Carina M. C. Lobley

Decision making in xia2

The basis for decision making in the program xia2 is described, alongside the framework to support these protocols. Where appropriate, applications of these protocols to interactive data processing are highlighted.

Structural Constraints on protein self-processing in L-aspartate-alpha-decarboxylase

Aspartate decarboxylase, which is translated as a pro‐protein, undergoes intramolecular self‐cleavage at Gly24–Ser25. We have determined the crystal structures of an unprocessed native precursor, in addition to Ala24 insertion, Ala26 insertion and Gly24→Ser, His11→Ala, Ser25→Ala, Ser25→Cys and Ser25→Thr mutants. Comparative analyses of the cleavage site reveal specific conformational constraints that govern self‐processing and demonstrate that considerable rearrangement must occur. We suggest that Thr57 Oγ and a water molecule form an ‘oxyanion hole’ that likely stabilizes the proposed oxyoxazolidine intermediate. Thr57 and this water molecule are probable catalytic residues able to support acid–base catalysis. The conformational freedom in the loop preceding the cleavage site appears to play a determining role in the reaction. The molecular mechanism of self‐processing, presented here, emphasizes the importance of stabilization of the oxyoxazolidine intermediate. Comparison of the structural features shows significant similarity to those in other self‐processing systems, and suggests that models of the cleavage site of such enzymes based on Ser→Ala or Ser→Thr mutants alone may lead to erroneous interpretations of the mechanism.

S-Adenosyl-S-carboxymethyl-L-homocysteine: a novel cofactor found in the putative tRNA-modifying enzyme CmoA.

The putative methyltransferase CmoA is involved in the nucleoside modification of transfer RNA. X-ray crystallography and mass spectrometry are used to show that it contains a novel SAM derivative, S-adenosyl-S-carboxymethyl-l-homocysteine, in which the donor methyl group is replaced by a carboxymethyl group.

Application of in situ diffraction in high-throughput structure determination platforms.

Macromolecular crystallography (MX) is the most powerful technique available to structural biologists to visualize in atomic detail the macromolecular machinery of the cell. Since the emergence of structural genomics initiatives, significant advances have been made in all key steps of the structure determination process. In particular, third-generation synchrotron sources and the application of highly automated approaches to data acquisition and analysis at these facilities have been the major factors in the rate of increase of macromolecular structures determined annually. A plethora of tools are now available to users of synchrotron beamlines to enable rapid and efficient evaluation of samples, collection of the best data, and in favorable cases structure solution in near real time. Here, we provide a short overview of the emerging use of collecting X-ray diffraction data directly from the crystallization experiment. These in situ experiments are now routinely available to users at a number of synchrotron MX beamlines. A practical guide to the use of the method on the MX suite of beamlines at Diamond Light Source is given.

pH-tuneable binding of 2'-phospho-ADP-ribose to ketopantoate reductase: a structural and calorimetric study.

A combined crystallographic, calorimetric and mutagenic study has been used to show how changes in pH give rise to two distinct binding modes of 2′-phospho-ADP-ribose to ketopantoate reductase.

Threonine 57 is required for the post-translational activation of Escherichia coli aspartate α-decarboxylase

Threonine 57 is identified as the key residue required for the post-translational activation of E. coli aspartate decarboxylase. The crystal structure of the site-directed mutant T57V is reported.

A generic protocol for protein crystal dehydration using the HC1b humidity controller

A generic protocol for investigating crystal dehydration is presented and tested with a set of protein crystal systems using the HC1b high-precision crystal humidifier/dehumidifier.

Structure of Escherichia coli aspartate α-decarboxylase Asn72Ala: probing the role of Asn72 in pyruvoyl cofactor formation

The crystal structure of the Asn72Ala site-directed mutant of Escherichia coli aspartate α-decarboxylase (ADC) has been determined at 1.7 Å resolution. The refined structure is consistent with the presence of a hydrolysis product serine in the active site in place of the pyruvoyl group required for catalysis, which suggests that the role of Asn72 is to protect the ester formed during ADC activation from hydrolysis. In previously determined structures of activated ADC, including the wild type and other site-directed mutants, the C-terminal region of the protein is disordered, but in the Asn72Ala mutant these residues are ordered owing to an interaction with the active site of the neighbouring symmetry-related multimer.

Structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC118

The structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC188 has been determined at 1.72 Å resolution. The structure was solved by molecular replacement, which identified the functional homodimer in the asymmetric unit. Despite only showing 57% sequence identity to its closest homologue, the structure adopted the typical α and β D-ribose 5-phosphate isomerase fold. Comparison to other related structures revealed high homology in the active site, allowing a model of the substrate-bound protein to be proposed. The determination of the structure was expedited by the use of in situ crystallization-plate screening on beamline I04-1 at Diamond Light Source to identify well diffracting protein crystals prior to routine cryocrystallography.

High-resolution structures of Lactobacillus salivarius transketolase in the presence and absence of thiamine pyrophosphate

The crystal structure of L. salivarius transketolase has been determined to high resolution in the presence and absence of thiamine pyrophosphate. The structures are presented with a brief comparison with other known transketolase structures.