SSGCID (Seattle Structural Genomics Center for Infectious Disease) is a NIAID-funded structural genomics effort targeting medically relevant proteins of class A and B pathogens. All the crystallography for SSGCID takes place here at Emerald Biostructures. While most structures of our targets can be solved by Molecular Replacement, a number of structure solutions require experimental phase information.
In order to obtain experimental phases, we recently have had very encouraging successes taking advantage of the strong anomalous signal that iodide has at CuKa-wavelength (f"=6.8e-), which is even stronger than the anomalous signal for selenium at the edge (f"=3.7e-). What was intended to be a test study yielded four new in-house phased structure within a month. As a start for this technique, we used a very bold approach: Crystals were transferred stepwise in a buffer containing up to 1M KI. The crystals were soaked in the high-iodide buffer for about 2 hours and then vitrified. Data were collected on our in-house diffractometer (Rigaku MicroMaxHF007 with a Saturn 944+ detector). Again, in a rather bold approach, we collected 360° of data in 0.25° frames with rather short exposures (~10s). Data were processed with XDS/XSCALE.
In our first case, a plasmid partition protein from Borrelia burgdorferi (BobuA.01478.a, pdb: 3k9g), crystallized in a tetragonal space group using NaCl as the precipitant, which was in two steps partially replaced with KI. A 25-fold redundant data set was collected, which showed an anomalous signal out to 2.25Å resolution. Seeding Phaser_EP with 2 strong sites, a total of 16 sites were eventually found and refined. After density modification with PARROT the electron density was readily interpretable (see picture at the end), and almost the entire model was built with BUCCANEER, which then could be extended with ARP/wARP. Further analyses revealed the importance of data redundancy: 180° of data yielded about the same results as 360°, while 90° of data would not yield the structure quite as readily. Other program packages, such as SHARP + ARP/wARP or PHENIX could also solve this structure easily.
In a second case, a deoxycytidine triphosphate deaminase from Anaplasma phagocytophilum (AnphA.00973.a, pdb 3km3), crystallized in a rhombohedral space group and yielded a 10-fold redundant data set up to 2.1Å resolution. Here, the anomalous signal was not as strong as in the previous case. Phaser_EP was seeded with 13 strong and weak iodide sites, eventually 16 sites were found and refined. Again, almost the entire model could be built automatically.
Within the past month, these two structures and another two structures could be solved using anomalous phase information from iodide. Structure #3 yields very well diffracting however twinned crystals; structure #4 only diffracted to 3Å, however phase combination with a weak molecular replacement solution easily yielded the structure. So far, we have only used rather short soaks with 1M KI. Further tests are planned to optimize for lower iodide concentrations.
These results indicated that iodide anomalous phasing can be used as the first shot for determining a new structure that requires experimental phases. With well diffracting crystals and high symmetry, structures can be built within hours after data collection. This technique has shown to work with a twinned data set; for another case a weak Molecular Replacement solution was of trememdous help. Adavantages of iodide soaks are:
- no growth of Se-Met labelled protein is necessary;
- strong anomalous signal in-house;
- high concentrations of iodide are possible during soaks;
- iodide can occupy both positively charged/polarized and hydrophobic pockets, less of a search than with heavy metals;
- it really works!
experimental electron density after in-house iodide phasing