This task allows you to run PHASER for molecular replacement, up to
and including building multi-subunit complexes, each component of
which is constructed from an ensemble of structures. Optionally, the
model can be:
Put through a few cycles of REFMAC refinement (option selected by default)
Symmetry matched to a reference PDB
Processed by running a coot script (e.g. to fill partial residues in a model that has been chainsawed).
Resolution range. Based on the expected LLGs, PHASER will try to
search with the best resolution. You may override the maximum
resolution that PHASER will attempt here.
Spacegroups. Where there is ambiguity, you can invite PHASER to
attempt MR in more than one spacegroup: data reduction generally
provides insight into the likely (but not certain!) pointgroup of the
crystal, with less reliable guidance as to the space group.
Free R set. Although PHASER does not take advantage of a FreeR set,
the optional REFMAC cycles which run at the end of this task should
be given the FreeR data object that you are using for this crystal
form.
The maximum likelihood approach in PHASER works best if it knows the
total scattering in the unit cell. The composition section allows you
to provide that information in one of three ways. Firstly, you may
choose to offer no information. In this case, PHASER will identify
the most probable solvent content for the resolution of data
provided, and infer the amount of scattering from that. Secondly, you
may provide an estimate of the total molecuar weight of protein
and/or nucleic acid in the crystal. In the event of non
crystallographic symmetry, this will be more than the sum of the
molecular weights of the macromolecular chains that you think you
have crystallized. Phaser will evaluate the number you provide here
and give feedback on the number of copies thereof that are most
consistent with the probable solvent content. Thirdly, you can
provide the sequences of all of the species you think to be present
in the crystal together with the number of copies of each that you
expect to form the crystallographic asymmetric unit. Again, PHASER
will provide feedback on the number of copies of your estimated
composition that are most likely based on the probable solvent
content.
Simple search with a single structure: If you have a single
structure that you want to position in the unit cell, then all you
have to do is provide that structure here :-). The user can specify a
subset of the structure to search with using the “coordinate
selection” syntax described
here. This may be useful if
the structure you are solving corresponds to one chain of a PDB that
you have downloaded, or if there are segments of the structure that
you think will be in a different conformation in the new crystal
(typically useful for N- and C-terminii or for known flexible loops).
The user must also specify either the sequence identity of the
search model to the species present in the crystal, or the expected
RMS coordinate similarity between them. These parameters are required
for PHASER’s maximum likelihood approach, although even reputable
crystallographers occasionally just provide the number 0.9 as
sequence identity
More complex MR search scenarios: Click the “Show list” button if
you want to:
Position multiple copies of a search molecule in the unit cell,
Build up a macromolecular hetero-oligomeric complex, and/or
Exploit ensembles (multiple superimposed search models for a
particular component of the asymmetric unit)
This will cause the expanded search model dialog to be displayed,
which provides control of the search models provided and the searches
to be carried out
The expanded search model dialog has two panels, namely:
• The search model tree (2.1), and
The search model detail (2.2)
The “search model tree” shows an overview of the search model(s) that
you have provided to the gui. This is made up of
“ensembles”, each of which is made up of
“structures”. What is displayed in the “search
model detail” depends on what is selected in the search model tree.
Ensembles: An ensemble corresponds to a unit of structure that can
be positioned in an MR run. It generally constitutes a “rigid body”
that is likely to be common between the search model(s) provided and
the structure for which the data have been collected. In the simplest
case, an ensemble can comprise a single structure. If this works
(which it will in > 90% of solvable cases), then more power to your
elbow :-). When an ensemble is selected in the search model tree, the
search model detail allows you to specify 1) The number of copies of
the ensemble to search for in this run, and 2) A short one word
alphanumeric identifier to associate with this ensemble.
Structures: If searching with a single structure in your ensemble
fails, then it may be that adding more than one (superimposed)
structure into your ensemble can help. The idea is that each
superimposed structure will have different strengths and weaknesses,
such that the search for the composite model (i.e. ensemble) may
succeed where searching for each constituent structure fails. N.B.
It is currently up to the user to superimpose the structures prior to
giving them to CCP4i2. When a “structure” is selected in the search
model tree, the search model detail allows/requires you to specify
similar properties to those described above for a
simple search model.
Adding and deleting ensembles and structures. To add an ensemble to
the search, click “+” and select “Add ensemble”. To add a structure
into an ensemble, select the ensemble in the search model tree, click
“+” and select “Add structure in ensemble”. To delete an ensemble or
structure, select it in the search model tree and click “-”
Searching for more than one copy. To search for more than one copy
of an ensemble, you will need to have clisked “Show list” to reveal
the Expanded search model dialog. The number of copies to search for
in this run is set be selecting the relevant ensemble in the search
model tree and then specifying the number to search fo r in the
search model detail panel.