The Crystallographic Refinement of TM1389- A methyl-transferase from Thermotoga maritima

1. The Crystallographic Refinement of TM1389- A methyl-transferase from Thermotoga maritima Rosanne Joseph SLAC Summer Intern Joint Center for Structural Genomics (JCSG) Stanford Synchrotron Radiation Laboratory, Menlo Park, CA, USA The JCSG is funded by the Protein Structure Initiative of the National Institutes of Health, National Institute of General Medical Sciences.SSRL operations is funded by DOE BES, and the SSRL Structural Molecular Biology program by DOE BER, NIH NCRR BTP and NIH NIGMS.

2. Crystallization and X-Ray Diffraction Screening TM1389 crystal TM1389/17314 Crystal of TM1389. The best diffracting crystals used for the structure determination were obtained using solutions containing 1M LiCl, 10%w/v PEG 6000, 0.1M citrate pH 5.0. For x-ray screening and data collection at liquid nitrogen temperatures, the crystals were treated with 10% ethylene glycol as a cryoprotectant.

3. X-Ray Data Collection 2.3 Ǻ • This is the x-ray diffraction image of a TM1389 crystal. Data were recorded to a resolution of 2.3 Ǻ taken at SSRL on Beamline 9-2. • During the data collection, an x-ray diffraction intensity for each reflection is recorded. These intensities are used to determine the atomic structure of the protein.

4. Automated Processing of X-Ray Diffraction Data from TM1389 X-Ray Diffraction Intensities Amino Acid Sequence Outline of High Throughput Processing Strategy Developed by JCSG for X-Ray Crystallographic Data Mosflm Scala SHARP ARP/wARP Autoindexing Diffraction Intensities Automated Model Building Scaling Reflection intensities Phasing Density Modification The XPLEO Web Server developed by Henry van den Bedem was used to fill five short three-residue amino acid in the gaps in our initial trace of the TM1389 polypeptide backbone and sidechains. http://smb.slac.stanford.edu/~vbedem

5. Refinement • The ccp4i program was used as a graphical interface for refinement of TM1389 with the program REFMAC. • Refinement is the procedure in which we manipulate the atomic coordinates and temperature factors in order to minimize the discrepancies between our observed x-ray diffraction intensities and intensities calculated from the model.

6. COOT-A New Molecular Graphics Program for Refinement A Screenshot of the Coot Graphics Window- A portion of the 2.3 Ǻ 2Fo-Fc electron density map (contoured at 1 σ) and superimposed on the structure of TM1389. Note the maps clearly indicate the location of the β-sheet. A Screenshot of the Coot Graphics Window in the vicinity of the S-adenosylhomocysteine cofactor (SAH)

7. Refinement Procedure-TM1389 • Used coot program to start refinement. Went through the protein to check for sidechains that were out of density. • Filled in all gaps using XPLEO. • Refined with Refmac. • Ran through MOLPROBITY (Jane Richardson’s structure validation server) to check for geometrically unfavorable sidechain orientations. Implemented COOT to correct these. • Implemented ARP/wARP and COOT to add waters and the SAH cofactor. http://kinemage.biochem.duke.edu/molprobity/index-king.html

8. X-Ray Data Collection and Refinement Statistics Xtal ID 16650 Xtal ID 17135

9. Overall Structure of the TM1389 A schematic representation of the overall structure and connectivity of the TM1389. The green circles symbolize α-helices while the purple triangles symbolize the β- sheets. TM1389 belongs to a general class of SAM-dependent methyltransferases containing a central seven-stranded β-sheet and several α-helices. The ribbon representation of the structure of TM1389. The crystal structure indicates the TM1389 is a homodimer shown in this view. The β-sheets on each monomer are shown in blue, the α-helices in cyan. The SAH cofactor is in pink.

10. Structural Homologues 1RI1-Mrna Cap (Guanine N-7) Methyltransferase 1VE3- Methyl Transferase, Sam Dependent Methyltransferase 1VLM- Sam-Dependent Methyltransferase 1Y8C- S-Adenosylmethionine-Dependent Methyltransferase 1XVA- Glycine N-Methyltransferase

11. Proposed Cofactor Binding Site Close- up view of the SAH cofactor (red) and proposed substrate binding site (white surface). The surface was computed with the program PASS that identifies potential binding sites on protein surfaces.

12. MB2097A Ribbon diagram of MB2097A which I refined earlier this summer.

13. Conclusion • I learned techniques and procedures for crystallographic refinement of protein structures. • I learned to implement computational procedures (Linux operating system, COOT computer graphics, PYMOL) for addressing specific scientific problems. • I learned how to identify features of protein structure (alpha-helices, beta-sheets) and identified a bound cofactor (S-adenosylhomocysteine) through x-ray crystallography. I would like to thank Herb Axelrod and Mike Soltis for giving me the opportunity to work in their lab. Herb patiently taught me the techniques used here.