|SAXS Data Collection and Analysis Protocol|
● Protein concentration for SAXS "rule of thumb": Concentration ≥ 100/MW. i.e. 50 kDaprotein, optimal concentration ≥ 100/50=2 mg/ml (*for SEC-SAXS, take dilution ratio (~3X) of SEC columns into consideration) - Typically prepare at least 500ul of > 5mg/ml.
● Buffer: 50 mM HEPES or Tris or phosphate buffer, pH ranging from 6-8 (if outside this range, consult beamline personnel for potential special measures), 150 mM NaCl (typical but up to 1M is permitted, can go higher but contrast will be affected), 1-2 % glycerol (higher concentrations can be done but column back pressures will increase), up to 5 mM DTT (or 1-2 mM TCEP)
● Columns for SEC-MALS-SAXS: Wyatt SEC column for MALS (ask Srinivas if you need specific column)
1. 010S5 100Å (MW range 100-100,000 Da)
2. 015S5 150Å (MW range 500-150,000 Da)
3. 030S5 300Å (MW range 5,000-1,250,000 Da)
Columns for SEC-SAXS: GE Healthcare SEC columns run at 0.75 ml/min elution rate (24ml - column volume) and discuss with beamline personnel to determine the most suitable column for your sample -
1. Superdex 75 10/300 (MW < 75,000 Da)
2. Superdex 200 Increase 10/300 (MW < 400,000 Da)
3. Superose 6 10/300 (MW < 5,000,000 Da)
● Quartz capillary flow-cell
○ lumen is 1.5 mm in diameter with a 10 um wall
● 0.5-1 s exposure every 2-3 seconds
● X-ray wavelength is 1.033Å, 12KeV (this seldom changes)
● Data recorded on Pilatus3 1M (Dectris) detector at a sample-to-detector distance of ~3.5 m. This covers a momentum transfer range of ~ 0.005 Å-1 < q < 0.4 Å-1 (see raw *.dat file for determining this for your specific data)
● Data usually collected at room temperature (discuss more specific needs with beamlinepersonnel).
● Normalized scattering data to the incident X-ray beam intensity
● Data are reduced to three column data q, I(q) and σI(q) via python script
● Switching buffer/equilibration
○ SEC-MALS-SAXS setup needs long equilibration (6h to overnight), which precludes too many buffer exchanges during a single run.
○ Split buffer in half so both inlet A and B can pump buffer (*Be prepared for enough buffer and bring them in two bottles in advance*). 2L is usually sufficient.
○ Change flow rate by 0.1 ml/min about every minute (after pressure levels out) as you stop the flow of one buffer and begin the flow of a new buffer
○ Once back up to 0.8 ml/min, equilibrate for at least 6 more hours
○ If equilibrating overnight, ramp up to 0.8 ml/min and equilibrate at that rate overnight
○ Make sure you clean the flow cell for MALS/DLS in between buffers, especially if there's more glycerol etc. (anything that could change refractive index)
○ ALSO: keep glycerol concentrations as low as possible (preferably 5%<).
● Injecting sample and starting HPLC run
○ Each run will take about 22 minutes. Have samples ready (*concentrate or dilute to appropriate concentration and spin down 10-15 minutes) so that near the end of one run you can: 1. clean the capillary
2. have the auto-sampler inject your sample just as the next run is about to start
○ Each run should be about done after 18 min - stop collecting SAXS data then and you will have 5 min to clean the capillary and put the new sample in the sample tray
■ If you think you will be running late, time can be extended
■ To change time, right click in Quat. pump, select Method, change time
○ Preparing your sample
■ Samples are injected from vials. 250 - 350 ul will be injected, but fill vials to ~50 ul more than the injection volume (900 ul is the upper limit)
○ Washing the system
■ Wash the capillary
● Do not have the HPLC and wash pump connected to the capillary at the same time
● Connect capillary to hamilton syringe pump (on desktop) and press wash
● When done, capillary can be reconnected to the HPLC (red outlet 1 line)
○ Make sure tube is inserted all the way (will feel resistance) and screw lines together
○ BE CAREFUL MANOUVERING THE PEEK TUBES CONNECTED TO THE CAPILLARY AS THE CAPILLARY WALLS ARE 10 MICRON QUARTZ AND THEREFORE EXTREMELY FRAGILE.
