1. Introduction to CCP4 programs for MR

 

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.

 

We will solve the hypF structure by molecular replacement, using several programs and approaches and the native 1gxu dataset to 1.3 A resolution, space group H32. The target has 91 residues and a Matthews calculation strongly suggests only one molecule in the asymmetric unit.

 

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.

 

1.1. Checking the data

 

We first use Sfcheck to check a few things about the data:

 

1.     Select Data Reduction and Analysis > Check Data Quality > Analysis with sfcheck to open the sfcheck task window.

2.     Enter a title.

3.     Make sure that Run Rampage to analyse structure geometry and Run Procheck to analyse structure geometry are unselected (we do not yet have any coordinates) and Run Sfcheck to analyse experimental data only is selected

4.     In the line MTZ in select the file hypF-1gxu-1gxt-HG_scaleit1.mtz

5.     Select the labels F FP1gxu, SIGF SIGFP1gxu and Free FREE

6.     Check that a suitable filename has been generated for Sfcheck Output PS

7.     Keep all defaults, and click Run -> Run Now.

 

Sfcheck produces a postscript file with some useful things (see under View Files from Job):

 

      Anisotropy of data (it is not very anisotropic)

      Overall B from Wilson plot of 21.9 A**2

      Pseudo-translation not detected (from analysis of the native Patterson map)

      Also check the log file - View Files from Job then View Job Results (new style) then click the Log File tab:

      This includes the results of a twinning test:   Perfect twinning test <I^2> / <I>^2 :  2.0573

      A value of 2.0 indicates untwinned data, whereas perfectly twinned data would have a second moment of 1.5

 

1.2. Choice of search models

 

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. We will return to Chainsaw later before running Phaser.

 

Notes on Sequence Alignment

 

There are many ways of approaching this, and the different tools will give slightly different assessments. The sequence identity depends on the definitions used (i.e. treatment of gaps and alignment length), the specific alignment technique, and whether bits have been chopped out of the model.

 

1.3. Molrep Run 1

 

We will use chain B of 1v3z as the search model.

 

1.     Select the Molecular Replacement module and open the Run Molrep - auto MR task window.

2.     Enter a title.

3.     Do molecular replacement should be already selected.

4.     For Data select the file hypF-1gxu-1gxt-HG_scaleit1.mtz

5.     Select the labels F FP1gxu and SIGF SIGFP1gxu

6.     For Model select the file 1v3z_B.pdb

7.     (Optional) You can use an upper resolution cut off of 3A to speed up the calculation, see folder Experimental Data.

8.     Keep all defaults, and click Run -> Run Now.

 

When the job has finished, look at the log file (View Files from Job -> View Job Results (new style) -> Log File tab). Note the following:

 

      Molrep automatically estimates:

 

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 folder Search Options in the Molrep GUI).

      Molrep checks whether or not an anisotropy correction is necessary:

 

INFO: Anisotropicy will not be used

 

      The first table is a list of peaks of the Cross Rotation Function (CRF), sorted according to their heights. This is followed by a plot showing which peaks are related.

      The second table shows the best Translation Function (TF) for each of the CRF peaks (scored according to the correlation coefficient * PKmax). Other TF solutions can be viewed in the file View Files from Job -> Output Files ... <proj_dir>_<job_no>_molrep.doc

      The final table gives a list of solutions, sorted according to the score.

      Molrep reports a contrast higher than 3.0. This contrast value suggests a correct solution.

 

1.4. Molrep Run 2

 

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.

 

1.     Select the previous job, and click ReRun Job

2.     Most of the parameters should be set correctly, but you should change the title, and the name of the Solution file, so that it is different from the first job.

3.     This time, input the target sequence file hypF_Ndom.seq in the Sequence box.

4.     Click Run -> Run Now

 

Look at the log file of this job.

 

      After a section about the input MTZ file, there are details of the sequence alignment between the target sequence you have supplied and the sequence of the search model (i.e. the PDB file).

      Molrep reports a sequence identity of about 30%. This is lower than other estimates because Molrep is more conservative in introducing gaps into the alignment.

      Molrep outputs tables for the CRF and TF as before.

      At this point it may not be apparent that the MR solution with the search model modifications has improved. The benefits of model preparation will become clearer when we refine the solutions.

 

1.5. Checking the solution

 

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.

  1. Select the Refinement module and open the Run Refmac5 task window.
  2. Enter a title.
  3. We will run Refmac with defaults, i.e. restrained refinement with no prior phase information.
  4. For MTZ in provide the file hypF-1gxu-1gxt-HG_scaleit1.mtz
  5. For PDB in provide the output PDB file from the first Molrep job.
  6. Leave everything else at defaults. Note this will run 10 cycles of restrained refinement.

