Index:
If all spin systems have been assigned uniquely to one type of amino acid, and all NOE's have been assigned, making sequential assignments is a very trivial thing. The sequential assignment buildup tool does contain some facilities to make it possible to combine spin systems sequentially, if these two conditions are not fully fulfilled. These facilities are summarized below:
1. Search profiles. It is possible to set up search profiles to search for not only assigned NOE's, but also for possible NOE assignments.
2. As the sequence of the protein is known to the system, it is possible to select only those spin systems that match the amino acid sequence. If the spin system has not been assigned uniquely to one amino acid, but just to a group of possible amino acids, all the selected names are used to match the sequence.
The term: NOE refers to any inter spin system effect, making it possible to build sequential assignments from fancy 3D or 4D experiments linking atoms across the peptide bond.
Note, the Pronto program links one or more spin systems together in one or more chains of sequential assignment. The chains can then later be merged, or they can be split up, if some of the assignments turn out to be wrong. The actual information about which atoms in which spin systems that are close to atoms in spin systems close in the sequence must be entered using the cross peak assignment tool.
The chains can be attached to one or more positions in the amino acid sequence. In many cases, when the length of a chain has reached a few residues, only one attachment site in the sequence is possible.
The sequential assignment buildup tool helps you keeping track of where in the sequence a chain can be attached.
Two examples of how to use the sequential assignment tool will be given below. The first case describes making sequential assignments using 2D spectra, the second case uses 3D spectra. In both cases, the database contains spin systems that have only been assigned to classes of amino acids. Only the aromatic residues, valines, alanines, isoleucines, and prolines have been uniquely assigned. Only a few of the NOE cross peaks between atoms in different spin systems have been assigned. The spin systems in these databases have been constructed so that the spin system number match the sequence number.
The sequential assignment starts by using one spin system as the seed for the search for sequential assignments. Open the sequential assignment catalog, create a new entry, zoom on that entry, and insert that spin system into that entry.
Figure 1 shows an example of how to start the sequential assignment using one spin system as the start of a sequential chain. Open the sequential assignment buildup tool, insert a 2D NOESY spectrum as the primary spectrum, and COSY and TOCSY spectra as secondary spectra. The sequential assignment containing the first spin system should be inserted as the sequential assignment to work with.
Figure 2 shows the layout of the sequential assignment buildup tool after inserting the sequential assignment entry containing one spin system, and inserting some 2D spectra.
The purpose of this example is to extend this sequential assignment towards the N-terminal of the protein, i.e. we start by looking for a spin system preceding spin system No. 39. To make the search more simple, we just look for sequential HN-HN and HA-HN effects.
The button at the top of the window, to the right of the button containing the sequential assignment entry, selects what we are going to search for. If this is set to: Pos -1: N terminus, the search operation will look for a spin system that can be attached to the N-terminal of the sequential chain. The spin system to look for is always numbered i. In this case, this means that the spin system already in the chain is numbered i+1. Already at this point, we can see how many isoleucines that are present in the amino acid sequence. These are listed at the bottom of the window. This is because spin system 39 has been assigned only to an isoleucine.
The search profile we are going to use is created above the middle of the window. Press the New button twice to insert two empty search entries. The first entry is used to look for sequential HN-HN effects, and the second entry looks for spin systems, whose Ha has an effect to the HN of spin system 39. The All CP button makes sure that we look for both assigned cross peaks, for unassigned cross peaks, and for peaks that have been assigned to something else. The Asg or Free SS button makes the search operation look for spin systems that either are unused or already a part of another sequential assignment chain. The Only matching Spin Systems button filters away those spin systems that do not match the amino acid sequence.
The search profiles can be saved in the database using the browser at the top of the window.
Each of the entries in the search profile contains one entry for each axis of the main spectrum. As the main spectrum in this case is a 2D spectrum, only the first two axis-entries are used in each search line. In each of the four axis-entries, only the Atom Name, Atom Index, chemical shift tolerance (Dev.), and the distance from the new spin system in the sequence, Seq. Dist., are used. The Atom Name entry should match the axis type, in this case all axes are proton axes. In the first entry in the profile list, the first axis is set to match the HN proton of the spin system we are looking for (Seq. Dist. is set to zero), with a tolerance of 0.03 ppm., and the second axis must match the HN proton of the spin system of the N-terminal of the chain, using the same tolerance. The second entry in the table makes the tool look for spin systems whose Ha have a link to the HN of spin system 39. It is important to understand the exact meaning of the entries used for the search. Using a wrong search profile will in most cases lead to a negative result (that is, no spin systems are found) when the Start Search button is pressed.
