Download the data from 1_intro.tgz
The target is an acylphosphatase-like domain of hydrogenase maturation factor HypF from E.coli, see Rosano et al., JMB, 321, 785 (2002). HypF-ACP sulphate and phosphate complexes have been deposited in PDB as 1gxt and 1gxu respectively.
Across various tutorials we will solve the hypF structure by molecular replacement, using several programs and approaches, and the native 1gxu dataset to 1.3 Å resolution in space group H32. The target has 91 residues and a Matthews calculation strongly suggests only one molecule in the asymmetric unit. In this tutorial we focus on MR using the program Molrep.
N.B. hypF-1gxu-1gxt-HG_scaleit1.mtz includes the data from 1gxu, 1gxt, the Hg derivative, and some experimental phases based on the Hg sites. Do not forget to select the correct mtz-columns (FP1gxu, SIGF SIGFP1gxu) each time you define the input mtz-file.
We first use Sfcheck to check a few things about the data:
Sfcheck produces a postscript file with some useful things (see under):
The target is an acylphosphatase-like domain. A search of the PDB reveals two acylphosphatases with a sequence identity to the target of about 31%, viz. 1v3z and 1w2i. Each has two chains in the asymmetric unit, either of which could be used as the basis of a search model.
Normally you would use something like Chainsaw at this point to prepare a search model from the template. As an exercise, we are going to try MR straightaway.
There are many ways of approaching this, and the different tools will give slightly different results. The sequence identity depends on the definitions used (i.e. treatment of gaps and alignment length), the specific alignment technique, and whether parts have been chopped out of the model.
We will use chain B of 1v3z as the search model.
When the job has finished, look at the log file (Log File tab). Note the following:→ →
INFO: expected number of models : 1 INFO: V_model: 61.6% (of asymm. part of u.c.)
which is correct. The estimate may be unreliable when there are many monomers in the asymmetric unit, in which case it can be set explicitly with the keyword NMON (see the folder Search Options)
INFO: Anisotropicy will not be used
In fact, we can make use of our knowledge of the target, and this will often improve the solution. The search model has a moderately low sequence identity with the target and therefore the majority of the side chains are incorrect. Molrep can make use of the target sequence to improve the search model.
Look at the log file of this job.
The positioned model can be submitted for a few cycles of automated refinement, then checked manually against 2mFo-DFc and mFo-DFc maps, using a graphics program such as Coot. Since we have a good resolution dataset, the model can also be passed to ARP/wARP for rebuilding. Refinement, validation and model re-building are covered in other tutorials
Here we will give a brief demonstration of how to refine the solution models using Refmac
When the job has completed, double-click on the job name in the job list window to open the results page. For this example, we are only interested in making a quick assessment of whether or not MR has worked. To do this we will look at the R/R-free values before and after the 10 cycles of refinement. These are listed in the Result table
Repeat the above steps using the output PDB file from the second Molrep job. Note, do not overwrite the MTZ and PDB output files from the first refinement job! Compare the R/R-free values for both jobs. You can clearly see that modifying the search model has greatly improved the results. Nevertheless, the best way to judge whether a solution is correct is to look at the electron density map. From the Refmac results page, you can launch Coot with the refined map and model loaded by clicking on the Coot button under Output Files
The Molrep solution is related to the deposited structure 1gxu by the symmetry operation -Y+2⁄3, X-Y+1⁄3, Z+1⁄3. Comparison of the structures in CCP4mg or Coot shows that the beta sheet and one of the two helices are well matched, but there are significant differences elsewhere.
In general, if we want to compare an MR solution to the deposited structure, then we need to take into account possible symmetry operations and possible changes of origin. Two solutions may be identical, even if it is not obvious from a quick look in a graphics program. This can be checked with the csymmatch utility:
The log file reports the symmetry operator and change of origin which give the best match, and a normalised score for the match is reported. The output PDB file has this transformation applied, and can be compared to the reference PDB file. Of course, usually we don't have a deposited structure to compare with, but the same process is useful to compare different MR solutions.