X-RAY CRYSTALLOGRAPHY 97
reference 19. A solvent - fl attened map (solvent fl attening techniques improve
electron density maps) was calculated at 2.8 - Å resolution and contoured
at 1.2 σ , and a 2| F 0 | − | Fc | composite OMIT map was prepared with CNS. Its
website at http://cns.csb.yale.edu/v1.1/ states that the Crystallography & NMR
System (CNS) is the result of an international collaborative effort among
several research groups. The program has been designed to provide a fl exible
multilevel hierarchical approach for the most commonly used algorithms
in macromolecular structure determination. For the Ca 2+ - ATPase crystallo-
graphic study, the refi ned temperature factors were 60.0 Å^2 for protein atoms,
48.7 Å^2 for bound water molecules, and 41.3 Å^2 for Ca 2+ ions. The assignment
of secondary structure was carried out with the program DSSP. The DSSP
program was designed by Wolfgang Kabsch and Chris Sander to standardize
secondary structure assignment. The DSSP database is a database of second-
ary structure assignments (and much more) for all protein entries in the
Protein Data Bank (PDB). Some refi nement statistics taken from reference
19 , Table 1 are the following: resolution range, 15 – 2.6 Å ; number of refl ections;
48,373; number of protein atoms, 7673; number of water molecules, 276; number
of calcium ions, 2. The calculated Rcryst factor (see equation 3.11 ) was 25.0%.
Using a test set comprised of 10% of the data for refi nement resulted in an
Rfree of 30.7%.
Mark Wilson and Axel Brunger published the 1.0 - Å crystal structure of
Ca2+ - bound calmodulin (PDB: 1EXR) in 2000.^20 Calmodulin (CaM) is a small
acidic protein of 148 amino acid residues weighing approximately 17 kDa.
CaM is present in all eukaryotic cells, serves as a primary receptor for intracel-
lular Ca 2+ , and is considered to be a switch in cellular processes controlled by
Ca2+ - dependent signaling pathways. Calmodulin will be discussed in more
detail in Section 6.3.2. Reference 20 presents a thorough X - ray crystallo-
graphic study of this small protein (about 1/10 the size of the Ca 2+ - ATPase
discussed previously in this section). The X - ray crystallographic data on the
calmodulin crystals were collected at the Stanford Stanford Synchrotron Radi-
ation Laboratory (SSRL). Essentially, Wilson and Brunger followed the same
processes for collecting and analyzing data as described above for Ca 2+ - ATPase.
Their detailed explanation of the data collection and multiple - stage refi ne-
ment of the calmodulin crystallographic model in reference 20 will be of inter-
est to those readers wishing to learn more about these techniques.
It is obvious from the foregoing description that solving the structure of
large proteins and other macromolecules requires technologically advanced
hardware and software. The National Institutes of Health are supporting the
Protein Structure Initiative, PSI (as discussed in Section 2.2.2 ). The mission of
the PSI is to make the three - dimensional, atomic - level structures of most
proteins easily available from knowledge of their amino acid sequences.
To this end, NIH is supporting the development of tools to accelerate experi-
mental structure prediction, whether by new technology in X - ray crystallo-
graphy or nuclear magnetic resonance (NMR). NMR will be discussed in
Section 3.4.