probably due to the fact that only
18water molecules have been included in the
model and consequently the solvent in the crystal has not been fully accounted for.
Fig. 70 Luzzati plot for the partially refined ApoLfN structure.
The position of the error lines equivalent to errors of 0. 1, 0.2, 0.3 and 0.4 A are indicated. Resolution is defmed in tenns of sine/A.
0.4 � 0.3
J§
et:
0.2 0.1 0.1 A 0.0 0 . 0 0 0 . 05 0 . 1 0 0 . 1 5 0 . 2 0 0 . 2 5 ResolutionThe agreement of the model with the data was quantitated by calculating real space correlation correlation coefficients using the program
0
(Jones et al., 1991). While most of the correlation coefficients are above 0.85 they are not as good as the correlation coefficients obtained for FeLfN (Section B3.2.8). There are several poorly defined regions including, residues 4, 38, 86, 134 - 143, 219 - 220, 260and 294 - 295 where the correlation coefficients fall below 0.75 and rebuilding is required. These are all on the surface of the molecule and the majority of the structure is believed to be correct.
Fig. 7 1 Mainchain real space correlation coefficients for ApoLfN.
!!. i j_ k �-strands
200 250 300
Preliminary analysis of the B values has also shown that although they are quite variable they are distributed in a similar manner to that found i n other N-terrninal
structures (Fig. 7 2). The average B value for ApoLfN is 3 1 .8
A2
which is actually lower than recorded for the other structures. This low average B value may be partly attributed to the fact that only eight cycles of B value refinement have been carried out and the correct B value for all of the atoms has not been reached. Despite the difference in the overall B value the main point to note is that in general residues with high average B values lie on the surface of the protein, in more mobile and poorly defined regions, while the residues with low average B values are mainly found in the interior of the protein and are involved in secondary structural motifs.Fig. 72 Plot of average mainchain B values against residue number.
80
_
�-strands0
50
100150
200
250
300Residue Number
The correctness of the structure was also examined by analysing the distribution of the dihedral angles. The geometry appears very good in that except for Ser 1 9 1 and Leu 299, which have been discussed during analysis of the FeLfN structure (Section B 3.2.8), all of the remaining residues lie within acceptable re
gi
ons on a Ramachandran plot (Fig. 73). This plot is actually better than expected andalthough the dihedral angles have not been restrained, refinement is incomplete and the dihedral angles may still reflect those of the strarting model.
B 4 . 2 . 5
Fig. 73 Ramachandran plot of mainchain phi/psi angles for ApoLfN.
The allowable regions, as reported by Ramakrishnan and Ramachandran (1965) are indicated by broken lines and glycines are marked by a square rather than a cross.
-90
+
···++· · ··.p:··--··· l:l··-.. �180 Cl •
-90
Analysis of the ApoLfN structure 0 Pili
+
180
As refmement of ApoLfN is not complete a detailed analysis of this structure is not warranted, however, several general features of the structure will be discussed. Conformation and crystal packing of ApolfN
A cartoon diagram of ApoLfN is shown in Fig. 7 4. The most notable feature of
this structure is that it is essentially identical to the structures of FeLfN and the N
lobe of Fe2Lf, ie. it is closed. The rms deviation between the two LfN structures
is only 0.30 A which is less than the rms deviation obtained when either of these
structures is compared with the N-lobe of Fe2Lf (fable 37). This is inspite of the
Fig. 7
4
Cartoon diagram of ApoLfNThis diagram was prepared using the program Ribbon (Richardson, 1985; Priestle, 1988). In this
representation a-helices are shown as pink spirals and �-strands as teal arrows.
Table 37 Comparison of the deviations between the three closed N-lobe structures.
