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Here are tutorials demonstrating the use of ccp4i2, as well as some of the newer CCP4 software, such as DIALS. Thanks to Ed Lowe for creating these tutorials.
The data for the data processing and SAD phasing tutorial is hosted externally, and linked to in the introduction to that tutorial.
The data for the molecular replacement and model building and refinement tutorials is bundled here: i2.tgz. Alternatively, just follow links at the top of the tutorials for each part.
Obviously these tutorials are to give an extensive flavour of ccp4i2, a more typical route through data processing, merging and phasing would be
For this practical you are presented with a set of images collected from a crystal of seleno-methionine containing CD44. These images constitute a dataset which ultimately allowed the structure of CD44 to be solved, despite showing some unwelcome pathologies. We will work through the process of indexing, integrating and scaling these data and by the end of the practical you should know how to spot some common problems with a dataset and how to extract an optimal dataset from a collection of images.
Data previously collected on native CD44 was observed to crystallize in spacegroup P212121 with cell constants a=48.88, b=77.27, c=87.67, α=β=γ=90°.
We expect there to be two protein molecules in the asymmetric unit so we will tell PARROT to try to find non-crystallographic symmetry (NCS) in our substructure model and apply any operators it finds to the density modification.
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:
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
1.2 Run PHASER for Molecular Replacement
1.3 Inspect the Output from the Molecular Replacement Job
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
2.2 Run PHASER for Molecular Replacement
2.3 Inspect the Output from the Molecular Replacement Job
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
3.2 Run PHASER for Molecular Replacement
3.3 Inspect the Output from the Molecular Replacement Job
cell content analysis
probability of input composition
translational ncs vector if any
input VRMS of members of the ensemble
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
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
number of clashes allowed
number of solutions accepted
increase in LLG for top solution
resolution for searches
expected difficulty of search
search order for ensembles (if more than one type)
cutoff selection changes (if any)
amalgamation (if any)
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.
Run Phaser again with the
anisotropy correction turned off. What effect does this have on
the structure solution?
In this practical we will continue working with the CD44 experimental phases we determined in the MAD/SAD phasing practical. We will begin where the previous practical finished, by inspecting a CD44 model which has been automatically built by the program Buccaneer.
The data for this tutorial may be found at cd44.tgz
|mdm2_unmerged.mtz||Unmerged MTZ file for the structure generated using Xia2-Dials task|
||A suitable search model.|
|4hg7.seq||Sequence file for the target structure
Stereo SMILES (CACTVS) and molecular graph
More i2 demos can be accessed as follows:
ccp4i2 > Utilities > Copy demo data to project
Quick descriptions are here:
ccp4i2 > File > Open browser window > link "Example data" (the second last line)