Introduction
This practical is closely based on
the tutorials written by Airlie McCoy, the author of PHASER and
slightly modified to make use of the ccp4i2 GUI.
During the practical you will learn
1) how to use ensembling to construct a search model and 2) how
to solve a heterodimeric complex 3) how to solve a
homo-oligomer from a monomer
The data used in this tutorial can be obtained here:
0. Getting Started with CCP4 GUI2
- Launch ccp4i2 by double
clicking on the icon on the desktop
- You will be presented with a
"Welcome" screen. Click on the link to "Start a new
crystallography project"
- In the "Name of
project/folder" field, enter MR. Click the "Select Directory"
button and browse to the directory where you unpacked the data
- A "Project Viewer" window
will open for project MR
1. MR using ensemble search models: TOXD
α-Dendrotoxin (TOXD, 7139Da)
is a small neurotoxin from green mamba venom. You have two
models for the structure. One is in the file 1BIK.pdb, which
contains the protein chain from PDB entry 1BIK, and the other is
in the file 1D0D_B.pdb, which contains chain B from PDB entry
1D0D. 1BIK is the structure of Bikunin, a serine protease
inhibitor from the human inter-α-inhibitor complex, with
sequence identity 37.7% to TOXD. 1DOD is the complex between
tick anticoagulant protein (chain A) and bovine pancreatic
trypsin inhibitor (BPTI, chain B). BPTI has a sequence identity
of 36.4% to TOXD. Note that models making up an ensemble must be
superimposed on each other, which has not yet been done with
these two structures.
1.1 Sumperimpose the two pdb files
that will make up the ensemble
- Launch Coot from ccp4i2 tool bar
or from Task Menu > "Model Building and Graphics" >
"Manual model building - COOT"
- From the File menu, select "File
> Open Coordinates" and browse to your project directory.
Open both 1BIK.pdb and 1D0D_B.pdb
- Are the two structures
superimposed? They will need to be
in order for PHASER to construct an ensemble from them.
- We can use COOT to superimpose
1BIK on 1D0D.
- Select "Calculate > SSM
Superpose"
- For the reference structure
select 1D0D_B.pdb and for the moving structure select 1BIK.pdb
- Click "Apply" and you should see
the two molecules superimposed on each other.
- Now save coordinates to ccp4i2
project MR
- In Coot menu bar select "File >
Save mol to CCP4i2"; check that molecule "0 ..." is selected;
press OK
- In Coot menu bar select "File >
Save mol to CCP4i2"; select molecule "1 ..."; press OK
- In Coot menu bar select "File >
Exit"
- You are now ready to carry out
molecular replacement with an ensemble.
1.2 Run PHASER for Molecular
Replacement
- From the "Task Menu" in ccp4i2,
select the "Molecular Replacement" section and launch the
"Expert Molecular Replacement - PHASER" task
- In the section marked
"Reflections" you will need to import an mtz file since this
project does not yet contain any reflections. Click the file
browser icon and select the file toxd.mtz. You can accept the
defaults you are offered when importing these data.
- In the section marked "Use Is or
Fs" select F. It is often
preferable to use Is, but this data file only contains Fs.
- We need to specify the
Composition of the asymmetric unit of our crystal to the best
of our understanding. We will do this by specifying a sequence
file for TOXD.
- In the composition section, click
the file browser icon and select the file toxd.seq. You can accept the defaults offered to you when
importing the sequence.
- In the section marked "Search
Model(s)" you will need to construct an ensemble from the two
structures you superimposed in section 1.1
- Click on the button marked "Show
list" to allow you to construct a search model from multiple
coordinate files. Here you have
the option to build one or more ensembles to use as search
models, each of which will contain one or more atomic models.
- In the menu just beneath the
"+" and "-" buttons, select "Coot output file 1", type in
sequence identity.
- Use "+" to
"add structure in ensemble". Select "Coot output file 2", type in sequence identity.
- In the end, the section "Search
model(s) should look similar to the image below
1.3 Inspect the Output from the
Molecular Replacement Job
- Open the "Results" tab for the
job you have just run.
- There are a number of nested
lists containing a great deal of information about the job
that has just run. Use this information and your knowledge of
molecular replacement to complete the following tasks:
- Write down the steps of structure
solution in the order in which they were taken. The section "Search strategy employed by PHASER"
should be helpful here.
- Look through the log file to find
the pieces of information listed in Table 1 at the bottom of
this tutorial.
- Has PHASER solved the structure?
