Experiences from the Structure Determination of Human Cytomegalovirus Protease

Abstract

Several obstacles were encountered and overcome during the structure determination of human cytomegalovirus protease. Dehydration of crystals, by exposing them to higher concentrations of the precipitant, reduced the mosaicity of the crystals and may have also resolved their microscopic twinning. The initial phase information was obtained with the selenomethionyl multiple-wavelength anomalous diffraction technique. However, site-specific mutagenesis was required to introduce extra Met residues into the protease. The phase information had to be improved by non-crystallographic symmetry averaging, initially among three 'crystal forms'. A change in the composition of the artificial mother liquor led to a significant improvement, from 3.0 and 2.0A resolution, in the diffraction quality of the crystals. The experiences reported here may prove useful to structure determination of other proteins.

Conserved mode of peptidomimetic inhibition and substrate recognition of human cytomegalovirus protease

“…The cause of this change in the dimer organization is currently unknown. Treatment of free enzyme crystals with Na 2 SO 4 also produced a re-organization of the dimer (and a significant improvement in diffraction quality) 3,11 . It appears that the herpesvirus protease dimer interface is capable of adopting alternative conformations, as the varicellar-zoster virus protease (which is a member of the herpesvirus family) shows an even larger reorganization 7 .…”

“…There are two dimers of the protease in the asymmetric unit. This unit cell is related to that of the free enzyme reported by us earlier 3,11 .…”

Post-crystallization treatments for improving diffraction quality of protein crystals

“…2; Heras et al, 2003). Crystals of DsbG dehydrated in this way exhibited a dramatic improvement in diffraction quality, with the pattern improving from 10 Å to beyond 2 Å resolution (Table 2) Combining dehydration with other post-crystallization treatments such as annealing, soaking, cryocooling or rehydration has also resulted in spectacular improvements in the diffraction quality and resolution of protein crystals (Table 2) (Izard et al, 1997;Tong et al, 1997;Pang et al, 2002;Abergel, 2004). For example, a recent publication reported the dramatic improvement in diffraction resolution of three protein crystals upon annealing and dehydration [Escherichia coli YbgL from 12 to 2.6 Å , E. coli YggV (HAM1) from 12 to 2.6 Å and Candida albicans 3-dehydroquinate dehydratase from no diffraction to 3 Å ].…”

Increasing the X-ray Diffraction Power of Protein Crystals by Dehydration: The Case of Bovine Serum Albumin and a Survey of Literature Data

“…Good results have been also obtained when protein crystals are mounted in a specific and adjustable stream of humidified gas, where it is possible to control the relative humidity [26,43–48,86–88]. Finally, crystal dehydration can also be performed by transferring the crystals into a dehydrating solution, which is the original mother liquor with a higher concentration of precipitant [24,27,50–70] or with a different dehydrating agent [49,71–85]. …”