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:

 

1.     Select the Symmetry match models task in module Coordinate Utilities.

2.     Enter the MR solution PDB file as the Work PDB in, and the deposited structure (1gxu) as Reference PDB in.

3.     Select Apply origin shift and hand correction and run.

 

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.

 

1.6. Chainsaw

 

Search models can also be prepared using Chainsaw. Chainsaw takes an external sequence alignment, which can be generated by many bioinformatics tools and/or manually adjusted. In this job, we will create a model based on chain B of 1v3z, using a previously prepared alignment to the target.

 

1. Select the Molecular Replacement module and open the Create Search Model task window in the Model Generation folder.

2. Enter a title.

3. Leave Create search model using Chainsaw unchanged.

4. Leave Prune non-conserved residues to gamma atom unchanged.

5. For PDB in select the file 1v3z_B.pdb

6. Use the sequence alignment format PIR and for Alignment in select the file 1v3z_B_to_target.pir

7. Click Run -> Run Now

 

Chainsaw produces a coordinate file 1v3z_B_chainsaw1.pdb which is an edited version of the input PDB file. 6 residues that do not align to the target sequence have been deleted. Of the rest, 34 have been left unchanged and 50 have had their side chains cut back to the gamma atom. The output PDB file uses the naming and numbering of the target sequence.

 

Have a look at the log file:

 

   At the top, the alignment used is confirmed.

   Then there is a listing of all the model residues, with the action applied (deleted, conserved, mutated).

   Finally, there is a summary of the changes made. This includes the estimated sequence identity. Note that this is not unique, but depends on the particular sequence alignment used.

 

Now repeat this exercise using the other search model, based on chain A of 1w2i. We can overlap the two models and use the ensemble as input to Phaser (in place of individual search models).

 

      For PDB in select the file 1w2i_A.pdb

      Use the sequence alignment format PIR and for Alignment in select the file 1w2i_A_to_target.pir

 

1.7. Aligning the models

 

These models can be aligned and the overlapped structures used as input to Phaser.

 

1.     Select the Coordinate Utilities module and open the Superpose Molecules task window.

2.     Enter a title.

3.     Change mode to Superpose using gesamt.

4.     Enter Moving 1w2i_A_chainsaw1.pdb

5.     Enter Fixed 1v3z_B_chainsaw1.pdb

6.     Enter PDB out 1w2i_A_to_1v3z_B_chainsaw1.pdb

7.     Click Run -> Run Now

 

The 1w2i_A_chainsaw1.pdb has been moved to overlap 1v3z_B_chainsaw1.pdb. The log file shows the transformation used, and gives an RMSD = 0.305 A between 84 C-alpha atoms of the superposed structures.

UPDATE: Ensemble models can also be generated using the new program Ensembler (Molecular Replacement->Model Generation->Ensembler).

1.8. Phaser

 

Using the superposed search models generated by Chainsaw, we will now use Phaser to solve hypF. Phaser is designed to use ensembles of models to improve the signal.

 

1.     Select the Molecular Replacement module and open the Phaser MR task window.

2.     Enter a title.

3.     Leave Mode for molecular replacement automated search unchanged.

4.     For MTZ in select the file hypF-1gxu-1gxt-HG_scaleit1.mtz, and select the labels F FP1gxu and SIGF SIGFP1gxu

5.     In the folder Define ensembles ..., enter the PDB #1 1v3z_B_chainsaw1.pdb. Set the similarity to be sequence identity 0.36

6.     To add another model click Add superimposed PDB file to the ensemble, enter the PDB #2 1w2i_A_to_1v3z_B_chainsaw1.pdb. Set the similarity to be sequence identity 0.38

7.     In the folder Define composition of the asymmetric unit, select Total scattering determined by components in asymmetric unit, and for the SEQ file select the file hypF_Ndom.seq, and leave Number in asymmetric unit 1 unchanged.

8.     In the folder Search parameters, select Perform search using ensemble1

9.     Click Run -> Run Now

 

Have a look at the log file.  The description below relates to an older version of Phaser therefore take it as a general explanation. The current Phaser will make two attempts at structure solution, with resolution limits of about 3A and 1.3A. Therefore the log file will contain two sets of tables.

 

       After details about the input parameters, there is information on the anisotropy correction used (compare to the output of Sfcheck above). This is followed by a Matthews coefficient calculation.

       Phaser then calculates a Fast Rotation Function (FRF). It finds 9 solutions greater than 75% of the top peak (this threshold can be changed with the option Rotation search peak selection in the folder Additional parameters).

       These peaks are passed to the Fast Translation Function (FTF). Detailed results for each rotation peak are given, followed by a summary table: Beware - these numbers may differ slightly for different versions of Phaser.   