2. Solving a heterodimeric complex using MR:
BETA/BLIP
β-Lactamase (BETA, 29kDa) is an
enzyme produced by various bacteria, and is of interest because
it is responsible for penicillin resistance, cleaving penicillin
at the β-lactam ring. There are many small molecule inhibitors
of BETA in clinical use, but bacteria can become resistant to
these as well. Streptomyces clavuligerus produces beta-lactamase
inhibitory protein (BLIP, 17.5kDa), which has been investigated
as an alternative to small molecule inhibitors, as it appears
more difficult for bacteria to become resistant to this form of
BETA inhibition. The structures of BETA and BLIP were originally
solved separately by experimental phasing methods. The crystal
structure of the complex between BETA and BLIP has been a test
case for molecular replacement because of the difficulty
encountered in the original structure solution. BETA, which
models 62% of the unit cell, is trivial to locate, but BLIP is
more difficult to find. The BLIP component was originally found
by testing a large number of potential orientations with a
translation function search, until one solution stood out from
the noise.
2.1 Consider the MR problem
- Import the reflection data for
BETA/BLIP into your ccp4i2 project.
- In the "Task menu" open the
"Import merged data,sequences,alignments or coordinates"
section launch the "Import merged reflection data" task.
- In the section marked "Reflection
data" click on the file browser icon and select the file
beta_blip_P3221.mtz
- Provide a crystal name and a
dataset name. These can be
anything, but giving them systematic names is usually a good
idea. I would suggest crystal1 and betablip
- Run the task.
- What spacegroup is reported by the
mtz file. Look in the input tab of
the job you have just run.
- If this structure had not already
been solved, would you know that this was the space-group? If
not, what other space-group(s) must you consider? Consider handedness and possible enantiomorphs.
2.2 Run PHASER for Molecular
Replacement
- From the "Task Menu" in ccp4i2,
select the "Molecular Replacement" section and launch the
"Molecular Replacement and refinement - PHASER" task
- In the "Use data from job"
section, select the "Import merged reflection data" job that
you ran in section 2.1
- In the section marked
"Composition" you need to describe the likely contents of the
asymmetric unit - in this case we expect to find one copy each
of BETA and BLIP.
- In the "Composition" section click
the "Show list" button.
- Click the file browser icon and
select the sequence file beta.seq. Make sure that the "Number
of copies in asymmetric unit" is set to 1 for this sequence.
- Click the "+" button to add an
additional sequence.
- Click on the file browser icon and
select the sequence file blip.seq. Make sure that the "Number
of copies in asymmetric unit" is set to 1 for this sequence.
- You must now define the search
model(s). This time we will be
searching for one ensemble after another (each containing a
single model) instead of using a single ensemble containing
multiple models.
- In the section marked "Search
model(s)" click on the "Show list" button. The first ensemble will be selected by default,
but will be populated with an incorrect model (it will have
assumed that we are still working with the same model as in
section 1).
- Click on the file browser icon
alongside the atomic model selection and select the file
beta.pdb. In the sequence identity box enter 1.0
- Click on the "+" icon and select
"Add ensemble" from the options presented.
- Select the model field for
Ensemble 2. Click on the file browser icon alongside the
atomic model selection and select the file blip.pdb. In the
sequence identity box enter 1.0
- Select the first ensemble (the
ensemble itself rather than the model it contains). Ensure
that you will be searching for 1 copy of this ensemble and
give the ensemble the name "BETA".
- Repeat the last step for the
second ensemble but give it the name "BLIP"
- Run the task
2.3 Inspect
the Output from the Molecular Replacement Job
- Open the Results tab for the job
you have just run. Use the information here and your knowledge
of Molecular Replacement to complete the following tasks:
- Write down the steps in the
structure solution in the order in which they were taken. How
are these steps different from the TOXD example?
- Find the pieces of information
listed in Table 1 at the bottom of this tutorial
- Which space group is the solution
in? Which other space groups were tested (if any)? The first translation function search would be
the best place to look for this.
- Why doesn't Phaser perform the
rotation function in the two enantiomorphic space groups?
- Which reflections in the data are
particularly important for deciding the translational symmetry
of the space-groups to search? Under what data collection
conditions might you not have recorded these important
reflections? Are there any other space-groups that you might
want to consider when solving BETA/BLIP?
- How big is the anisotropic
correction for the data? How does this compare to TOXD?
- Has PHASER solved the structure?
3. Solving a homo-oligomeric complex: HICA (If you have time)
Carbonic anhydrase is an enzyme that
assists rapid inter-conversion of carbon dioxide and water into
carbonic acid, protons and bicarbonate ions to aid removal of
carbon dioxide from the blood in respiration. This ancient
enzyme has three distinct classes; alpha, beta and gamma.
Carbonic anhydrase from mammals belong to the alpha class, the
plant enzymes belong to the beta class, while the enzyme from
methane-producing thermophillic bacteria forms the gamma class.
