4. Using Phased RF and TF

 

Project directory:      4_saptf

Reflection data:         1tj3.mtz

Structure files:           1s2oA.pdb, 1s2oA_dom1.pdb, 1s2oA_dom2.pdb

Sequence file:                        1tj3.seq

 

4.1. Introduction

 

This tutorial is based on a crystal structure of sucrose-phosphatase (spp) from Synechocystis sp. pcc6803 in a closed conformation, PDB code 1tj3. The crystal space group is P6522 and data resolution 2.8A.

 

There are several homologous structures available in both the open and closed conformations. For this exercise we will choose an open-conformation model with 100% sequence identity, PDB code 1s2o. The file 1s2oA.pdb contains chain A of the crystal structure. Check with Coot that there are two obvious domains, which are also provided in separate files, 1s2oA_dom1.pdb and 1s2oA_dom2.pdb.

 

This example demonstrates the MR search using electron density maps from partial structure. Three different protocols for this type of search are implemented in Molrep (see comments in 4). These methods can be really useful for large structures containing several domains.

 

This particular example is an easy case. In particular, for Phaser it is a very straightforward run with two ensembles corresponding to two different domains.

 

4.2. Checking the data

 

– Matthews suggests one molecule in the asymmetric unit.

 

– Sfcheck shows no particular problems with data.

 

– There is a space group ambiguity. The space group cannot be selected from the absences alone; P65 2 2 and P61 2 2 would both require that the only reflections observed along the c* axis are 0 0 6n. In real experiment, you should check both space groups and see which one gives the best result for the translation search. If you are a very cautious person, you may wish to check all space groups consistent with the point group - in this case P6 2 2, P61 2 2, P65 2 2, P62 2 2, P64 2 2, and P63 2 2.

 

In Molrep interface, the space group can be selected in "Search Options > Used SG" menu. Note that in the "MR using Phases" mode this menu is not active.

 

4.3. Structure solution

 

1. Try to solve the structure using Molrep and the complete model, s2oA.pdb

 

2. Refine the MR solution

 

Automatic MR using the whole 1so2 model finds the solution, but the subsequent refinement does not improve the R-factors much, and the maps show no reasonable density for the small domain. However a clear density for the first domain is a good sign.

 

Therefore in the second attempt we use individual domains as search models. In this process, the phases from the partial model containing the larger domain are used in the search for the smaller domain.

 

3. Position the first domain, 1s2oA_dom1.pdb.

      From solvent content analysis, Molrep will assume that there are two copies of the model in the asymmetric unit. Set the correct number of copies explicitly to save some time: "Search Options > Number of copies to find 1"

 

4. Refine the partial model and compare refinement statistics with (2)

 

5. Position the second domain using phases from partial structure

      Select "MR Using Phases"

      Select output mtz-file from (4) as input Data; mtz-columns will be set automatically to FWT and PHWT

      Select the second domain 1s2oA_dom2.pdb as the Model

      Select output pdb-file from (4) as Fixed model

 

6. Refine the structure and check with Coot that the whole structure matches the maps

 

4.4. Notes on Spherically Averaged Phased Translation Function (SAPTF)

 

The standard Molrep protocol uses phase information from the fixed model in the Translation Function only. This is a preferred procedure until 1/3 of the structure is positioned. (You can try it: select "Molecular replacement", use the model from step (3) or (4) as Fixed model and search for the second domain.)

 

In (5), the phase information was used in both RF and TF. The 2Fo-Fc and Fo-Fc map coefficients were generated by Refmac after refinement of the structure containing the first domain. Molrep calculated one of these maps and set electron density to zero in the area occupied by the partial structure. Then Molrep uses the structure amplitudes from the masked map in conventional Patterson based RF,  and both amplitudes and phases in Phased TF.

 

In the Molecular Replacement with SAPTF, the phase information is used directly in both RF and TF steps but now the order of steps is changed and an additional Phased TF step is added to confirm and adjust the initial positions:

– SAPTF: overlap function is calculated between spherically averaged density around this point and the model density. Some of SAPTF peaks correspond to a correct position of the model regardless of the model orientation.

– Phased Rotation Functions with the centres of rotation at SAPTF peaks.

– Phased Translation function to refine the position of the molecule.

 

4.5. Finding solution for the second domain using SAPTF

 

7. Rerun (5) with an additional change to default:

      Search Options > Search protocol > SAPTF + Local Phased RF + Phased TF

 

8. Rerun (5) with another protocol:

      Search Options > Search protocol > SAPTF + Local RF + Phased TF

 

Examine log-files from steps (7,8) to see at which stage the correct solution clearly emerged from noise.

 

9. Optionally, refine the structures (7, 8).