 

   Translation Function Table
   --------------------------
   SET ROT*deep   Top   (Z)     Second   (Z)      Third   (Z) Ensemble SpaceGroup 
     1   1       19.9  5.46          -     -          -     - ensemble H 3 2      
     1   2       -5.9  5.28       -8.7  4.29      -11.3  4.43 ensemble H 3 2      
     1   3      -11.6  5.40      -19.6  4.74      -19.6  4.75 ensemble H 3 2      
     1   4          -     -          -     -          -     - ensemble H 3 2      
     1   5      -15.4  4.63      -24.2  4.59      -31.0  4.64 ensemble H 3 2      
     1   6      -15.6  4.45      -18.6  4.61      -26.9  4.54 ensemble H 3 2      
     1   7          -     -          -     -          -     - ensemble H 3 2      
     1   8          -     -          -     -          -     - ensemble H 3 2      
     1   9      -19.8  4.64      -27.2  4.69          -     - ensemble H 3 2      
     1  10*     -10.6  5.30          -     -          -     - ensemble H 3 2      
     1  11*     -46.6  4.63      -48.8  4.79          -     - ensemble H 3 2      
     1  12*     -19.3  4.82          -     -          -     - ensemble H 3 2      
     1  13*         -     -          -     -          -     - ensemble H 3 2      
     1  14*     -25.4  4.86      -35.8  4.79          -     - ensemble H 3 2      
     1  15*         -     -          -     -          -     - ensemble H 3 2      
     1  16*         -     -          -     -          -     - ensemble H 3 2      
     1  17*         -     -          -     -          -     - ensemble H 3 2      
     1  18*         -     -          -     -          -     - ensemble H 3 2      
     1  19*     -25.3  4.87      -31.3  4.92          -     - ensemble H 3 2      
     1  20*     -16.9  4.97          -     -          -     - ensemble H 3 2      
     1  21*     -20.7  5.60          -     -          -     - ensemble H 3 2      
     1  22*         -     -          -     -          -     - ensemble H 3 2      
     1  23*     -16.9  5.17          -     -          -     - ensemble H 3 2      
     1  24*         -     -          -     -          -     - ensemble H 3 2      
     1  25*     -21.8  4.91          -     -          -     - ensemble H 3 2      
     1  26*         -     -          -     -          -     - ensemble H 3 2      
     1  27*         -     -          -     -          -     - ensemble H 3 2 

The first trial (based on the 1st peak of the FRF) gives a clear solution, with a good Z-score, and a single significant peak of the FTF.

       Next is a check on packing for this good solution. Phaser finds 2 clashes between a C-alpha and a C-alpha of a symmetry-related molecule. Because the threshold is set to 4 clashes in total (5% of trace atoms), this solution is accepted.

       Finally, Phaser refines the MR solution, and displays the improvement in the log-likelihood gain (LLG).

       Phaser outputs a .sol file containing the MR solution, a .pdb file containing the correctly positioned model, and .mtz file containing the original data plus a calculated structure factor from the model and columns of map coefficients.

 

Checking the solution:

 

       Direct comparison of the Phaser solution and the deposited structure 1gxu using Coot may or may not be possible. This is because the spacegroup H32 has two possible origins (see $CHTML/alternate_origins.html). If both structures will be on the same origin, the comparison will show that the beta sheet and one of the two helices are well matched, but there are significant differences elsewhere.

       The solution .pdb and .mtz files can be loaded to Coot (use Coot button in the Qt result page) to inspect the model against the 2Fo-Fc map. This shows good agreement in most places, but also highlights problem areas.

       Do 20 cycles of restrained refinement in REFMAC (Run Refmac5 task in module Refinement) and check the model and maps.

       Optionally, run ACORN which removes phase bias (Acorn task in module Program List).

       Optionally, rebuild in arp/warp using the ACORN phases as restraints.

 

1.9. MrBUMP

 

You have now prepared three search models based on 1v3z, and used Molrep and Phaser to do the molecular replacement. These steps, and the initial discovery of 1v3z and other related proteins, are automated in the program MrBUMP.

 

1.     Depending on what you want to do, MrBUMP can make use of web-based services. The following tutorial deliberately does not make use of the web, so that it can be run anywhere. At the end of the tutorial, there are suggestions for web-based options. The use of a few local PDB template files also means that the tutorial is fairly quick. Beware that a full run of MrBUMP might take longer than is reasonable for a tutorial.

