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 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.

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. TM1389/17314 Crystallization and X-Ray Diffraction Screening TM1389 crystal

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. X-Ray Data Collection 2.3 Ǻ

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. Automated Processing of X-Ray Diffraction Data from TM1389 Mosflm Autoindexing Diffraction Intensities Scala Scaling Reflection intensities SHARP Phasing Density Modification ARP/wARP Automated Model Building Amino Acid Sequence X-Ray Diffraction Intensities Outline of High Throughput Processing Strategy Developed by JCSG for X-Ray Crystallographic Data

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. Refinement

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)

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.

X-Ray Data Collection and Refinement Statistics Xtal ID Xtal ID 17135

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. 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. Overall Structure of the TM1389

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

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. Proposed Cofactor Binding Site

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

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.