Members of these different classes share very little sequence or
structural similarity. The alpha enzyme is a monomer and the
gamma enzyme is trimeric. The beta enzyme can be a dimer,
tetramer, hexamer or octamer. Haemophilus
influenzae β-carbonic anhydrase
(HICA,2a8d) is an allosteric protein. The model you have for
this structure is E. coli β-carbonic anhydrase, which has 61% sequence
identity to HICA. NB. This is a
computationally demanding task so don't worry if on your
particular machine it fails or takes an unacceptably long time
to run - try running the same task on a more powerful machine at
a later time.
3.1 Consider the MR problem
- Import the reflection data for
HICA into your ccp4i2 project.
- In the "Task menu" open the
"Import merged data,sequences,alignments or coordinates"
section launch the "Import merged reflection data" task.
- In the section marked "Reflection
data" click on the file browser icon and select the file
fast_2a8d.mtz
- Provide a crystal name and a
dataset name. These can be
anything, but giving them systematic names is usually a good
idea. I would suggest crystal1 and hica
- Run the task.
- From the Task menu, select the
"Reflection data tools" section and launch the "Estimate cell
content" task.
- In the section marked "Cell
parameters taken from reflection data" select the reflections
from the import data job you just ran.
- In the section marked "Calculate
molecular weight from" click on the file browser icon and
select the sequence file fast_2a8d.seq
- The Matthews calculation will be
carried out automatically.
- How many monomers of β-carbonic
anhydrase can fit in the asymmetric unit? Which of these
possibilities is most probable? Which of these are possible?
What are the oligomeric associations that could correspond
with the possible asymmetric unit contents? Consider the application of crystal symmetry.
3.2 Run PHASER for Molecular
Replacement
- From the "Task Menu" in ccp4i2,
select the "Molecular Replacement" section and launch the
"Molecular Replacement and refinement - PHASER" task
- In the "Use data from job"
section, select the "Import merged reflection data" job that
you ran in section 3.1
- In the "Composition" section,
select the fast_2a8d sequence. In the field for the number of
copies in the asymmetric unit, enter the number of copies you
think should be present in the asymmetric unit based on
section 3.1
- In the "Search model(s)" section,
you will need to define a single search model but tell PHASER
to search for multiple copies of it.
- Click on the file browser icon and
select the file fast_1i6p.pdb
- In the "Sequence identity" field
enter 0.61
- Click on "Show list" and select
the ensemble rather than the atomic model that it contains
- Tell PHASER to find the same
number of copies that you entered in the "Composition" field
above.
- Run the task (this job will take
longer to run - this is normal when there are many copies to
find)
3.3 Inspect
the Output from the Molecular Replacement Job
- Write down the steps in the
structure solution in the order in which they were taken.
- Find the pieces of information
listed in Table 1
- Has Phaser solved the structure?
- How many molecules
are there in the asymmetric unit?
Table 1
|
cell content analysis
|
probability of input
composition
|
|
anisotropy correction
|
anisotropic B-factor
|
|
translational ncs
|
translational ncs vector if
any
|
|
ensembling
|
input VRMS of members of
the ensemble
|
|
rotation function
|
selection criteria for
rescoring fast RF orientations with full RF
number selected for
rescoring with full RF
highest RFZ in full RF
final (purging) selection
criteria
number selected for TF
|
|
translation function
|
selection criteria for
rescoring fast TF positions with full TF
number selected for
rescoring with full TF
highest TFZ in full TF
number of TFZ > 8
final (purging) selection
criteria
number selected for packing
|
|
packing function
|
number of clashes allowed
number of solutions
accepted
|
|
refinement
|
increase in LLG for top
solution
refined VRMS
TFZ equivalent
|
|
automated MR
decision making
|
resolution for searches
expected difficulty of
search
search order for ensembles
(if more than one type)
cutoff selection changes
(if any)
amalgamation (if any)
|
4. Advanced
If you have time revisit the TOXD
and BETA-BLIP examples and look at the following exercises. These should help you understand how the choices
you made in the worked examples influence the outcome of
Molecular Replacement.
4.1 TOXD
- Run PHASER again without using
ensembling, using just 1BIK or 1D0D as a search model. What
are the LLG values of the final solutions? What are the
Z-scores of the translation functions? Was ensembling a good
idea?
- Run PHASER again using the two
pdb files before superposition as search models. What does
PHASER report?
4.2 BETA-BLIP
Run Phaser again with the
anisotropy correction turned off. What effect does this have on
the structure solution?
Return to the top of this section
. You should unpack this
archive into a directory where you have space (users at Diamond,
beware the limited quota of your home directory!)