Norifumi Muraki

In macromolecular structure determination using X-ray diffraction from multiple crystals, the presence of different structures (structural polymorphs) necessitates the classification of the diffraction data for appropriate structural analysis. Hierarchical clustering analysis (HCA) is a promising technique that has so far been used to extract isomorphous data, mainly for single-structure determination. Although in principle the use of HCA can be extended to detect polymorphs, the absence of a reference to define the threshold used to group the isomorphous data sets (the `isomorphic threshold') poses a challenge. Here, unit-cell-based and intensity-based HCAs have been applied to data sets for apo trypsin and inhibitor-bound trypsin that were mixed post data acquisition to investigate the efficacy of HCA in classifying polymorphous data sets. Single-step intensity-based HCA successfully classified polymorphs with a certain `isomorphic threshold'. In data sets for several samples containing an unknown degree of structural heterogeneity, polymorphs could be identified by intensity-based HCA using the suggested `isomorphic threshold'. Polymorphs were also detected in single crystals using data collected using the continuous helical scheme. These findings are expected to facilitate the determination of multiple structural snapshots by exploiting automated data collection and analysis.

In macromolecular structure determination using X-ray diffraction from multiple crystals, the presence of different structures (structural polymorphs) necessitates the classification of diffraction data for appropriate structural analysis. Hierarchical clustering analysis (HCA) is a promising technique that has so far been used to extract isomorphous data, mainly for single structure determination. Although in principle the use of HCA can be extended to detect polymorphs, the absence of a reference for defining a ‘similarity’ threshold used for grouping the isomorphous datasets poses a challenge. Here, we have applied unit cell-based and intensity-based HCAs to the datasets of apo-trypsin and inhibitor-bound trypsin that were mixed post-data acquisition to investigate how effective HCA is in classifying polymorphous datasets. Single-step intensity-based HCA successfully classified polymorphs with a certain ‘similarity’ threshold. In datasets of several samples containing an unknown degree of structural heterogeneity, polymorphs could be identified by intensity-based HCA using the suggested ‘similarity’ threshold. Polymorphs were also detected in single crystals using the data collected by the continuous helical scan method. These findings are expected to facilitate the determination of multiple structural snapshots by exploiting automated data collection and analysis. Synopsis Single-step intensity-based hierarchical clustering is demonstrated to allow the detection of structural polymorphs in the diffraction datasets obtained from multiple crystals. By splitting the datasets collected by continuous helical scan into several chunks, both inter and intra-crystal polymorphs can be successfully analyzed.

We have determined the crystal structure of thermoglobin (AaTgb) from a hyperthermophilic bacterium Aquifex aeolicus. Tyrosine and glutamine at the B10 and E7 position, respectively, are conserved in AaTgb as are the case of single domain hemoglobins (sdHbs). While the binding affinity of O₂ or CO is affected by the replacement of Tyr29 in the distal heme pocket, wild type and Y29F variant of AaTgb show a similar binding affinity of imidazole.

The crystal structures of the conserved region domains of HtaA and HtaB, which act as heme binding/transport proteins in the heme uptake machinery in Corynebacterium glutamicum, are determined for the first time. The molecular mechanism of heme transfer among these proteins is proposed based on the spectroscopic and structural analyses.

Glucoamylases are widely used commercially to produce glucose syrup from starch. The starch-binding domain (SBD) of glucoamylase fromAspergillus nigeris a small globular protein containing a disulfide bond. The structure ofA. nigerSBD has been determined by NMR, but the conformation surrounding the disulfide bond was unclear. Therefore, X-ray crystal structural analysis was used to attempt to clarify the conformation of this region. The SBD was purified from anEscherichia coli-based expression system and crystallized at 293 K. The initial phase was determined by the molecular-replacement method, and the asymmetric unit of the crystal contained four protomers, two of which were related by a noncrystallographic twofold axis. Finally, the structure was solved at 2.0 Å resolution. The SBD consisted of seven β-strands and eight loops, and the conformation surrounding the disulfide bond was determined from a clear electron-density map. Comparison of X-ray- and NMR-determined structures of the free SBD showed no significant difference in the conformation of each β-strand, but the conformations of the loops containing the disulfide bond and the L5 loop were different. In particular, the difference in the position of the C^αatom of Cys509 between the X-ray- and NMR-determined structures was 13.3 Å. In addition, theBfactors of the amino-acid residues surrounding the disulfide bond are higher than those of other residues. Therefore, the conformation surrounding the disulfide bond is suggested to be highly flexible.

Corynebacteria contain a heme uptake system encoded in hmuTUV genes, in which HmuT protein acts as a heme binding protein to transport heme to the cognate transporter HmuUV. The crystal structure of HmuT from Corynebacterium glutamicum (CgHmuT) reveals that heme is accommodated in the central cleft with His141 and Tyr240 as the axial ligands and that Tyr240 forms a hydrogen bond with Arg242. In this work, the crystal structures of H141A, Y240A, and R242A mutants were determined to understand the role of these residues for the heme binding of CgHmuT. Overall and heme environmental structures of these mutants were similar to those of the wild type, suggesting that there is little conformational change in the heme-binding cleft during heme transport reaction with binding and the dissociation of heme. A loss of one axial ligand or the hydrogen bonding interaction with Tyr240 resulted in an increase in the redox potential of the heme for CgHmuT to be reduced by dithionite, though the wild type was not reduced under physiological conditions. These results suggest that the heme environmental structure stabilizes the ferric heme binding in CgHmuT, which will be responsible for efficient heme uptake under aerobic conditions where Corynebacteria grow.

Ferredoxin-NAD(P)⁺ reductase (FNR) is a key enzyme that catalyzes the photoreduction of NAD(P)⁺ to generate NAD(P)H during the final step of the photosynthetic electron-transport chain. FNR from the green sulfur bacterium Chlorobium tepidum is a homodimeric enzyme with a molecular weight of 90 kDa; it shares a high level of amino-acid sequence identity to thioredoxin reductase rather than to conventional plant-type FNRs. In order to understand the structural basis of the ferredoxin-dependency of this unique photosynthetic FNR, C. tepidum FNR has been heterologously expressed, purified and crystallized in two forms. Form I crystals belong to space group C222₁ and contain one dimer in the asymmetric unit, while form II crystals belong to space group P4₁22 or P4₃22. Diffraction data were collected from a form I crystal to 2.4 Å resolution on the synchrotron-radiation beamline NW12 at the Photon Factory.