Hi everyone,
My name is Brennan Bonnet and I am doing my Master’s project on SAD phasing of
proteins using xenon gas.
I plan on doing several proteins but first I am trying to get everything
working smoothly on lysozyme since it is easy to grow, diffracts well, and is
already known to bind xenon atoms (PDB entry 1C10, 3 sites @ 8bar pressure).
Put simply, my method involves growing the crystals, mounting them in
cryoloops, cryoprotection (if required), pressurization by xenon gas using the
Hampton Xenon Chamber and quickly freezing them using liquid nitrogen with only
a couple of seconds between depressurization and freezing.
I have chosen pressures and durations of pressurization based on previous work
which indicates that suitable derivatives may be produced using pressures
between 1-100 bar and that binding should be complete within minutes. (see Use
of Noble Gases Xenon and Krypton as Heavy Atoms in Protein Structure
Determination by M. Schiltz, R. Fourme, and Thierry Prangé for a summary).
Therefore I have chosen to use pressures between 7-28bar (100-400psi) for a
duration of 15 minutes. After processing using XDS and solving with Phenix,
the results show occupancies <0.1 which indicate no xenon binding or at least
nothing that “sticks out”. The general trend is that increased xenon pressure
results in a stronger anomalous signal and I have attached a table below with
some processed data from XDS.
I plan on trying a couple of other things. I have been collecting at 7keV but
plan to try 6keV in order to get closer to the xenon L-edges and get a better
anomalous signal. I also plan on trying longer pressurization times up to an
hour to hopefully get better occupancy.
Has anyone had any success with this method or any sort of “Aha!” moment? Help
in the matter would be much appreciated.
Set Pressure Energy Anom Max Anom Total Anom
Max / Anom Total Rmeas Resolution Last shell I/σ(I)
(psi) (keV)
(%)
(Å)
Sep7 100 7 2.111 1.352
1.561390533 4 2.03
13.9
Sep7 160 7 1.935 1.244
1.555466238 4.7 2.03
12.17
Sep7 200 7 2.454 1.293
1.897911833 3.4 2.03
11.01
Jul28 200 7 3.13
1.749 1.789594054 2.2 2.03
17.6
Sep7 280 7 2.375 1.54
1.542207792 3.1 2.03
14.33
Jul28 400 7 3.082 1.649
1.869011522 3.5 2.03
14.29
Thanks,
~Brennan~
