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Human carbonic anhydrase IX (CA IX) expression is upregulated in hypoxic solid tumours, promoting cell survival and metastasis. This observation has made CA IX a target for the development of CA isoform‐selective inhibitors. To enable structural studies of CA IX–inhibitor complexes using X‐ray and neutron crystallography, a CA IX surface variant (CA IXSV; the catalytic domain with six surface amino‐acid substitutions) has been developed that can be routinely crystallized. Here, the preparation of protiated (H/H), H/D‐exchanged (H/D) and deuterated (D/D) CA IXSV for crystallographic studies and their structural comparison are described. Four CA IXSV X‐ray crystal structures are compared: two H/H crystal forms, an H/D crystal form and a D/D crystal form. The overall active‐site organization in each version is essentially the same, with only minor positional changes in active‐site solvent, which may be owing to deuteration and/or resolution differences. Analysis of the crystal contacts and packing reveals different arrangements of CA IXSV compared with previous reports. To our knowledge, this is the first report comparing three different deuterium‐labelled crystal structures of the same protein, marking an important step in validating the active‐site structure of CA IXSV for neutron protein crystallography. [ABSTRACT FROM AUTHOR]

This work presents a comparison of the crystal packing of three eukaryotic membrane proteins: human aquaporin 1, human aquaporin 5 and a spinach plasma membrane aquaporin. All were purified from expression constructs both with and without affinity tags. With the exception of tagged aquaporin 1, all constructs yielded crystals. Two significant effects of the affinity tags were observed: crystals containing a tag typically diffracted to lower resolution than those from constructs encoding the protein sequence alone and constructs without a tag frequently produced crystals that suffered from merohedral twinning. Twinning is a challenging crystallographic problem that can seriously hinder solution of the structure. Thus, for integral membrane proteins, the addition of an affinity tag may help to disrupt the approximate symmetry of the protein and thereby reduce or avoid merohedral twinning.