[ { "id": "authors:96z2z-hsn94", "collection": "authors", "collection_id": "96z2z-hsn94", "cite_using_url": "https://authors.library.caltech.edu/records/96z2z-hsn94", "type": "monograph", "title": "Modeling the Correlation between Z and B in an X-ray Crystal Structure Refinement", "author": [ { "family_name": "Buscagan", "given_name": "Trixia M.", "orcid": "0000-0001-8242-9203", "clpid": "Buscagan-Trixia-M" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "abstract": "
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We have examined how the refined B-factor changes as a function of Z (the atomic number of a scatterer) at the sulfur site of the [4Fe:4S] cluster of the nitrogenase iron protein by refinement. A simple model is developed that quantitatively captures the observed relationship between Z and B, based on a Gaussian electron density distribution with a constant electron density at the position of the scatterer. From this analysis, the fractional changes in B and Z are found to be similar. The utility of B-factor refinement to potentially distinguish atom types reflects the Z dependence of X-ray atomic scattering factors; the weaker dependence of electron atomic scattering factors on Z implies that distinctions between refined values of B in an electron scattering structure will be less sensitive to the atomic identity of a scatterer than for the case with X-ray-diffraction. This behavior provides an example of the complementary information that can be extracted from different types of scattering studies.

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", "doi": "10.1101/2023.07.04.547724", "pmcid": "PMC10350028", "publisher": "Cold Spring Harbor Laboratory Press", "publication_date": "2023-07-04" }, { "id": "authors:41rhx-h9m88", "collection": "authors", "collection_id": "41rhx-h9m88", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20220607-424749000", "type": "monograph", "title": "Anaerobic single particle cryoEM of nitrogenase", "author": [ { "family_name": "Warmack", "given_name": "Rebeccah A.", "orcid": "0000-0002-9612-0511", "clpid": "Warmack-Rebeccah-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "abstract": "The enzyme nitrogenase catalyzes the reduction of dinitrogen to ammonia during biological nitrogen fixation through a mechanism involving the ATP dependent interaction of two component proteins adopting multiple conformational states. To date, high resolution structural information has been provided by X-ray crystallography, which restricts the states that can be accessed to those that can be crystallized. Cryo-electron microscopy (cryoEM) presents a new opportunity for structural characterization of nitrogenase solution structures, and may yield new information on the mechanism of nitrogenase by revealing structures of transient or heterogeneous states. In this study, we present single particle cryoEM structures of the MoFe-nitrogenase endogenously isolated from Azotobacter vinelandii. To maintain the fully reduced cluster states of this oxygen sensitive protein, we prepared samples within an anaerobic chamber and employed specialized conditions to minimize partial disordering of the \u03b1-subunit at the air-water interface during freezing. Under these conditions, cryoEM structures of the as-isolated MoFe-protein and stabilized MoFe-protein-Fe-protein ADP-AlF4-complex were generally found to closely resemble their corresponding X-ray crystallographic structures. The cryoEM structures did reveal disordering in regions of the MoFe-protein \u03b1-subunit reminiscent of that observed previously for the \u0394nifB MoFe-protein lacking the FeMo-cofactor, suggesting that this disorder may reflect functionally relevant dynamics, as well as the possibility of asymmetric binding of the Fe-protein to the MoFe-protein in solution. The methods presented here pave the way toward the capture and interrogation of turnover-relevant nitrogenase states by cryoEM.", "doi": "10.1101/2022.06.04.494841", "publication_date": "2022-06-05" }, { "id": "authors:37mvw-kze83", "collection": "authors", "collection_id": "37mvw-kze83", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20220511-226093100", "type": "monograph", "title": "Selenocyanate Derived