Automated resonance assignment with FLYA (EMBO 2013)

We want to determine the resonance assignments and the structure of a protein using the program CYANA.

Experimental input data

The protein sequence is stored in three-letter code in the file 'demo.seq'.

The following spectra have been measured:

• [¹H,¹³C]-HSQC, called 'C13H1' in FLYA
• [¹H,¹⁵N]-HSQC, called 'N15H1' in FLYA
• HNCA
• HN(CO)CA, called 'HN_CO_CA' in FLYA
• HNCO
• HN(CA)CO, called 'HN_CA_CO' in FLYA
• CBCANH
• CBCACONH
• HBHACONH
• HCCH-TOCSY, called 'HCCH24' in FLYA
• HCCH-COSY, called 'HCCH7' in FLYA
• C(CO)NH, called 'C_CO_NH' in FLYA
• HC(CO)NH, called 'HC_CO_NH' in FLYA

Peak lists in XEASY format that have been prepared by automatic peak picking with the program NMRView are stored in files XXX.peaks, where XXX denotes the FLYA spectrum type.

Each peak list starts with a header that defines the experiment type and the order of dimensions. For instance, for HNCA.peaks:

# Number of dimensions 3
#FORMAT xeasy3D
#INAME 1 HN
#INAME 2 C
#INAME 3 N
#SPECTRUM HNCA  HN C N
      5   6.475  58.033  98.548 1 U   2.769E+02  0.000E+00 e 0     0     0     0
      6   6.476  62.123  98.126 1 U   2.571E+01  0.000E+00 e 0     0     0     0
      7   6.475  54.017  98.159 1 U   2.547E+01  0.000E+00 e 0     0     0     0

The first line specifies the number of dimensions (3 in this case). The '#SPECTRUM' lines gives the experiment type (HNCA, which refers to the corresponding experiment definition in the CYANA library), followed by an identifier for each dimension of the peak list (HN C N) that specifies which chemical shift is stored in the corresponding dimension of the peak list. These labels must match those in the corresponding experiment definition in the general CYANA library (see below). After the '#SPECTRUM' line follows one line for every peak. For example, the first peak in the 'HNCA.peaks' list has

• Peak number 5
• HN chemical shift 6.475
• C (CA) chemical shift 58.033
• N chemical shift 98.548

The other data are irrelevant for automated chemical shift assignment with FLYA. In particular, the peak volume or intensity (2.769E+02) is not used by the algorithm.

FLYA initialization script

The CYANA commands to run the automated assignment calculation are stored in two CYANA scripts or "macros".

One has the fixed name 'init.cya' and is executed automatically each time CYANA is started. It can also be called any one wants to reinitialize the program. It contains normally at least two commands that read the CYANA library and the protein sequence:

cyanalib
read demo.seq

The command 'cyanalib' reads the standard CYANA library. When you start CYANA, the program reads the library and displays the full path name of the library file. You can open the standard library file to inspect, for example, the NMR experiment definitions that define which expected peaks are generated by FLYA. For instance, the definition for the HNCA spectrum (search for 'HNCA' is

SPECTRUM HNCA  HN N C
 0.980  HN:H_AMI  N:N_AM*  C_A*  C_BYL  C:C_ALI
 0.800  HN:H_AMI  N:N_AMI  C_BYL  C_ALI  N_AMI  C:C_ALI

The first line corresponds to the '#SPECTRUM' line in the peak list. It specifies the experiment name and a label for the atoms that are detected in each dimension of the spectrum. The number of labels defines the dimensionality of the experiment (3 in case of HNCA).

Each line below defines a (formal) magnetization transfer pathway that gives rise to an expected peak. in the case of HNCA there are two lines, corresponding to the intraresidual and sequential peak. For instance, the definition for the intraresidual peak starts with the probability to observe the peak (0.980), followed by a series of atom types, e.g. H_AMI for amide proton etc. An expected peak is generated for each molecular fragment in which these atom types occur connected by single covalent bonds. The atoms whose chemical shifts appear in the dimensions.