A tutorial for learning and teaching macromolecular crystallography

Five experiments have been designed to be used for teaching macromolecular crystallography. The three proteins used in this tutorial are all commercially available; they can be easily and reproducibly crystallized and mounted for diffraction data collection. For each of the five experiments the raw images and the processed data of a sample diffraction data set as well as the refined coordinates and phases are provided for teaching the steps of data processing and structure determination.


Experiment 3: Molecular Replacement on monoclinic Lysozyme
Lysozyme is a 129 amino acid enzyme that dissolves bacterial cell walls by catalyzing the hydrolysis of 1,4-β-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in the peptidoglycan layer and between N-acetyl-D-glucosamine residues in chitodextrins. It is abundant in a number of secreted fluids, such as tears, saliva and mucus.
Lysozyme is also present in cytoplasmic granules of the polymorphonuclear neutrophils (Voet et al., 2006). Large amounts of lysozyme can also be found for instance in egg whites. The crystal structure of hen egg-white lysozyme (HEWL) based on crystals belonging to the tetragonal space group P4 3 2 1 2, was the first enzyme structure published (Blake et al., 1965). Over the years, HEWL has been crystallized in many different crystal forms (for an overview see Brinkmann et al., 2006) and has become a standard object for methods developments but also for teaching purposes.
In this experiment, the structure of monoclinic HEWL is determined by Molecular Replacement (MR) using the structure of tetragonal HEWL as a search model. MR is a method to determine a structure in cases where a similar structure is already known. If the similar structure can be correctly oriented and positioned in the unit cell of the structure to be solved, it can be used as a starting point for phase calculation and refinement. Currently, about two thirds of all new structures deposited with the PDB (Berman et al., 2000) are solved using MR (Long et al., 2008).

Data Processing
The data were indexed, integrated and scaled using the program XDS (Kabsch, 1993). XDS is able to recognize compressed images, therefore it is not necessary to unzip the data before using

• integration 2 nd run of XDS
After determination of space group and cell parameters all images will be integrated and corrections for radiation damage, absorption, detector etc. will be calculated in a second XDS run.

DEFPIX
defines the trusted region of the detector, recognizes and removes shaded areas, and eliminates regions outside the resolution range defined by the user. XPLAN helps planning data collection. Typically, one or a few data images are collected initially and processed by XDS. XPLAN reports the completeness of data that could be expected for various starting angles and total crystal rotation.
Warning: If data were initially processed for a crystal with unknown cell constants and space group, the reported results will refer to space group P1. XSCALE writes out a *.ahkl file, which can be converted with XDSCONV to be used within the CCP4-suite (Collaborative Computational Project, 1994) or other programs.

Structure Solution
The structure can be solved using the SAD-protocol (run in the advanced version) of AUTO-RICKSHAW: the EMBL-Hamburg automated crystal structure determination platform (Panjikar et al., 2005). AUTO-RICKSHAW can be accessed from outside EMBL under www.emblhamburg.de/AutoRickshaw/LICENSE (a free registration may be required, please follow the instructions on the web page). In the following the automatically generated summary of AUTO-RICKSHAW is printed together with the results of the structure determination: The structure was solved using the MR-protocol of Auto-Rickshaw with tetragonal HEWL (PDB entry 193L, Vaney et al., 1996) as a starting model. The input diffraction data (file XDS_ASCII.HKL) were uploaded and then prepared and converted using programs of the CCP4-suite. The molecular replacement step was done using MOLREP (Vagin and Teplyakov, 1997) with a resolution cut-off of 4 Å to find the two molecules in the asymmetric unit. Despite a very high initial R-factor of 73% (correlation coefficient 43%), the solution was correct as was demonstrated by subsequent refinement. This was performed to a resolution of 3.0 Å using the program CNS (Bruenger et al., 1998) in four consecutive steps: rigid body refinement, a minimisation step, B-factor refinement and a second minimisation step. At this point the R-and R free -values were 24.9 and 33.5%, respectively. Further refinement was done in REFMAC5 using all available data to R-and R free -values of 28.3 and 31.5%. The model was completed and further modified using COOT and refined using REFMAC5. Figure 6 shows the final electron density with some nitrate ions clearly visible. For more detailed information see the AUTO-RICKSHAW output (directory experiment3/autorickshaw). Figure 3 shows the cartoon representation of the two molecules in the asymmetric unit. Clear electron density can be found where the nitrate ions are bound (see Figure 4).