.aax1803 Diamond Proposal Number(s): [19832] Open Access Show Abstract ▼ Abstract: Quinol-dependent nitric oxide reductases (qNORs) are membrane-integrated, iron-containing enzymes of the denitrification pathway, which catalyze the reduction of nitric oxide (NO) to the major ozone destroying gas nitrous oxide (N2O). Cryo–electron microscopy structures of active qNOR from Alcaligenes xylosoxidans and an activity-enhancing mutant have been determined to be at local resolutions of 3.7 and 3.2 Å, respectively. They unexpectedly reveal a dimeric conformation (also confirmed for qNOR from Neisseria meningitidis) and define the active-site configuration, with a clear water channel from the cytoplasm. Structure-based mutagenesis has identified key residues involved in proton transport and substrate delivery to the active site of qNORs. The proton supply direction differs from cytochrome c–dependent NOR (cNOR), where water molecules from the cytoplasm serve as a proton source similar to those from cytochrome c oxidase.	Aug 2019

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