The result of starting the search is shown in Figure 3. The possible spin systems are listed below the Start Search button. The field to the left of the table contains some information: The number after Expr: is the number in the search profile that is matched with this spin system, and the letter A means that the cross peak listed two fields to the right already has been assigned to match the two protons, and the letter P means that this cross peak is a possible assignment between the two atoms in the two spin systems. It can be seen from the table that spin system 38 is the only spin system for which both entries in the search profile are fulfilled. Selecting one of the entries in the result table that contains a reference to spin system 38 will make the sequence list at the bottom only show the possibilities in the sequence where the combination of spin system 38 (assigned as a valine) followed by spin system 39 (assigned as an isoleucine) match.
This is shown in Figure 4. It can be seen, that the combination: Val-Ile occurs only once in the sequence of this protein. This example also shows that it is important to start with a spin system that has been assigned uniquely to one amino acid type. This makes it much faster to build a sequential assignment chain that can be attached to one position in the sequence only. Press the Make sequential button to connect spin system 38 to the sequential assignment, select the sequence entry at the bottom, and connect the sequential assignment to that part of the sequence by pressing the Connect to Sequence button. The Make sequential button must be pressed for each spin system added to the sequential assignment, whereas the Connect to Sequence should be pressed only once for each sequential assignment chain, unless of course you need to change the sequence assignment at a later stage.
Select: Pos -1: N terminus and press Start Search to look for the next spin system in the chain.
Figure 5 shows the result of starting the search for a third member of this sequential chain. The result of this search is one spin system only. This spin system has not been assigned to one amino acid type only, but if the spin system is assumed to be a lysine, then it will fit in as number 37 in the amino acid sequence.
Of course, all these search operations should be combined with the use of the cross peak assignment tool and the contouring facilities to check out the possible assignments suggested by the search operations. You can also plot two spin systems simultaneously and look for possible sequential effects between them.
Making the same sequential assignments as shown in the preceding section using 3D, 15N-HMQC-NOESY spectra is a much simpler operation. Especially the HN-HN effects are very reliable.
Figure 6 shows the setup for a search operation involving only HN-HN effects between neighbouring residues. We include a search on both sides of the "diagonal". A tolerance of 0.5 ppm has been used in the 15N dimension. It is important to remember, that the Seq. Dist. value used in the 15N dimension must be the same as the one used in the dimension containing protons bound to an 15N atom. The first entry in the profile looks for effects from the new spin system to the last spin system of the sequential chain and the second entry looks for effects from the last spin system in the chain to the new spin system.
The result of performing the search is shown in Figure 7. Four entries are listed in the result browser. You can see that the table only contains one entry for the effect between Val-38 and Ile-39, but two effects between Ile-39 and Leu-40. There will always be a doubt about a sequential HN-HN effect as to whether it goes to the next or previous residue. The sequential relationship between Val-38 and Ile-39 could be further illustrated by including the condition: N/HN in the last residue to the Ha of the new spin system.
The attachment of the spin system to the chain, and the attachment of the chain to the sequence is done as in the 2D case.
The third residue in this chain is searched simply by selecting Pos -1 after having attached residue 38 to the chain and pressing Start Search. This is shown in Figure 8. The most likely candidate from this search is spin system 37. The search finds both the effect from 38 to 37 and from 37 to 38.
The alignment of the peaks between these two residues can be verified by clicking at the cross peak field containing the text: CP 2742 A and pressing the Cross Peak entry of the Set menu of the contouring setup window. The other cross peak: CP 2727 is then selected, and the Cross Peak entry of the Add menu is selected. Pressing Make Contouring gives a plot as shown in Figure 9.
This chapter has described some simple examples of how to use the sequential assignment tool. A lot of other possibilities are possible, especially if you have spectra linking residues via couplings over the peptide bond.
It is also possible to use this tool to look for effects between residues further away than the neighbouring residue. This is especially useful for proteins with a lot of helix structures.
Last updated: 14-JAN-1997