Structures rms deviation
ApoLfN and FeLfN
0.30 A
ApoLfN and Fe2Lf N-lobe
o.34 A
FeLfN and Fe2Lf N-lobe
o.36 A
Like FeLfN, ApoLfN is also slightly more 'open' than the N-lobe of Fe2Lf, however it is certainly not wide open like the N-lobe of ApoLf. This is the first structure of a supposed iron free lactoferrin where the N-lobe has been found to be closed. Possible reasons for the closed structure may depend on the origin of the density in the iron site which remains unexplained at present (see below). A second intriguing question concerns the crystal packing. If the structures of ApoLfN and FeLfN are so similar, as indicated here, why have they crystallised in different space groups and why is the packing of the molecules in the two unit cells so different? As shown in Fig. 7 Sa the molecules in the FeLfN unit cell are packed in a 'back to back' configuration while the molecules in the ApoLfN unit cell are packed in a 'front to front' configuration (Fig. 75b) and the back of the each molecule is exposed to the solvent (indicated by an arrow for the purple ApoLfN molecule). Admittedly the position of residues
3 1 3 - 333
has not been determined in ApoLfN and these will probably occupy some of the space in this channel, butthe orientation of the molecules in the two unit cells is still quite different. It will be interesting to determine the position of the remaining residues in ApoLfN as these may hold the key to the difference in crystal packing even though almost identical crystallisation conditions were used. Perhaps these residues have yet another orientation in ApoLfN? Initial attempts to build these residues into the structure have not been successful because of the lack of well defmed density and the wish to be confident about the position of the residues to avoid incorrectly
Fig. 75 Comaparison of the crystal packing in ApoLfN and FeLfN.
Fig. 75a shows the packing of the molecules in FeLfN and Fig. 75b shows the packing in
ApoLfN. Fig. 75a
biasing the structure. It is also possible that they are disordered, however, and this question may only be resolved by collection of a higher resolution data set or a derivative data set. Unfortunately limitations on the time available have not permitted this work to be completed in this study.
The binding site in ApoLfN
Besides being closed the ApoLfN structure is distinguished by the presence of
extra density within the metal binding site (Fig. 76). As the protein sample used to
prepare these crystals was colourless and supposed to be metal free the presence of
this density was most u nexpected. At the end of round three of refinement an attempt was made to fit two water molecules into the density. When the position of the two water molecules was not restrained during refinement one of them was moved out of the metal binding site and the B values of both changed rather dramatically. This suggested that the water molecules had been placed in
unfavourable positions. Both water molecules were therefore removed from the model and a further eight cycles of refinement were carried out at the end of round
four. A new 2Fobs -Fcaic map was created at the end of these extra cycles �nd the
density at the metal site analysed. The photograph in Fig. 76 was taken at this
time. Clearly 'something' is in the iron site but the nature of the density is unknown. Comparison of the level of density with other parts of the structure showed that it was similar to that of a well defined water molecule, although its shape shows that it is clearly not a single water molecule. To date no viable alternative h as been obtained but several possible reasons for this den sity have been considered. Firstly it is possible that the metal binding site is partially occupied by iron and carbonate and that these moieties are responsible for the density. This however, seems unlikely as the crystals do not appear even faintly
pink yet -25% occupancy would be required to account for the level of density
seen in the binding site (estimated by comparison of the level of electron density for a well defined water molecule in ApoLfN with one in FeLfN). Another alternative is that some other metal ion has bound together with a single water molecule, or even an anion such as azide may be present. It is hoped that further analysis of these crystals may allow this density to be accounted for.
Besides having extra density, the iron site also differs in the orientation of the iron binding ligands. If FeLfN is reorientated to match ApoLfN and the iron sites are
overlaid (Fig. 77) it is clear that differences exist. Although the extra, unaccounted
8 4 . 3
Fig. 76 ApoLfN metal binding site showing the unassigned electron density.
The four ligands involved in metal binding are shown together with the unassigned density. The
2Fobs - Fcalc
map has been contoured at l .Ocr in this instance.position of at least aspartic acid 60 and histidine 253 appear to be real. The sidechain density for these two ligands is distinct from the unaccounted density and the sidechains are quite well defined. The position of the two tyrosines is more likely to be erroneous as their density merges with that of the extra density and the sidechains are obviously distorted in an attempt to move into the latter. Despite these problems in accurately positioning the ligands there does appear to be a real difference in their position. Once the origin of the density in the metal binding site has been determined it may be possible to account for the differences in geometry seen here.