○ Loading your sample
■ At the beginning of each new sample set, set a program for that sample set
● In ASTRA (Wyatt's MALS software):
○ Create new sequence file, type number of samples and name them
○ Set the sample property 1) name, 2) choose method (MALS-dRI-SAXS), 3) set time (22 mins) for each sample
● In Chemstation (Agilent's HPLC software):
○ Use the sample entry window to select positions in sample tray and name them
○ Set the method SEC_constflow for each sample
■ For each sample, put the vial in the proper position in the tray for that sample.
■ Watch the autosampler pick up the vial, aspirate the sample, and replace the vial
■ Once the vial has been replaced and the robot moves out of the way, remove the vial and check that a reasonable amount of sample has been aspirated to ensure proper functioning of the autosampler. Begin station search (make sure capillary has been cleaned and is hooked back up to HPLC)
○ Data from HPLC runs are saved in pre-determined folder (usually date followed by PI last name).
COMPUTER WORKFLOW: SAXS
○ On the computer called Rodin, setup the labview program that records the intensity of the incident and transmitted x-ray beams. Beamline personnel will give you detailed instructions for how to do this before the start of the experiment.
○ On the medm** interface that controls the detector, update file name and change the file counter field (next file) to 1. Make sure the name you type here matches the one entered in the labview program above.
■ **for the medm interface you have to click in box and leave mouse in box while typing. Must press enter before moving mouse. Make sure these fields are updated before you proceed with data acquisition!!!***
■ Once these things are updated, can start collecting data. This will open and close the shutter exposing the sample to x-rays for 0.5 to 1 s periods every 2-3 seconds. Do make sure the shutter allowing beam into hutch D is open before commencing data acquisition.
DATA PROCESSING AT THE BEAMLINE
• On the SAXS data analysis computer you will use BioXTAS RAW to reduce the detector images (.tif) to one dimensional scattering profiles (.dats) and do preliminary processing.
o RAW is a full-featured GUI-based SAXS analysis program that can do data reduction and has specialized tools for processing SEC-SAXS data. It can do analysis all the way from basic background subtraction to Guinier fits, molecular weight calculation, IFT calculation (P(r) function) using GNOM, envelope calculation using DAMMIF/N, electron density calculation using DENSS, and deconvolution of SEC-SAXS data using EFA.
o Full written tutorials, video tutorials, a manual, and instructions for how to install RAW on your own computer can be obtained from the RAW website.
users may find the section on
o Beamline scientists will provide hands-on training with RAW at the beamline
o RAW is developed and maintained by Jesse Hopkins, a beamline scientist at BioCAT. Bugs reports, feature requests, and other RAW questions should be sent to him.
• In addition to RAW we also provide the ATSAS package (latest version), EMAN2 (for averaging electron densities produced by DENSS), pyMOL and Chimera on the primary data analysis computer.
• There is an additional identically configured computer available for data analysis at the beamline.
DATA PROCESSING AT HOME
• Users will take home all unprocessed data: .tif files and log files, as well as a RAW .cfg file. In addition, they will take home any data processed at the beamline.
• In order to carry out basic processing (images into one dimensional scattering profiles) users will need to install RAW
o IMPORTANT: You need version 1.4.0 or greater to process (image) data collected at BioCAT.
