Backbone sequential assigment tutorial - NMR Spectroscopy ...

proton (H) of (mostly) every NH groups presents in the protein. One needs a 15 N labeled protein for
this experiment. Therefore, every peak in this spectrum, represents the fingerprint of each amino acid
making up the protein. Note that not only the backbone amide groups are seen, but also side chain NH
groups (from Arg, Asn, Gln, His and Trp, sometimes Lys) can also be present in this spectrum.
CI2 sequential backbone assignment tutorial
Thanks to several Utrecht NMR Research group lab members
[file:///home/student/STRUCT_BIOL/ASSIGNMENT/html/assignment.html]
Once NMR spectra have been recorded and processed, the following steps are to assign the peaks in
your spectra. In this tutorial, you will assign the sequential backbone atoms of a protein, CI2
(Chymotrypsin Inhibitor type 2), using 2 3D-NMR experiments, HN(CO)CACB and HNCACB, and a
2D-NMR experiment, 15 N- 1 H HSQC. SPARKY is a NMR spectra visualisation and analysis program
that has been developed to assist in NMR structure determination of proteins, DNA and RNA. We will
use this software for the assignment of the protein.
When a double labeled sample is available; both 15 N and 13 C labeled; it is then possible to extend the
dimension of recording. This is called 3D-NMR or triple resonance experiments. Spectra produced
after recording these 3D-NMR experiments are used to assign the peaks present in the HSQC
spectrum. Roughly speaking, two complementary 3D-NMR experiments are recorded, HNCACB (or
equivalent CBCANH) which will give you the chemical shift information for each amide (H N
and N H
)
peak in the HSQC spectrum of the residue (lets call it residue i) and the chemical shift information of
the alpha (CA) and beta (CB) carbons of the residue (residue i) and of the previous residue (residue
i-1).
Note: To run this practical make sure that SPARKY is installed on your system, otherwise
download it from the SPARKY website.
A few helpfull links for this practical:
PDB entry of 2CI2
Manual of Sparky
Structure Determination by NMR
Questions & assignments are indicated in bold and with a *. Please hand in the answers at the end of
the practical.
CI2 is a protein composed of 64 amino acids. The amino acid sequence is displayed below.
The second spectrum, HNcoCACB (or equivalent CBCAcoNH) will also give the chemical shift
information for each amide (H N
and N H
) peak in the HSQC spectrum of the residue (residue i) but
only the chemical shift information of the alpha (CA) and beta (CB) carbons of the previous residue
(residue i-1). Hence, you can use the information from this spectrum to assign which CA and CB are of
the residue and of the previous residue in the HNCACB (or CBCANH).
Sequence of CI2:
1-10
LEU LYS THR GLU TRP PRO GLU LEU VAL GLY
11-20
LYS SER VAL GLU GLU ALA LYS LYS VAL ILE
21-30
LEU GLN ASP LYS PRO GLU ALA GLN ILE ILE
31-40
VAL LEU PRO VAL GLY THR ILE VAL THR MET
41-50
GLU TYR ARG ILE ASP ARG VAL ARG LEU PHE
51-60
VAL ASP LYS LEU ASP ASN ILE ALA GLN VAL
61-64
PRO ARG VAL GLY
In the 15 N- 1 H HSQC spectrum, every peak connects the chemical shifts of the nitrogen (N) and the
The use of 13 C spectrum makes the protein assignment much easier for two reasons:
Addition of the 13 C dimension reduces the overlap of the peaks that often occurs in the 2D
spectra.
Since the chemical shift of the CA and CB are specific for each amino acid, it is thus possible,

just with these 2 carbon resonances, to identify each amino acid and use them for the sequential
backbone assignment.
The average chemical shifts for the carbons alpha and beta for each amino acid is shown in the next
picture. The values have been taken from the BioMagResBank. A nice tool that includes this picture is
available:
Go to the directory cd ~/STRUCT_BIOL/ASSIGNMENT/shift/ and type
/usr/bin/wish in your terminal, then source shift.tcl, followed by
hh_shift_table. Click on the button H/C to alter between proton and carbon chemical
shifts.
To open the project for this exercise go to File -> Project/Open project and
select assign.proj (located in the /home/course*/STRUCT_BIOL
/ASSIGNMENT/sparkydata/Projects/ directory). Three spectra should appear on
your screen; an HSQC of CI2 and the 3D spectra; HN(CO)CACB and HNCACB. The HSQC
spectrum has two scrollbars; so you can view the spectrum even when you are zoomed in.
Use the shortcuts zi and zo for zooming in and out, or use the zoom pointer mode. Both 3D spectra
have an extra scrollbar at the bottom, so you can go through the 3 rd dimension (of the cube). In the
HSQC spectrum, peaks correspond to all the amides of CI2. The 3D spectra are put in such a way that
we see the 1 H- 13 C plane of the cube. Pick a peak by typing F8 and clicking on a peak.
When you select this peak in the HSQC and center on it - type vc (view center) - you will see the CA
and CB carbons of this residue (and the one before) in the 3D spectra. All the spectra are connected, so
when you will move through one of them, the other ones will also move. This is made possible in
View -> More -> Synchronize. You will see that the 15 N, 13 C and 1 H axis are
connected.
Specific View options of each spectrum can be accessed by the shortcut vt or View -> View
options. The menu can also be accessed by right-clicking inside the spectrum. Contour levels can
be adjusted by the shortcut ct or View -> More -> Contours Levels. These
levels do not have to be changed anymore.
First you have to peak-pick the HSQC spectrum. The pointer mode should be selected as
find/add peaks. Now you can put peaks into the spectrum by hand. The program can also
do automatic peak picking by dragging a box over the spectrum. Sparky will automatically recognise
the peaks according to the current contour levels. When you put a peak by hand; the peak can be
centered by the shortcut pc (peak center) and integrated by pi (peak integrate). The last one can be
handy for overlapping peaks; Sparky will fit the lineshape with two or more peaks.
* How many (backbone & side-chain!) peaks do you expect to find in your 1 H- 15 N HSQC
spectrum. Compare this to the number of peaks found by SPARKY
Now the peaks have been picked; find the corresponding CA and CB values in the 3D spectra. Since
the 3D experiment data that we have are really of good signal/noise ratio and well dispersed, we can
do a restricted automatic peak picking. First select all the peaks in your HSQC and then open the
Restricted Peak Picking panel (kr, or Extension -> Restricted Peak Pick).
Select to find peaks in the HNCACB using peaks in the HSQC; the ranges are alright; then pick the
peaks.
* How many peaks do you expect to find in your HNCACB? Does the amount of found peaks
match that number; if not try to explain the difference. What about the HN(CO)CACB?
Analyzing the NMR spectra of CI2 with the software SPARKY
Open a new terminal and type sparky to start the assignment software.
You can make a so called 'strip plot' from the 3D experiments. This is a handy tool for the assignment.
First select all the peaks in your HSQC and then open the Strip Plot panel (sp or Extension
-> Strip Plot Peak Pick). Select the 3D spectra in Show -> Select
Strip Spectra. Mark the HNCACB and HN(CO)CACB. Leave the axis order as HCN, click
on OK and close the panel. Then make the strips by Show -> Add Selected Peak
Strip. The strips are put next to each other and you are able to see which carbons belong to the
residue itself and which belong to the previous one.
* Use the chemical shift statistics (shift.tcl) to find some specific residues like Threonine (T),

