X-ray data processing
Prepare data
Convert h5 format to cbf format: XDS can't operate with h5 format images, so you need to convert the image to cbf format before the process. On macOS, eiger2cbf is a useful program to do this.
eiger2cbf path/to/your/h5_master_data -- get number of frames
eiger2cbf path/to/your/h5_master_data N out.cbf -- write N-th frame to out.cbf
eiger2cbf path/to/your/h5_master_data N -- write N-th frame to STDOUT
eiger2cbf path/to/your/h5_master_data N:M out -- write N to M-th frames to outNNNNNN.cbf
For example:
eiger2cbf path/to/your/h5_master_data 1:720 out # convert all the 720 images to cbf format
X-Ray data processing
Process with XDS
Install XDSGUI according to this instrucment.
Run the XDSGUI in the terminal.
cd path/to/your/images xdsgui
Load the image and generate the XDS.INP file:
Load your imageModify the XDS.INP file to meet your needs, then click
Run XDS
.:::caution
If you collect your data with 10U2 beamline in SSRF, you should modify the
ROTATION_AXIS
parameter to0 -1 0
.:::
Check the result in page CORRECT
Process with Xia2
There are two ways to use Xia2: use Xia2 in CCP4, or install the latest xia2/DIALS bundle according to this website.
Process your data with it:
cd path/to/images xia2 pipeline=dials-aimless ./ goniometer.axes=0.000000,1.000000,0.000000 xia2.settings.resolution.d_min=2
Process with autoPROC
- Request a license and install autoPORC according to this website.
Process your data with it:
cd path/to/images process -I . -d autoproc -R 50 2 > out.log #save the result in subdirectory named autoproc, resolution vary from 2 to 50, save the log to out.log file.
Structure determination
Valid the data
Find the
XDS_ASCII.HKL
file in the autoproc directory as the diffraction file.Valid the data with Phenix Xtriage, put the diffraction file in, and run.
Molecular replacement
Find the
XDS_ASCII.HKL
file in the autoproc directory as the diffraction file.Get the template model:
- Blast the sequence of your protein with the PDB database, select the proper structure, and download the PDB file.
- After downloading the template model, you need to edit it with PyMOL to remove the redundant copies and water molecules.
Determine how many copies are in ASU(asymmetric unit):
You can get the number from the result of the Xtriage
Or you can use the cell content analysis tool in CCP4.
Note
You can determine the number of copies through the Solvent content, generally, the solvent content of most biological macromolecule crystals is between 40% and 60%.Do molecular replacement with Phenix Phaser-MR.
Build your model with Phenix AutoBuild: input the result of molecular replacement, and run.
AutoBuildCheck the model from AutoBuild in Coot and refine it manually.