Collaborative Computational Project No. 4
Software for Macromolecular X-Ray Crystallography

CCP4 Tutorials

Basic phasing tutorials


Download tutorials and data from here: phasing.tgz

It's not likely that you will be able to go through all these exercises in two hours. Therefore, go straight to the one which is the most interesting for you. Examples 1-4 and 6-8 are about Molecular Replacement, example 5 demonstrates the Crank pipeline for experimental phasing and example 6 is about the combination of the two techniques. Example 4 illustrates the fitting of a model into electron density maps in the context of MR structure solution, but the same technique may be used for experimental phases.

Exercise 1 is an introduction to the MR programs in CCP4 and to the CCP4I interface. Exercise 4 uses Molrep and requires some bookkeeping, but is a useful step-by-step procedure and will allow you to see changes in the maps as the structure solution progresses. Exercises 2, 3 and 6 are from the Phaser web-site and are probably the most important ones although not very difficult with the modern version of Phaser. If you are confident enough with Molrep or Phaser, try example 7 - it may not be entirely straightforward and its description does not contain step-by-step instructions. Example 8 illustrates a frequent problem with the space group assignment in the presence of pseudo-symmetry and twinning and how an incorrect assignment may affect MR structure solution. Example 5 contains step by step instructions on how to use the Crank pipeline in the default mode for experimental phasing.

It is recommended to set up a new ccp4i project for each of the examples and set the project directory as indicated below. Project directories contain all necessary data for corresponding exercises. They are located in the directory ccp4_phasing which can be downloaded from the above link.

1. Introduction to CCP4 programs for MR

Solution of hypF structure using Molrep, Phaser or MrBUMP.
The structure contains one protein molecule in the asymmetric unit.
Project directory:      1_intro
Details

2. BETA/BLIP (a protein-protein complex)

Solution of Beta-lactamase:BLIP complex using Phaser
Project directory:      2_complex
Details

3. TOXD (using ensemble models)

TOXD structure solution with ensembles in Phaser.
Project directory:      3_ensemble
Details

4. Using Phased RF and TF

Positioning the large and small domains of sucrose-phosphatase using conventional Patterson search and search in electron density map, respectively.
Project directory:      4_saptf
Details

5. Crank pipeline for experimental phasing

Automated structure solution using Crank pipeline and inflection point data.
Project directory:      5_crank_infl
Details

6. Combined MR-SAD

MR-SAD tutorial with S-SAD lysozyme and goat alpha-lactalbumin as model
Project directory:      6_mr_sad
Details

7. Structure containing several domains

Location of six domains in the AU constituting two molecules of mth685 protein.
Project directory:      7_domains
Details

8. Revising point group assignment

Twinning interfering with pseudosymmetry as well as low data quality make it possible to "solve" this structure in an incorrect orthorhombic space group.  The procedures that led to both false and true solutions are demonstrated including symmetry assignment and data merging.
Project directory:      8_symm
Details

9. An example of false solution

A simple example demonstrating a high contrast false solution and the importance of search model modification.
Project directory:      9_easy
Details

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Integration and data processing

Here is a collection of tutorials starting from diffraction images. These will develop experience with data processing software and show some tips/tricks for ways to obtain good quality reduced datasets. However, you need not stop at that point. In some cases the tutorials also go on to phasing and structure solution.

These tutorials were developed, with permission, from the X-ray Tutorial at Helmholtz-Zentrum Berlin.

If you are unfamiliar with Linux commands please ask for help to unpack the datasets. It is not completely straightforward!

Experiment 1: S-SAD on bovine Insulin

Data part 1
Data part 2

Data processing using iMosflm
Experimental phasing with CRANK
Details

Experiment 3: Molecular Replacement on monoclinic Lysozyme

Data part 1
Data part 2
Data part 3

Data processing using iMosflm
Molecular replacement with MrBUMP
Details

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Multi-crystal processing

Here are three tutorials related to the processing of data from multiple crystals using BLEND.

  1. Guide to getting started with BLEND using data from insulin crystals.
  2. A more involved investigation into the balance between completeness and non-isomorphism when merging multiple lysozyme datasets.
  3. SAD phasing with multiple, individually incomplete datasets.

BLEND tutorials

Details

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Refinement with ligands and covalent links


Download and unpack tutorial data jligand.tgz to any convenient location.
Paths of files and directories will be given relative to this location.
A copy of this page will be available locally at jligand/JL_tutorial.html.

1. Using links available from CCP4 dictionary

Description:
Adding glycan to a protein crystal structure and refinement of the completed structure

Project directory:         jligand/1_glycan/
Reflection data:            4gos.mtz
Incomplete structure:   model.pdb

Details
Check your results

2. Using a new ligand description

Description:
Refinement of a new structure from the PDB that contains a ligand missing in the CCP4 dictionary

Project directory:         jligand/2_newligand/
Reflection data:            4do4.mtz
Structure:                     4do4.pdb

Details

3. Using a new ligand and link descriptions

Description:
Additional dictionary is generated defining two entries:
– covalent link between PLP and LYS and
– isolated ligand formed by covalently bound PLP and Methyl-ASP
A structure containing two forms of bound PLP is completed and refined.

Project directory:         jligand/3_newlink/
Reflection data:            1ajs.mtz
Incomplete structure:   model.pdb

Details

Related web resources

Ligand Expo at RCSB PDB
http://ligand-expo.rcsb.org/

The PRODRG Server
http://davapc1.bioch.dundee.ac.uk/prodrg/

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More MX tutorials

EPS-CCP4 school
http://www.ccp4.ac.uk/schools/APS-school/index.php

Phaser tutorials
http://www.phaser.cimr.cam.ac.uk/index.php/Tutorials

Tutorials on Helmholtz Zentrum Berlin website
http://www.helmholtz-berlin.de/forschung/oe/em/soft-matter/forschung/bessy-mx/tutorial/index_en.html

The iMosflm tutorial
HTML and PDF

Tutorials distributed with CCP4
$CCP4/examples/tutorial/html/index.html
$CCP4/mr_tutorial_2006/mr_tutorial_first.html (example 1 in the Basic phasing collection)
$CCP4/examples/mr_tutorial_2006/mr_tutorial_advanced.html

Molrep tutorial
http://www.ysbl.york.ac.uk/~alexei/downloads/tutorial_MR.tar.gz


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