Calnexin is a membrane anchored lectin involved in the control of protein trafficking through the endoplasmic reticulum and promotes correct folding of glycoproteins. We have crystallized the luminal domain of calnexin, which has a molecular weight of 45,00, in two crystal forms. One form is tetragonal with one molecule per asymmetric unit and is very temperature sensitive. The second form appears orthorhombic, but we have not yet obtained crystals large enough to characterize on a rotating anode source. We have developed conditions for flash freezing ithe first tetragonal crystals for data collection. With the conventional source the crystals diffract weakly beyond about 4 A resolution, although we observe some reflections up to 3.3 A resolution. We have collected a full dataset to 4 A resolution and are in the process of screening for useful heavy atom derivatives. Based on the experience of other crystallographers, we hope that at CHESS the available crystals will show diffraction beyond 3.0A and lead to a well determined 3-D structure of this extremely interesting and important protein. We had hoped to test the diffraction limits and collect data to beyond 3 A resolution during our time at CHESS, but were unable to collect any useful data as a result of a combination of factors. First, our crystal transport was not successful and most of the crystals were accidentally frozen and cracked on thawing at CHESS. Some of the crystals looked all right optically, but when tested did not diffract beyond the resolution that we see on a anode. Most showed split replections indicative of small cracks. These problems were compounded by the fact that the small refrigerators near the beam lines were inadequate for maintaining the crystals and crystal quality dropped rapidly. We were also unfortunate in that our time coincided with a number of extended technical difficulties with the beam. The combination of down time and crystal damage in transport and at the beamline prevented any useful data collection on this trip. The crystal transport problem will be solved by freezing crystals in advance and transporting and storing them at liquid nitrogen temperatures. This should render the crystals stable to transport and storage while at CHESS and should allow evaluation of diffraction limits and collection of the best data possible from these crystals.
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