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Please ignore the links to individual datasets at the top of each of the sections below and instead take the unpacked data from the following location: /dls/i02/data/2015/mx13273-1/bag_training/.
This tutorial introduces data processing using the newly developed integration program DIALS. The tutorial is located at the DIALS website, but there is no need to download the dataset from the link there, as it has been unpacked for you locally.
Click here to visit the DIALS tutorial site: Processing in Detail
Here is a collection of tutorials starting from diffraction images. These will develop experience with data processing software and show some tips/tricks for ways to obtain good quality reduced datasets. However, you need not stop at that point. In some cases the tutorials also go on to phasing and structure solution.
These tutorials were developed, with permission, from the X-ray Tutorial at Helmholtz-Zentrum Berlin.
If you are unfamiliar with Linux commands please ask for help to unpack the datasets. It is not completely straightforward!
This tutorial originally comes from the Phaser Wiki.
The files for this tutorial can be found at: 2_complex.tgz
|Structure files:||beta.pdb, blip.pdb|
|Sequence files:||beta.seq, blip.seq|
This tutorial originally comes from the Phaser Wiki.
The files for this tutorial can be found at: 3_ensemble.tgz
|Structure files:||1BIK.pdb, 1D0D_B.pdb|
This is an easy but not completely straightforward MR example. Illustrates importance of model editing.
Assume that 4hg7.mtz presents your experimental data. Try to solve and refine this structure starting from 1t4e.pdb and using any tools you like.
The asymmetric unit of the template structure 1t4e contains two molecules of a fragment of a human oncoprotein MDM. The target structure 4hg7 contains a complex of an almost (but not completely!) identical fragment of the same protein with the ligand.
Fragments are very similar, same initial sequence, model 16-111 (+ 5 residues of expression tag, invisible in the structure), target 17-108. So at a first glance there is no need to modify the model.
The ligand is as follows:
A clear density for this ligand will indicate that the structure is solved. At this point you may also exercise with Coot > Calculate > Ligand Builder to generate description of this ligand and to refine the complex.
(1) Find the files listed above in the tutorial directory.
(2) How many molecules are there in the asymmetric unit of the target structure? To answer this question, use files 4hg7.seq and 4hg7.mtz and Cell Content Analysis task in CCP4I (CCP4I > Molecular Replacement > Analysis > Cell Content Analysis).
(3) Extract chain A from the file 1t4e.pdb into 1t4e_A.pdb. (CCP4I > Molecular Replacement > Edit PDB Model File. Change selections in the second line of the task menu to "pdbset" and "perform selection for output PDB file". Other selections should be clear)
(4) Run molrep (don't use sequence- assuming it's the same protein, and almost same length constructs) and examine the table at the end of the log-file. Is the molrep solution convincing? Try refinement. Did it go smoothly and produce interpretable density? Do you see the ligand density?
(5) Run phaser. Did you notice any unusual message? Refine the model. Do you think phaser found a solution?
(6) Ways to proceed: Phaser: increase phaser's tolerance to clashes, or remove three C-terminal residues from the model.
Molrep - use sequence now. Or remove three C-terminal residues.
(7) Compare solution from (4) or (5) with the solution from (6) using Csymmatch (CCP4I > Program List > csymmatch; define input files and tick the box "Apply origin correction and hand correction).
(8) Overlap the structures in Coot. See the shift between the right and wrong solution. Go to Draw>cell and symmetry>show symmetry>symmetry by molecule-display as Calpha s - and see why 3 residues made such a big difference.
This example shows how a small modification of the model can make a big difference.
*) like cloning in a way, when two-three truncated residues could make the construct soluble.
Download the data from ccp4_modelbuilding.tgz
|AMPLE generated Molecular Replacement model:||ample.pdb|
|Sequence file(s):||cMybCEBP_beta-seq.fasta (protein/DNA), cMybCEBP_beta-protein_seq.fasta (protein only) and cMybCEBP_beta-DNA_seq.fasta (DNA only)|
The goal of this tutorial is to illustrate how to use various automated density modification and model building tools to improve upon an initial partial model for a target structure produced by molecular replacement (MR). Molecular replacement can often succeed with only a partial or fragment search model. It then becomes necessary to build upon this model to give a final structure. This can be done manually using programs like Coot and Refmac, however, effective use of density modification and automated model building tools can make the task of completion a lot easier.
Here we will use several tools from CCP4, SHELX and ARP/wARP to demonstrate how to build up a final model from an inital model procduced by the AMPLE molecular replacement pipeline in CCP4. AMPLE uses ab initio modeling to derive ensemble search models for target structures that may not have suitable homologues available for use in MR. From an initial ensemble of search models it will produce several truncated forms of the same ensemble, truncating away the most variable sections in steps right down to a low variance core which may only represent a fragment of the target structure.
The target structure we will try to complete is the crystal structure of a protein-DNA complex. It is a mechanism of c-Myb-C/EBP beta cooperation from separated sites on a promoter. The protein component of the target is a coiled-coil structure and AMPLE has been used to generate search models for this and use them in MR. It has produced a solution (ample.pdb) which is made up of several short helical fragments which scoring indicates are positioned correctly.
Download and unpack the file ccp4_modelbuilding.tgz (linked to above) in a suitable location. In ccp4i create a new project with the directory ccp4_modelbuilding as the project directory. Give the project a suitable name. Select this project from the "Change Project" menu.
The first step is to refine the positioned model to produce an electron density map that we can examine. This will help us to further confirm the correctness of the solution and give us a starting map for model building.
Now that we are more confident that the MR solution is correct we will try to use Buccaneer to produce a more complete model for the protein part of the target structure.
We are going to run SHELXE to do density modifcation and c-alpha tracing. Before we can proceed we need to determine the solvent content of our unit cell. SHELXE is quite sensitive to this parameter so we need to make an accurate estimate. To do this we will use the program Matthews_coef
We will now run SHELXE twice, firstly assuming a 2 copies in the asymmetric unit and a solvent content of 53.51% and secondly with 1 copy in the asymmetric unit and a solvent content of 76.75%.
The density modification and phase improvement from SHELXE has given us a much better map to attempt model building in. Currently, the DNA and protein need to be built in separate steps. With a good map, the best approach is the build the DNA first and then the protein with the DNA fixed in place. The DNA can be built with Nautilus or with ARP/wARP. We will use ARP/wARP here but you should also try using Nautilus in your own time.
Our final step will be to use Buccaneer again to build the protein structure, but this time fixing the DNA in place. We will also use the SHELXE output map to build into.