The C₁subunit of α-crustacyanin: thede novophasing of the crystal structure of a 40 kDa homodimeric protein using the anomalous scattering from S atoms combined with direct methods

Abstract

The previously unknown crystal structure of the C1 subunit of the carotenoid‐binding protein α‐crustacyanin has been determined using the anomalous scattering available at 1.77 Å wavelength to determine the partial structure of the S atoms intrinsic to the native protein. The resulting `heavy‐atom' phases, in conjunction with near‐atomic resolution (dmin = 1.15 Å) data, were then used to initiate successful structure determination using a direct‐methods approach. This is, to the authors' knowledge, the first time such a small anomalous signal (∼1%) has been used to aid the determination of a macromolecular structure. As well as the structure itself, the methods used during data collection and those used in the elucidation of the sulfur `heavy‐atom' partial structure are described here. As predicted, the C1 subunit adopts a tertiary structure typical of the lipocalin superfamily: an eight‐stranded antiparallel β‐barrel with a repeated +1 topology. The β‐barrel has a calyx shape with the two molecules in the asymmetric unit interacting in such a way that the open ends of each calyx face each other, although they do not form a single elongated pocket. A comparison of this structure with those of other members of the lipocalin superfamily has allowed speculation as to the nature of carotenoid binding by the protein.

Recent advances in theCRANKsoftware suite for experimental phasing

“…A total of 132 data sets were used and were composed of 78 data sets from the Joint Center for Structural Genomics (JCSG; http://www.jcsg.org/), 1vjn, 1vjr, 1vjz, 1vk4, 1vkm, 1vlm, 1vqr, 1z82, 1zy9, 1zyb, 2a2m, 2a2o, 2a3n, 2a6b, 2aml, 2avn, 2b8m, 2etd, 2etj, 2ets, 2etv, 2evr, 2f4p, 2fea, 2ffj, 2fg0, 2fg9, 2fna, 2fqp, 2fur, 2fzt, 2g0t, 2g42, 2gc9, 2nlv, 2nuj, 2nwv, 2o08, 2o1q, 2o2x, 2o2z, 2o3l, 2o62, 2o7t, 2o8q, 2obp, 2oc5, 2od5, 2od6, 2oh3, 2okc, 2okf, 2ooj, 2opk, 2osd, 2otm, 2ozg, 2ozj, 2p10, 2p4o, 2p7h, 2p7i, 2p97, 2pg3, 2pg4, 2pgc, 2pim, 2pn1, 2pnk, 2ppv, 2pr7, 2prr, 2prv, 2prx, 2pv4, 2pw4, 2b78 and 2b79; 23 data sets from Mueller-Dieckmann et al (2007), 2g4h, 2g4i, 2g4j, 2g4k, 2g4p, 2g4q, 2g4l, 2g4n, 2g4o, 2g4r, 2g4s, 2g4t, 2g4u, 2g4v, 2g4w, 2g4x, 2g4y, 2g4z, 2ill, 2g51, 2g52, 2g54 and 2g55; and 31 from various other individual data-set contributors, 1e42 (Owen, Vallis et al, 2000), 1e6i (Owen, Ornaghi et al, 2000), 1hf8 (Ford et al, 2001), 2ahy (Shi et al, 2006), 2hba (J.-H. Cho, S. Sato, E. Y. Kim, H Schindelin & D. P. Raleigh, unpublished work), 2o0h (Sun et al, 2007), 2rkk (Xiao et al, 2008), 3bpj (L. Nedyalkova, B. Hong, W. Tempel, F. MacKenzie, C. H. Arrowsmith, A. M. Edwards, J. Weigelt, A. Bochkarev & H. Park, unpublished work), 2fdn (Dauter et al, 1997), 1of3 (Boraston et al, 2003), 1i4u (Gordon et al, 2001), 1dw9 (Walsh et al, 2000), 1v0o (Holton et al, 2003), 1fse (Ducros et al, 2001), 1xib (Carrell et al, 1989), 1fj2 (Devedjiev et al, 2000), 1h29 (Matias et al, 2002), 1c8u (Jia et al, 2000), 1lvy (Schiltz et al, 1997), 1lz8 (Dauter et al, 1999), 1e3m (Lamers et al, 2000), 1ga1 (Dauter et al, 2001), 1djl (White et al, 2000), 1dtx (Skarzynski, 1992), 1dpx (Weiss, 2001), 1mso (Smith et al, 2003), 1ocy …”

SAD at home: solving the structure of oxalate decarboxylase with the anomalous signal from manganese using X-ray data collected on a home source

“…However, it was not until comparatively recently that more challenging macromolecular structures were being routinely solved by the SAD technique using relatively weak anomalous signals (Jasko  lski, 1996;Dauter et al, 2000Dauter et al, , 2002Liu et al, 2000;de Graaff et al, 2001;Gordon et al, 2001;Weiss et al, 2001;Lemke et al, 2002;Li et al, 2002;Debreczeni et al, 2003;Dodson, 2003;Ramagopal et al, 2003;Uso  n et al, 2003;Yang et al, 2003). Clearly, these advances have been driven by a combination of improved instrumentation and the development of powerful software algorithms to process and analyse the X-ray data.…”

ACORN in CCP4 and its applications

“…This structure (Gordon et al, 2001) contained 362 residues (3382 non-H atoms) in the asymmetric unit with space group P2 1 2 1 2 1 . The native data were collected to 1.15 A Ê resolution and anomalous scattering data to 1.77 A Ê .…”