From: Brennan Bonnet
Date: 4 November 2011 21:41
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~
----------
From: Albert Guskov
Date: 4 November 2011 21:41
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~
----------
From: Albert Guskov
Hi Brennan,
we did some time ago a kind of similar experiment, where
we used Xe and Kr to contour the channels within Photosystem II (see Gabdulkhakov et al, Structure. 2009 Sep 9;17(9):1223-34 for the pressure, wavelength and derivatization time values we used)
However before doing this we went the same way you're going now - we tried this technique with lysozyme crystals and as I remember it worked fantastically well and we could solve the structure by SAD within few minutes at the beam line.
From your description it seems you're on the right scale of time and pressure and according to your table you have detectable anomalous signal.
Are you sure you've calculated your anomalous map correctly? Have you solved the structure with SAD or first with MR?
Best regards,
Albert
Albert GUSKOV (Dr) | Research Fellow | Division of Structural & Computational Biology | Nanyang Technological University
Proteos 7-01, Biopolis Drive 61, Singapore 138673 Tel: (65) 6586-9690 GMT+8h
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