Se-Incorporation into the Nitrogenase Fe Protein Cluster", "author": [ { "family_name": "Buscagan", "given_name": "Trixia M.", "orcid": "0000-0001-8242-9203", "clpid": "Buscagan-Trixia-M" }, { "family_name": "Kaiser", "given_name": "Jens T.", "orcid": "0000-0002-5948-5212", "clpid": "Kaiser-Jens-T" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "abstract": "The nitrogenase Fe protein mediates ATP-dependent electron transfer to the nitrogenase MoFe protein during nitrogen fixation, in addition to catalyzing MoFe protein independent substrate (CO\u2082) reduction and facilitating MoFe protein metallocluster biosynthesis. The precise role(s) of the Fe protein Fe\u2084S\u2084 cluster in some of these processes remains ill-defined. Herein, we report crystallographic data demonstrating ATP-dependent chalcogenide exchange at the Fe\u2084S\u2084 cluster of the nitrogenase Fe protein when potassium selenocyanate is used as the selenium source. The observed chalcogenide exchange illustrates that this Fe\u2084S\u2084 cluster is capable of core substitution reactions under certain conditions, adding to the Fe protein's repertoire of unique properties.", "doi": "10.1101/2022.04.29.490034", "publication_date": "2022-05-02" }, { "id": "authors:vezvm-wdv47", "collection": "authors", "collection_id": "vezvm-wdv47", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200210-132609996", "type": "monograph", "title": "A fast genetically encoded fluorescent sensor for faithful in vivo acetylcholine detection in mice, fish, worms and flies", "author": [ { "family_name": "Borden", "given_name": "Philip M.", "clpid": "Borden-P-M" }, { "family_name": "Shivange", "given_name": "Amol V.", "clpid": "Shivange-A-V" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Fan", "given_name": "Chengcheng", "orcid": "0000-0003-4213-5758", "clpid": "Fan-Chengcheng" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "abstract": "Here we design and optimize a genetically encoded fluorescent indicator, iAChSnFR, for the ubiquitous neurotransmitter acetylcholine, based on a bacterial periplasmic binding protein. iAChSnFR shows large fluorescence changes, rapid rise and decay kinetics, and insensitivity to most cholinergic drugs. iAChSnFR revealed large transients in a variety of slice and in vivo preparations in mouse, fish, fly and worm. iAChSnFR will be useful for the study of acetylcholine in all organisms.", "doi": "10.1101/2020.02.07.939504", "publication_date": "2020-02-08" }, { "id": "authors:ctt4r-4zn75", "collection": "authors", "collection_id": "ctt4r-4zn75", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20191111-072609203", "type": "monograph", "title": "Crosslinking of nucleotide binding domains improves the coupling efficiency of an ABC transporter", "author": [ { "family_name": "Fan", "given_name": "Chengcheng", "orcid": "0000-0003-4213-5758", "clpid": "Fan-Chengcheng" }, { "family_name": "Kaiser", "given_name": "Jens T.", "orcid": "0000-0002-5948-5212", "clpid": "Kaiser-J-T" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "abstract": "ATP Binding Cassette (ABC) transporters often exhibit significant basal ATPase activity in the absence of transported substrates. To investigate the factors that contribute to this inefficient coupling of ATP hydrolysis to transport, we characterized the structures and functions of variants of the bacterial Atm1 homolog from Novosphingobium aromaticivorans (NaAtm1), including forms with disulfide crosslinks between the nucleotide binding domains. Unexpectedly, disulfide crosslinked variants of NaAtm1 reconstituted into proteoliposomes not only transported oxidized glutathione, but also exhibited more efficient coupling of ATP hydrolysis to GSSG transport than the native transporter. These observations suggest that enhanced conformational dynamics of reconstituted NaAtm1 may contribute to the inefficient use of ATP. Understanding the origins of this uncoupled ATPase activity, and reducing the impact through disulfide crosslinking or other protocols, will be critical for the detailed dissection of ABC transporter mechanism to assure that the ATP dependent steps are indeed relevant to substrate translocation.", "doi": "10.1101/836676", "publication_date": "2019-11-09" } ]