Serine (S), Alanine (A) or Glycine (G) and write down their 1 H N
, 15 N, 13 CA and 13 CB
frequencies. These residues can be used as starting point for your assignments.
By looking at the previous carbons of the residue; you can already guess what kind of residue it will be
according to the statistics; you can map this in the sequence of CI2. Select a peak in the strip plot and
go to one of your 3D spectra. Type vc (view center) and you can see this strip in your actual spectra.
phi and psi backbone angles, their averages can reliably be used as angular restraints for the protein whose
structure is being studied. These predictions will be indicated with Good.
The flow chart of the TALOS program is shown in the figure below
Now delete all strips and select one of the starting peaks in your HSQC. Make the strips of only this
peak in the Strip Plot Panel. Now select the peaks from the previous residue (visible in both
experiments) in the HNCACB and try to find matching peaks ( sm or Find -> Add
Strips Matching Peaks). In this way it should be possible to go through the sequence
and assign all the HSQC peaks.
* Try to assign a stretch of about 5 residues in the protein and write down their 1 H N
, 15 N, 13 CA
and 13 CB frequencies. Remember that one can see the prolines only from the next residue and
that the first residue can most of the time not be found.
Use of backbone chemical shift information for predicting dihedral angles
The following figure shows the graphical display of the TALOS output.
Chemical shifts of backbone atoms in proteins are very sensitive to local conformation, and homologous
amino acid sequences show quite similar patterns of secondary chemical shifts. The inverse of this relation is
used to search a database for triplets of adjacent residues with secondary chemical shifts and sequence
similarity which provide the best match to the query triplet of interest. The database contains 13 CA, 13 CB,
13 C',
1 Ha and
15 N chemical shifts for 20 proteins for which a high resolution X-ray structure is available.
The computer program TALOS was developed to search this database for strings of residues with chemical
shift and residue type homology. The relative importance of the weighting factors attached to the secondary
chemical shifts of the five types of resonances relative to that of sequence similarity was optimized
empirically. TALOS yields the 10 triplets which have the closest similarity in secondary chemical shift and
amino acid sequence to those of the query sequence. If the central residues in these 10 triplets exhibit similar

Go to the ~/STRUCT_BIOL/ASSIGNMENT/talos_ci2 directory. Look at the file called
ci2d_shift.tab. This file contains the chemical shifts of the CA, CB, C, N and H N
nuclei of each
amino acid. Type in the terminal talos -help for a short description of TALOS. Then type
talos.tcl -in ci2d_shift.tab. TALOS will start the predictions and will save them in
the directory /pred. For a summary of the results, type vina.tcl -in
ci2d_shift.tab -ref ci2.pdb -auto. That will create a summary file called
pred.tab. You can then see the results of the prediction by typing rama.tcl -in
ci2d_shift.tab -ref ci2.pdb.
* How many residues have a helical conformation? And how many are located in a beta-strand?
Open the crystal structure of CI2 in a graphical program like molmol: molmol ci2.pdb. Select all
atoms Prop > Select All > Atom. Since in a crystal structure there are usually no
H-atoms, we will have to calculate them: Calc > Atom > H*. In order to calculate the secondary
structure elements and to show a ribbon presentation of the structure, do the following: Attr >
Style > Bond > invisible and click on ribbon in the panel on the right. You can label
the structure with the residue numbers by clicking on label num.
To calculate the backbone angles in the crystal structure, write the following lines in the command-line:
SelectAngle ':*@PHI,PSI' followed by CalcAngle.
* Compare the predicted secondary structure elements & predicted dihedral angles (labeled with
Good and in green) extracted from the NMR data with TALOS to the ones of the crystal structure.