Transcript The Structures of Two Homologs from Agrobacterium

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.
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.
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 xray diffraction intensity for each
reflection is recorded. These
intensities are used to determine
the atomic structure of the protein.
Automated Processing of X-Ray
Diffraction Data from TM1389
X-Ray Diffraction
Intensities
Outline of High Throughput Processing Strategy Amino Acid Sequence
Developed by JCSG for X-Ray Crystallographic Data
Mosflm
Scala
SHARP
Autoindexing
Scaling
Reflection
intensities
Phasing
Density
Modification
Diffraction
Intensities
ARP/wARP
Automated
Model
Building
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
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.
COOT-A New Molecular Graphics Program for
Refinement
A Screenshot of the Coot Graphics
Window- A portion of the 2.3 Ǻ 2FoFc electron density map (contoured
at 1 σ) and superimposed on the
structure of TM1389. Note the maps
clearly indicate the location of the βsheet.
A Screenshot of the Coot Graphics
Window in the vicinity of the Sadenosylhomocysteine 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.
http://kinemage.biochem.duke.edu/molprobity/index-king.html
•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 16650
Xtal ID 17135
Overall Structure of the TM1389
A schematic
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.
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 sevenstranded β-sheet and several
α-helices.
Structural Homologues
1RI1- Mrna Cap (Guanine N-7)
Methyltransferase
1VE3- Methyl Transferase, Sam
Dependent Methyltransferase
1Y8C- S-AdenosylmethionineDependent Methyltransferase
1VLM- Sam-Dependent
Methyltransferase
1XVA- Glycine NMethyltransferase
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.
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.