 

2.     Select the Molecular Replacement module and open the Run MrBUMP task window.

3.     Enter a title.

4.     Leave Program Mode Model search and Molecular Replacement unchanged.

5.     For SEQ in select the file hypF_Ndom.seq

6.     For MTZ in select the file hypF-1gxu-1gxt-HG_scaleit1.mtz, and select the labels F FP1gxu, SIGF SIGFP1gxu and Free FREE

7.     Leave the rest of the files folder unchanged, and move to the Template Search Options folder.

8.     Un-check Do a FASTA search for possible template models. Instead we are going to use some known local templates.

9.     Un-check Update local copies of search databases

10. Select Multiple alignment program Mafft if available

11. Un-check all Additional search methods, i.e. SCOP, PQS and SSM

12. The folder User specified search models will have opened. Because we have switched off all search options, we are required to use local files. Click on Add PDB file 3 times to add 3 local PDB files. The first file is 1w2i_A.pdb and Chain identifier A. The second file is 1v3z_B.pdb and Chain identifier B. The third file is 2acy.pdb and Chain identifier A.

13. In the folder Search Model Preparation Options, keep the default which is to use Molrep, Chainsaw and Sculptor. This means there will be 9 search models in total. Turn one or two off to make the job quicker.

14. In the folder Molecular Replacement and Refinement Options, keep Molrep and switch off Phaser. If you want, you can use Phaser instead of Molrep or both.

15. In the folder Model Building and Phase Improvement, select the model building programs to try after MR and refinement. By default Buccaneer is set but depending on your installation you may be able to try ARP/wARP and c-alpha tracing with SHELXE as well. Model building can help determine if MR has been successful.

16. Click Run -> Run Now

 

After a few minutes, have a look at the MrBUMP log file (do not wait for the job to finish).

 

       At the top, it echoes the options selected.

       Under Target Information, it estimates that there is 1 molecule in the target asymmetric unit.

       Under Template Model Search Results, it lists the three local files entered. They are named "loc0", "loc1", "loc2" for internal use.

       Under Search Model Preparation Results, details of the Molrep, Chainsaw and Sculptor methods are given.

       Finally, the section Molecular Replacement and Refinement gives details for every MR job tried.

 

By default, it will finish when it finds a solution. For example, it may finish with model loc1_B_MOLREP, which corresponds to template 1v3z_B.pdb with a search model created with the Molrep editing features. The Rfree drops from 0.549 to 0.436 (precise numbers may vary!) indicating that the MR solution is refinable, and likely to be correct. If you want to try all search models in MR (a good idea unless you are in a rush), select Finish when all of the search models have been tried in MR in the folder Molecular Replacement and Refinement Options.

 

If there are no problems accessing web-based services, then you can search for templates rather than use local PDB files. Run as above, with the following differences:

 

1.     In the folder Template Search Options, check Do a FASTA search for possible template models.

2.     Check Run the FASTA search locally. This refers just to the search step - the PDB files are still downloaded from the web.

3.     Check all of the Additional search methods, i.e. SCOP, PQS and SSM

4.     Do not enter anything into the folder User specified search models.

 

For comparison, here are some example results from MrBUMP (you may not get exactly the same):

 

PDB chain   

sequence identity   

source / release date

Rfree from MrBUMP

1w2i_B

0.310

OCA - released Apr 2005

chainsaw 0.447 molrep 0.442

1w2i_A

0.310

OCA

chainsaw 0.471 molrep 0.527

1v3z_B

0.310

OCA - released Mar 2005

chainsaw 0.430 molrep 0.453

1v3z_A

0.310

OCA

chainsaw 0.474 molrep 0.470

2bje_G

0.287

OCA - released Nov 2005

chainsaw 0.458 molrep 0.442

2bje_E

0.287

OCA

chainsaw 0.468 molrep 0.486

2bje_C

0.287

OCA

chainsaw 0.491 molrep 0.481

2bje_A

0.287

OCA

chainsaw 0.448 molrep 0.443

2bjd_B

0.287

OCA - released Nov 2005

chainsaw 0.468 molrep 0.529

2bjd_A

0.287

OCA

chainsaw 0.544 molrep 0.466

1y9o_A

0.275

OCA - released Jan 2006 (NMR)

(not tried)

1ulr_A

0.286

OCA - released Nov 2004

chainsaw 0.476 molrep 0.471

2acy_A

0.264

SSM - released Nov 1997

           (authors tried) 

chainsaw 0.539 molrep 0.564

 

1.10. Other search models for hypF

 

Another possible search model is chain A of 1w2i. This is a different structure of the same protein as 1v3z. You may try repeating the above steps using 1w2i_A.pdb as the search model.

You should find that this is more difficult! Modifying the search model using the target sequence is now necessary. Adjusting the resolution limits also helps.

Check your solutions against those produced from 1v3z_B.