• Once you've installed RAW, make sure to load in your config file ("SAXS.cfg") BEFORE loading any images.
o This part of the RAW tutorial covers loading config files and how to process batch-mode samples: https://bioxtas-raw.readthedocs.io/en/latest/tutorial/s1_basic.html
o This part of the RAW tutorial covers basic SEC-SAXS data processing: https://bioxtas-raw.readthedocs.io/en/latest/tutorial/s1_sec.html
• If you'd rather use another data processing program (like Primus or ScAtter), simply load your .cfg file into RAW, select all of your images in the 'File' Control Panel and click 'Quick Reduce'. This will generate .dat files from the images without loading them into RAW. These .dats are compatible with most popular SAXS software packages.
SAXS PROCESSING METHOD
● A standard batch-mode data set for a well behaved globular protein might be processed using RAW as:
► MW calculation to verify data quality and oligomeric state
► Evaluating the potential ambiguity of a 3D reconstruction using AMBIMETER to verify that a reconstruction is worth doing
► 3D reconstruction with ab initio bead models using DAMMIF/N and average these models using DAMAVER or cluster these models using DAMCLUST
• 3D reconstruction with ab initio electron density models using DENSS and EMAN2
• For more on what RAW can do, see the full tutorial: https://bioxtas-raw.readthedocs.io/en/latest/tutorial.html and tutorial videos: https://bioxtas-raw.readthedocs.io/en/latest/videos.html
• Alternatively, you can use Primus or Scatter for the processing described above, except for the electron density reconstruction.
● A standard SEC-SAXS data set for a well behaved globular protein might be processed using RAW as:
• Load the SEC curve in RAW, and use plots of the Rg and MW across the peak to determine what regions to process
• Testing buffer regions before and after the peak, create a subtracted scattering profile
• Carry out the steps for batch-mode processing above starting with Guinier analysis.
● P(r) can be used to calculate ab initio models using GASBOR, part of the ATSAS package, which is more reliable with higher quality high-q data (meaning > q = 0.25-0.3) and for non-globular proteins.
● I-TASSER (or Phyre2 or Swiss-model) is used to build a homology model (if you don't have any structural information for your protein, which is then fit to the model using SUPCOMB)
● SymmDock can be used to generate models with P2 and P4 symmetry (based on Phyre2 and I-TASSER model) ***This symmetry stuff is necessary if you are trying to figure out what oligomeric state the protein is in. This may not be necessary for everyone***
● CRYSOL used to calculate theoretical scattering curves of P1 (starting model), P2, and P4 homology models and compare to experimental data (*do this before you dock your model to ab initio envelope*)
○ This will generate a .fit file containing a theoretical scattering curve of the pdb file fit to the experimental data (dat file) (the first three columns are q, Iexp(q) and Ifit(q)), and used for plotting). These .fit files can be loaded directly into RAW.
● SUPCOMB will superimpose envelopes to homology models
○ supcomb **.pdb ****.pdb
○ Output file will be ****r.pdb
○ **reference(homology) model
○ ****moving (envelopes) model
COMPUTER WORKFLOW: MALS
○ In ASTRA (Wyatt's software for SEC in-line MALS):
■ Open an experiment (your file)
■ Open Experiments tab on the bottom left
■ Configuration -> generic pump (on left side panel)
● Set flow rate, (default is 0.8 but you should remember to change this to the real flow rate for any given experiment).
■ Procedures -> baselines (on left)
● First click autofind baselines
● Click through baselines and make sure all baselines are defined (if they are not good you have to define them; click once at start point and, hold and drag it to right side to define the baseline and range)
● Click Apply
■ In the top toolbar, Experiment -> configuration -> band broadening
● Define an area
● Click Perform fit
● Manually define peaks if needed
● Click Apply
■ Click Peaks on left side panel
● Choose what peaks (UV curve) you will be working with (better to choose range above baseline)
● Click Apply
■ Click Molecular Mass & Radius from LS on left side panel
● De-select detectors that had bad baselines
● Click Apply
● Can scroll through peaks to see MW for each peak from MALS
■ Click Rh from QELS on left side panel
● Can scroll through peak and see Rh from QELS (DLS)