Computational protocol: Classifying the metal dependence of uncharacterized nitrogenases

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Protocol publication

[…] NifD homologs were obtained from the NCBI non-redundant database through BLASTp searches using NifD, VnfD, and AnfD proteins from A. vinelandii as queries (YP_002797379, YP_002797497, and YP_002801974, respectively). In the case of NifD searches, sequences were compiled using Pattern Hit Initiated BLAST (PHI-BLAST) specifying the NifD conserved “CXRS” amino acid pattern, where X is variable. In addition, a length criterion of 300–700 amino acids was imposed by the [slen] command.Sequences were aligned using the EMBL ClustalW2 server with default parameters (Gonnet weight matrix) and manipulated with Jalview (; ) and ClustalX (). Sequences were examined for known catalytic residues as reported in . Phylogenies were generated using the PhyML webserver () and calculated using the maximum likelihood method (; ) using Shimodaira–Hasegawa-like aLRT supports and the LG substitution matrix (). The resulting phylogram was projected using Fig Tree version 1.3.1, and naming accomplished using the REFGEN/TREENAMER online web server (). Sequence alignments for each of the protein classes used in this study are available upon request from the authors. Analysis of amino acid conservation was judged by the presence of a * (conserved) or a colon above the alignment in ClustalW corresponding to amino groups of strongly similar properties (scoring >0.5 in the Gonnet PAM 250 matrix). [...] Models of NifD and NifD homologs were generated through sequence submission to the iterative threading assembly refinement (I-TASSER) server (, ; ). The top model based on C-score () was selected for further analysis. The C-scores are derived from calculating convergence of intermediate structures that are produced during the I-TASSER run and range from -5 to 2 with high scores signifying models with high confidence (). Structures were visualized using PyMol. Models were selected in an effort to obtain a representative diversity from each nitrogenase lineage. In the case of uncharacterized nitrogenases, these included nitrogenase associated with the archaeal methanogen Methanocaldococcus infernus ME (YP_003615674.1), an uncultured archaeal methanotroph believed to belong to the ANME-2c clade (ADF27322.1; ), and the firmicutes Candidatus Desulforudis audaxviator MP104C (YP_001716346.1) and Syntrophothermus lipocalidus DSM 12680 (YP_003703435.1). A NifD homolog from a ANME-1 phylotype () was not included in this analysis as the sequence lacks ligand binding residues and is likely a “Nif-like” protein which are conserved in methanogenic archaea (). [...] Inferred structures were compared using an “all against all” comparison performed by the ProCKSI server. The relatedness of structures was calculated with standardized distance matrix derived from the Vorolign V-score () and hierarchical clustering accomplished using the Complete Link (furthest neighbor) method () with the Clustering Calculator server.Active site volumetric calculations were performed on the CASTp server with a probe radius of 1.4 Å (). Cavities were manually inspected in each case to ensure correspondence with the nitrogenase metal cofactor-binding pocket. In order to identify putative conserved active site second shell residues, representative nitrogenase structures from the Protein Data Bank (PDB; ) and those created through homology modeling were structurally aligned with the Pymol program. The amino acids within 5 Å of the metal cofactor in the A. vinelandii 1M1N structure (), including homocitrate, were selected for analysis. […]

Pipeline specifications

Software tools BLASTP, Clustal W, Jalview, PhyML, I-TASSER, PyMOL, ProCKSI, Vorolign, CASTp
Applications Phylogenetics, Protein structure analysis
Chemicals Ammonia, Molybdenum, Vanadium