LACS

Introduction

Linear analysis of chemical shift (LACS) provides an independent means for absolute referencing of protein chemical shifts.  LACS is routinely run on all chemical shift sets submitted to the BioMagResBank (BMRB). 

In the contest of protein structure determination chemical shifts (CS) play a key role as reporters of secondary structure.  Backbone chemical shift derived dihedral angles can be obtained through programs such as TALOS+ and are used as constraints in the structure determination process. The backbone shift set (CA, CB, CO, HN, N, HA) is also used in the molecular fragment selection (MFR) for CS-Rosetta structure prediction protocol.  Here fragments are selected from the protein databank (PDB) that have the same predicted CS as the query sequence.  Successful use of chemical shift for secondary structure inference requires the correct referencing.  The LACS server gives independent CS offsets for CA, CB, CO and HA and allows for effective correction of chemical shift sets derived from [U-¹³C,¹⁵N] and from [U-²H,¹³C,¹⁵N].  The latter labeling scheme may show distinct degrees of offset for CA and CB due to the different number of bound deuterons which affect the value of the carbon to different degree.   

Running LACS

LACS can be run on the native server using an edited version of the chemical shift file in bmrb 2.1 format, the user's email and project name.  The server returns the results very rapidly, the chemical shift file format is stringent and the output is of straight forward interpretation. 

The items returned are:

i) a text file with the nucleus specific offsets from the linear fit of the secondary CS  (e. g. deltaCA vs. deltaCA-deltaCB), obtained by subtracting the random coil value from the atom of interest.

ii) plots of the secondary CS outliers that may reveal incorrectly assigned residues.

A running bmrb file and text output example are provided for user's convenience, adhering to the format should provide smooth operation of the server.

Example BMRB input for LACS

 _Mol_residue_sequence
;
MLIYKDIFTDDELSSDSFPM
KLVDDLVYEFKGKHVVRKEG
EIVLAGSNPSAEEGAEDDGS
DEHVERGIDIVLNHKLVEMN
CYEDASMFKAYIKKFMKNVI
DHMEKNNRDKADVDAFKKKI
QGWVVSLLAKDRFKNLAFFI
GERAAEGAENGQVAIIEYRD
VDGTEVPTLMLVKEAIIEEK
CLE
;


  loop_
    _Residue_seq_code
    _Residue_label
    _Chem_shift_ambiguity_code
4    1     MET     C     C    170.742     .     1
5    1     MET     CA     C    54.828     .     1
6    1     MET     CB     C    32.579     .     1
7    2     LEU     H     H    8.934     .     1
8    2     LEU     HD1     H    0.688     .     1

Example LACS ouput

The CA correction of -0.19 ppm is found (last line).

data_LACS

         #################################
         #    LACS Output Information    #
         #################################

############################################################
#              LACS Designator Definition                  #
#                                                          #
#   Index Value                    Definition              #
#                                                          #
#        0                          Outliers               #
#        1                        Normal points            #
#                                                          # 
############################################################            


save_LACS_CACB_CA_output
 _LACS_plot.Sf_category                   LACS_output
 _LACS_plot.Input_file_name               "data.txt"

 _LACS_plot.X_coord_name                  CA-CB
 _LACS_plot.Y_coord_name                  CA
 _LACS_plot.Line_1_terminator_val_x_1     -6.35
 _LACS_plot.Line_1_terminator_val_y_1     -2.37
 _LACS_plot.Line_1_terminator_val_x_2      1.99
 _LACS_plot.Line_1_terminator_val_y_2      0.93
 _LACS_plot.Line_2_terminator_val_x_1     -2.00
 _LACS_plot.Line_2_terminator_val_y_1     -1.18
 _LACS_plot.Line_2_terminator_val_x_2      5.76
 _LACS_plot.Line_2_terminator_val_y_2      4.33
 _LACS_plot.Y_axis_chem_shift_offset      -0.19

Practical Experience with Running LACS in the NESG

In the NESG the IUPAC referencing is obtained using internal 50 uM DSS and setting the DSS methyl peak to 0.00 ppm at the temperature of interest.  The spectrometer temperature is calibrated using a neat methanol sample in the 10 - 40 °C range.  Under these conditions, backbone CS of double labeled protein samples derived from triple resonance experiments alone typically show small deviations 0.1 - 0.3 ppm in ¹³C and 0.01 - 0.03 ppm in ¹H.  The final CS set derived from double labeled samples filtered through higher resolution [¹H,¹³C]-HSQC tends to give offsets at the lower extreme of this range.  On a smaller statistical sampling of triply labeled proteins we found higher offset values (0.4 - 0.8 ppm) in CA and CB.  Offsett correction prior to TALOS or MFR runs are therefore highly recommended especially for the treatment of triply labeled samples.