Welcome to the home page of BMB 695a,
Macromolecular Crystallography (Spring 2018)!

The UMass protein crystallography home page is found at xtal.biochem.umass.edu

This year's journal club is organized by Scott Garman. We meet Fridays at noon in 745E LGRT.

This year, we will process some x-ray diffraction data, solve a couple of molecular replacement problems, experimentally phase electron density maps, and build into electron density maps.

 

1. Installing the needed software:

A. Download the ccp4 suite of crystallographic programs. Version 7.0 is the current version. This package will be the workhorse of our class this semester.

• Follow the instructions for installing the package. Different operating systems will have different installations.
• If you are installing on a laptop, make sure you have enough memory and storage space available.
• Beware of spaces in directories and file names. Unix does not allow spaces in file and directory names. The CCP4i graphic interface won't run correctly if you have spaces anywhere in your directory tree where you are running CCP4.
• Run the install tests in CCP4 to make sure your installation is working.

B. Download and install Coot.

• Coot is now packaged with the CCP4 suite.

• You can also install a standalone version.
• If you are a Mac user, there is a Mac OS X build on Bill Scott's website here. His site on scientific computing on OS X (here) is tremendously useful for getting started.
• If you are a Microsoft Windows user, there is a build for that OS here.
• If you are a Linux user, there are binaries available from the main Coot page here.
• Once you have installed Coot, open the program, from which you can download coordinates and structure factors from the Protein Data Bank (PDB). Or download your favorite coordinates from the PDB, save them as a text file, and open it from the file menu in Coot.
• Make sure you can open coordinates and maps in Coot. RefMac from the CCP4 suite (see section A above) will calculate maps from structure factor files.

C. Download and install the Phenix package.

• The homepage for the suite is here. You must register as a user to get the password to download the software.
• The Phenix suite does many of the same things as the CCP4 suite (experimental phasing, molecular replacement, refinement, etc.) using different algorithms.

 

2. Test the software by processing X-ray diffraction images

The first test we will try is to process diffraction images into intensites.

See if you can process the following frames into a scaled reflection file. The frames were collected in house on our old Rigaku rotating Cu anode (wavelength λ=1.5418 Å) with the RaxisIV+ detector set to 170 mm detector distance and a beam center of 150.6 mm in x and 150.3 mm in y. Try iMosflm (included in the ccp4 package) to process the images. The goal is to extract the intensities of the whole h, k, l reflection set.

• Here are the images (in a 1.3 GB file, so make sure you have disk space and a fast connection!)
• They are compressed with zip for faster download, so the first thing you need to do after downloading is to decompress them, depending on your operating system. Try double clicking on the file to uncompress the file into an "images" folder.
• Put the images folder in a sensible place on your computer. Do not have any spaces in your directory tree. Do not put them 20 folders deep.
• Create another directory for putting the processing results.
• See if you can open the iMosflm program in the CCP4 package.
• See if you can load the images into the image viewer in iMosflm.
• See if you can process the images.

Some issues to consider:

• What is the correct space group? Getting this right is critical if we are going to get the right molecular replacement solution.
• There are several space groups consistent with the unit cell parameters of the unknown crystal. See if you can sort out which of the possible space groups is correct based upon scaling and comparing symmetry mates.
• Pay particular attention to the translational component of the space group (the subscripted number in the space group name). This is important if we are going to solve the structure.

Work on the images, and then we will discuss some strategies for processing diffraction data.

During class on February 9th, we decided that the data scaled into space group P3121 or its mirror image P3221. Let's see if we can now find out where the molecule sits in the unit cell.

 

3. Get phases from molecular replacement

Here is a pdb file for molecular replacement. See if you can figure out where the molecule falls in the unit cell. The search object is 99% identical to the unknown object we are looking for in the box, so the search in principle should be easy.

Some questions to consider before we start:

• How many objects are we looking for?
• What is the best search object, the monomer or the dimer?
• Which of the two mirror image space groups is the right one? We need to check both.
• What program are we going to use for molecular replacement? AMoRe, MolRep, Phenix, and Phaser are all reasonable choices.
• Note: The ccp4 molecular replacement tutorials are excellent.
• Some programs (e.g. MolRep) want the sequence of the unknown. Here is a fasta sequence file: GLA.fasta

Starting with the scaled data set (containing h, k, l, Fobs, σF), see if you can get started with molecular replacement, which we will work on in class on Feb 16th.

During class on Feb 16th, we took the scaled reflections data and the pdb file above and did molecular replacement in Phaser, leading to a model with a dimer in the asymmetric unit in space group P3221. We checked the packing of the molecule relative to symmetry equivalents, which all seemed to pack nicely into a three-dimensional lattice. The R-factor was still 50% after molecular replacement, so we still have work to do to refine the structure down to a reasonable R-factor. See if you can do the molecular replacement on your own, and we will move into refinement in the next class on Feb 23rd.

 

4. Build into electron density

Now that we have amplitudes and phases, we can calculate electron density maps for the unknown. Take the output coordinates from molecular replacement, run 10 cycles of rigid body refinement in Refmac, and calculate maps from the output coordinates. Some questions at this stage:

• How do we know the molecular replacement was successful?
• Do the molecules pack into the unit cell without too much collisions?
• How do we avoid model bias? The maps are likely to look quite a bit like the search object at this point.
• What do we need to fix to get to a publishable structure?

 


 

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