The refinement of water molecules, temperature factors and occupancy

The bulk of the solvent molecules within a crystal will be disordered and lie in solvent channels between the protein molecules. The contribution of this bulk solvent towards X- ray scattering can be determined. A smaller proportion of the water molecules will be ordered and should be included in the refined structure. The starting model for the refinement of Thermus-MnSOD-N3- was the native Thermus-MnSOD, including water

molecules with refined positions, temperature factors, and occupancy. The inclusion of too many waters from the native structure in early rounds of refinement is likely to bias the starting model toward the native structure and decrease the amount of novel information that can be derived from the azide-derivative X-ray data sets. During the refinement of

Thermus-MnSOD-N3-, the positions, temperature factors and the occupancy of water

molecules were further refined. The results of this refinement are that within the submitted structure of Thermus-MnSOD-N3- there are water molecules that have: full occupancy and

low temperature factors; full occupancy and high temperature factors; low occupancy and low temperature factors; and low occupancy and high temperature factors; refer to Table 10. The treatment of waters in the structure is sub-optimal and there is likely fitting of data to structure rather than fitting structure to data.

B-factor <25 B-factor >25 Total # of H2O 202 → 139 + \ 63 - \

↓

Occupancy > 80% 135 \+ 76 + + 59 - + Occupancy < 80% 67 \ - 64 + - 3 - - Table 10. The distribution of occupancy and temperature factors of ordered solvent within Thermus-MnSOD-N3-.

Table 10 shows the 202 solvent molecules of Thermus-MnSOD-N3- categorised by

temperature factor and occupancy. The threshold for B-factors was chosen to put roughly one third of water molecules into the high temperature factor category. Waters with low temperature factors and high occupancy are indicated by (++). Waters with either low occupancy or high temperature factors are indicated by (- +) or (+ -). Low quality water molecules with low occupancy and high temperature factors are shown by (- -). Less than half the water molecules fall into the (++) category.

Typically, ordered water molecules are built into spherical peaks of electron density at suitable hydrogen bonding distances from the protein and other water molecules. In early rounds of refinement only the first shell of hydration, within about 3.5 Å of the peptide, can be built reliably. During further refinement, if experimental data warrant it, more water molecules can be added.

Initially as iterative refinement between map interpretation and refinement proceeds, the signal-to-noise ratio in maps improves. Eventually, however, the amount of novel structural information that can be derived from electron density maps decreases due to increased model bias. In later cycles of refinement the size of density peaks associated with novel solvent molecules will decrease. In final rounds of refinement when there is very little influence of X-ray derived information on electron density maps, water peaks will become indistinguishable from noise. If care is not taken during the refinement process, water molecules can be added incorrectly to ambiguous density peaks that are the result of Fourier ripples or side chains in multiple conformations. During automated

refinement by computer programs the position of a water molecule within a protein structure is less restrained by bond distances and angles than the atoms of the peptide and during refinement have greater degrees of freedom.

The temperature factors and occupancies are specific to individual atoms and are targets for refinement within the structure. These variables are correlated (Bhat, 1989) and they can both can act as “fudge factors” to make an individual atom agree with the X-ray data without altering the Cartesian position of the atom. Both occupancy and temperature factors are measures of the proportion of time that an “average” atom is at a point within the unit cell. In the interpretation of electron density maps a water molecule with low occupancy is almost indistinguishable from of a water molecule with a high temperature factor (Kundrot & Richards, 1987).

In high- and medium-resolution structures it is more sensible to refine temperature factors than occupancy. The majority of protein atoms have limited movement and most atoms will have 100% occupancy and low temperature factors. Subsequently regions with some conformational freedom, such as the long flexible side chains of surface lysines and arginines, can be built with full occupancy and high temperatures factors. Well defined water molecules will have low temperature factors and poorly defined waters will have high temperature factors.

Well-defined water molecules should have temperature factors that are comparable to surrounding atoms after temperature-factor refinement. If the temperature factor of an individual water atom is distinctly higher than surrounding atoms it may indicate that the solvent molecule should be removed as it is may be derived from a false peak. The temperature factors of a structure should vary smoothly over the whole structure. The analysis of temperature factors can give an indication of how much individual atoms of a structure contribute to the calculated X-ray data. Atoms with very high temperature factors essentially contribute very little to the calculated structure factors, especially for high- resolution data. This is particularly relevant to older structures where the structure factors have not been submitted.

Alternatively, water molecules can have their occupancy refined. It has been suggested to manually set occupancy based on the interpretation of the electron density for possible water molecules (Jensen, 1990). This approach can also be used for waters that are clearly split between two conformations. Split occupancy waters will manifest away from peptide as two peaks closer than hydrogen-bonding distances or as dumb-bell shaped projections

in electron-density maps. The refinement of water occupancy becomes viable only with ultra high resolution data. After occupancy refinement, waters that have low occupancy should be removed as they are not relevant to the bio-molecule or are the result of Fourier ripples. Protein crystals contain large amounts of solvent, sometimes more than 60% of the volume of the crystal (Matthews, 1968). The water molecules close to the protein will have some ordered qualities created by hydrogen bonding partners and electrostatic forces. Ordered waters will have interpretable electron density.

The optimal methods for building of solvent in protein structures are much debated at present (Brown & Ramaswamy, 2007, Kleywegt, 2000) as it was at the time the structure of Thermus-MnSOD-N3- was solved (Kleywegt & Jones, 1995, Kundrot & Richards,

1987).