Week Two: BioSAXS

I accompanied Professor Andresen to the Cornell High Energy Synchrotron Source (CHESS) to run SAXS experiments on various nucleosome arrays. Friday and a large part of Saturday, we modified the experimental setup. We elongated the tubing in the hutch, inserted a beam stop, switched the detector from a 100K pixel to a 200K and began with an X-ray of 200 by 200 micrometers. 

Inside the hutch, overall view of the setup. The long tube farthest to the left was exchanged with the tube on the wall behind to lower the q value. The X-ray beam enters at the far right.

Further to the right the 200K detector can be seen which is the box on the end of the tubing. Right before the detector is a glass plate that the beam stop is placed on. **Fun Fact: Professor Andresen, as a graduate student, painted the maroon square behind the detector.

Then a camera was set up to look at the sample inside the beam mostly to ensure that the sample was centered at the beam and to look for possible air bubbles or aggregates in the sample. At this point the guards and elements of the tube were varied to get the best beam signal throughout the tube with minimal scattering due to the sides of the tubes. Silver behenate was used as a calibration of the beam to find rough values for q to see what range we were working with. All of this was changed in the hopes of lowing the q value, which is the momentum transfer or scattering vector, to a low of 0.004. After the physical setup was chosen and implemented, all the different elements had to be synced with the computer outside the hutch (the hutch is the room where the X-rays are present during experiments).This was what took the most time because so many variables were changed since the last time the G1 hutch was operating. 

Operating system outside of hutch including the real time values of I3 and I2 which give an idea of the strength of the X-rays inside the hutch, the camera monitor that is looking at the sample, and all of the programs that control the beam, guards, sample etc. 

In the image above, Professor Andresen was closing the hutch door and turning the key. This is where the key is placed and the X-rays are turned on. After searching the hutch (pressing two buttons on either side of the hutch and ensuring no one is inside) the door is shut, key is taken and placed in the Station G1 control and the black buttons are pressed on the top right corner of the control box. 

 The element that gave the most trouble was the beam stop which was not in the correct place for running initally and also had different components that continually got in the way of the X-ray beam. This caused the I3 signal to be very low (~16) until it was fixed and showed a signal of ~200. After many hours of centering the beam and correcting the scattering from the sides, we began taking data from our samples. 
We began with the Trimer A which is three nucleosomes together because it is the smallest set of nucleosomes we will look at this weekend. First, distilled water is sent through the sample cell (about 3 times), then ethanol is sent through (about 2 times). At this point the sample cell is mostly clean so we turned on the air to dry the area for about 30 seconds minimum. After all that, we inserted a buffer, closed the hutch, and ran the buffer. This gives us a base line to subtract out any systematic problems against the actual samples. 

View of where the sample is inserted. Between the two plates, a small clear sample cell can be seen. This is where the samples, buffers, water and alcohol is placed and also where the air comes out. The camera is on the opposite side of the beam.  

We ran everything for 5 seconds with 20 images with a ten second break and then once more. After the buffer, we cleaned the sample cell with water, alcohol, and air once more and put the sample inside. We repeated the set up of 5 seconds, 20 images twice for the sample. This procedure was followed for all of Trimer A which included three dilutions (1x, 2x, 4x) for buffers of 10, 50, 100 and 200. 
Today, we are running samples of higher nucleosome arrays. Tetramers and dodecamers (4 and 12 nucleosome arrays respectfully) are much larger structures so the set up had to be modified so that the scattering angles could be measured. This is because larger objects give smaller scattering angles and smaller objects give larger scattering angles. We had some issues with a peak coming off in the z direction from the beam but by changing the guards and the beam stop, this was corrected. Because we are working with the other samples, we lower the beam size to 100 micrometers in the z direction which also lower our intensity so we will be taking longer time intervals when we run.