Crystal structures of monoclinic and triclinic formsThe monoclinic and triclinic crystals are distinct units but always grow stacked on top of each other. The stacking direction in each case is the c*-axis (i.e., the (0 0 1) face-normal), which has about the same d-spacing: 105 Å and 101 Å, respectively. It is an open question if the crystals form separately and then stick together, or whether one form crystallizes fi rst and the other grows on top, using the fi rst as a kind of template (akin to epitaxial growth). In any case, so far it has proven impossible to separate them. The Agilent SuperNova A diffractometer was used to examine these crystal aggregates. CrysAlisPro software was found to be effective in separating out the monoclinic and triclinic domains. Data for each domain were processed. Each crystal form was then solved and refi ned in the CCP4 program suite. The results demonstrate that the data for each domain is of suffi cient quality to yield clear and unambiguous crystal structure solutions.

IntroductionHuman hookworm infection is a major cause of anemia and malnutrition of adults and children in the developing world. The major pathology occurs as a consequence of adult hookworms that feed on blood in the human small intestine. As part of on-going efforts to control hookworm infection, the Human Hookworm Vaccine Initiative has identifi ed candidate vaccine antigens from the infective L3 larval stages and adult stages of the parasite. Adult stage antigens include the cytosolic glutathione-S-transferases (GSTs). In addition to their antigenic properties, the GSTs have been shown to have a high affi nity for heme and other metal porphyrins. This is considered to be an adaptation related to the blood-feeding activity of the parasite. In order to clarify the role of hookworm GSTs as possible drug and vaccine targets, we have undertaken X-ray structural analysis of Na-GST-1 from the human hookworm parasite N. americanus.

Agilent SuperNova A diffractometer

Diffraction image showing the superposition of spots from the two crystal domains

CrysAlisPro software was used to fi nd the two domains. …

… and the data for each domain were processed separately.

EwaldPro ScreenshotsTriclinic form in white, reduced monoclinic form in red. View looking down b* on left, c* on right. The two domains appear to line up along c*, which is the crystal stacking direction.

Na-GST-1-Na-porphyrin

Three Crystal Forms of Glutathione S-Transferase from Necator Americanus (Na-GST-1)Chris Ceccarelli, Agilent Technologies, Inc., 5301 Stevens Creek Blvd., Santa Clara, CA 95051Oluwatoyin A. Asojo, National School of Tropical Medicine, Baylor College of Medicine, 1102 Bates Ave., Houston, TX 77030

Structure AnalysisThe active form of this enzyme is a homodimer. The four homodimers found in the three ASUs of these crystals have virtually the same tertiary structure. The pairwise superposition RMS differences for all main-chain atoms do not exceed 0.8 Å. Variability in protein preparation and purifi cation, and differences in pH or type and concentration of crystallization reagents, notably the addition of inhibitors, causes the polymorphism of this enzyme.

Agilent Xcalibur PX Ultra diffractometer

Reconstructed (h0l) and (0kl) precession images showing spot resolution along c* (vertical axis)

The crystal structure of the orthorhombic form was solved by molecular replacement in PHASER (A. J. McCoy et al., J. Appl. Cryst. (2007), 40, 658-674) and is being refi ned in REFMAC (G.N. Murshudov et al., Acta Cryst. (1997), D53, 240-255). The current agreement indices are R(F) = 24% and Rfree = 33% for one homodimer and 17 water molecules in the ASU.

Na-GST-1-C2221

Orthorhombic C2221

Monoclinic C2 Triclinic P1

Web: www.agilent.com/chem/xrdEmail: xrdapplications@agilent.com

Crystal structure of the orthorhombic formThe orthorhombic form crystallizes either in space group C2221 with one homodimer per ASU or in space group P212121 with two homodimers per ASU. The cell dimensions are about the same, 48 x 80 x 200 Å. The long axis was resolved quite easily using only a 100 mm detector distance by collecting narrow frames (0.2° frame width) and by making use of a key feature of the automated data-collection strategy routine of CrysAlisPro. The strategy was constrained to position the four-circle Kappa goniometer such that the long axis was in the detector plane of as many of the diffraction images as possible. One or two other orientations were required in order to collect a complete data set. While the P centered crystal form can be collected and indexed on other setups, the process was laborious and required visually attempting to mount the crystals to avoid the thinnest face as well as increasing the detector distance to 200 mm. We were unable to integrate a complete data set of the C centered crystals without using the automated longest axis fi xed strategy of CrysAlisPro.

The crystal structures of the monoclinic and triclinic forms were solved by molecular replacement in PHASER and are being refi ned in PHENIX ( P.V. Afonine et al., Acta Cryst. (2012), D68, 352-367) and REFMAC. For the monoclinic form, the current agreement indices are R(F) = 23% and Rfree = 28% for one homodimer and 28 water molecules in the ASU. For the triclinic form, the current agreement indices are R(F) = 22% and Rfree = 28% for two homodimers and 63 waters in the ASU.

crystal structure solutions.

1903-11_Agilent_Poster_ACA_Chris Ceccarelli 1_1 (JH)_AW HR.indd 1 16/07/2013 16:49