1. Introduction to ABACUS

 

ABACUS (Applied BACUS) is a novel approach for protein structure determination that has been applied successfully for more than 20 NESG targets. ABACUS is characterized by use of BACUS, a procedure for automated probabilistic interpretation of NOESY spectra in terms of unassigned proton chemical shifts based on the known information on "connectivity" between proton resonances. BACUS is used in both the resonance assignment and structure calculation steps. The ABACUS is distinguished from conventional approaches to NMR structure determination mostly by its resonance assignment strategy (see Fig.1.1A).

Figure 1.1A.  Flowchart of resonance assignmnent by ABACUS. 

 

Figure 1.1B. Schematic description of two types of molecular fragments: traditional spin-system (AA-fragment) include all the atoms belonging to the same residue; PB-fragment includes all the atoms from one residue except the backbone amide group, plus the amide group from the next residue in the protein

 

Some features /advantages of the ABACUS protocol:

•   It does not rely on sequential connectivities from less sensitive experiments such as HNCACB indispensable for most traditional sequential assignment procedures;
• Inter-residue sequential connectivities are established mainly from NOE data, which saves time at a later stage in “troubleshooting” NOE and resonance assignments.;
• Probabilistic nature of the ABACUS procedure provides measure of reliability of assignments, and therefore one can obtain a partial, yet highly reliable assignment (even when the NMR data are sub-optimal) with the knowledge of where to focus manual intervention;
• It can make use of partial spin-systems;
• It can efficiently identify manual errors in the input peak lists;

 

1.1. NMR spectra required for ABACUS.

The spectra typically needed for ABACUS approach are most conveniently separated into 3 groups: NH-rooted, the CH-rooted and the aromatic (also CH-rooted).  Table 1 shows the optimal set of NMR spectra. This, of course, is neither an exclusive or exhaustive list. For example, a simultaneous CN-NOESY could be recorded instead of three different ones listed in the table. In case there are very few aromatic residues in a protein, to collect only one aromatic spectrum, namely aromatic NOESY, could be enough for assignment of aromatic resonances.

 

Table 1. ABACUS optimal set of experiments

 

NH-rooted	CH-rooted	Aromatic
15N-HSQC	13C-CT-HSQC	13C-HSQC-aro
HNCO	13C-HSQC	H(C)CH-TOCSY-aro
HNCA	H(C)CH-TOCSY	(H)CCH-TOCSY-aro
CBCA(CO)NH	(H)CCH-TOCSY	13C-NOESY-HSQC-aro
HBHA(CO)NH	13C-NOESY-HSQC
15N-NOESY-HSQC
CCCONH-TOCSY (optional)
H(CCCO)NH-TOCSY (optional)

 

 

1.2. Spin-system identification strategy.

The resonance assignment procedure starts from grouping resonances in spin systems (PB-, or peptide bond, fragments) comprising correlated resonances from the side chain of residuei and the NH resonances of residue i+1 (see Figure1.1B). The uncompleted HN-rooted PB spin-systems, which include resonances of                                                    atoms only, are called bPB-fragments in this manual.

 

Spin-system identification in ABACUS approach consists of 3 main steps.

 

     1. On the first step, bPB-fragments are collected from high sensitivity NMR correlation experiments (such as HNCO, CBCA(CO)NH, and HBHA(CO)NH) that transfer magnetization via the intervening peptide bond (see Figure 4.1A).

 

     2. On the second step, completion of bPB-fragments with side-chain aliphatic resonances as well as identification of additional spin-systems (lacking HN resonances) is performed using HCCH-TOCSY and 13C-NOESY spectra (see Figure 4.1B)  

 

      3. Finally, spin-system validation and correction is performed. This step allows one to find mistakes made during spectra peak-picking and to correct the mistakes by going back to the spectra.

 

For each spin-system, 20 scores S(T) were calculated during the validation (see Figure 1.2). Here T corresponds to amino acid type, and T=A,R,D,…, and V, respectively. The score evaluate goodness-of-fit of the spin-system resonances to those observed in BMRB data base. If the best score , where , is too low, it means that either the spin-system has very unusual chemical shifts or the spin-system does not make sense and need to be corrected.

 

Figure 1.2. Spin-system scoring.

                     

Were:

T is one of 20 amino acid types, i.e. T=A,R,D,….,V;

 stands for chemical shifts that make up spin-system; (here i=1,. .,N_ω;  )

 is average chemical shift observed in BMRB database for an atom X in a template residue of amino acid type T; ( for example, if T = A, then X could be  and , respectively)

 is standard deviation for a chemical shifts observed in BMRB database for an atom X in a template residue of type T;

M^T is one of possible mapping of the spin-system resonances  on the atoms of a template residue of type T;

 is resonance mapped to atom X in the mapping M^T;

1.3. Fragment assignment by FMC procedure.

Sequence-specific assignment of PB-fragments is achieved using a Fragment Monte Carlo (FMC) stochastic search procedure. The scoring function used in the FMC procedure is based on both fragment amino acid typing (matching the spin system to amino acid types) and fragment contact map (reflecting which residue is next to which) derived from HNCA data and the analysis of NOEs interpreted by BACUS (see Figure 1.3)

 

 FMC procedure performs probabilistic assignment of PB-fragments. The assignment probabilities are calculated by Simulated Annealing (SA) or Replica Exchange Method (REM) Monte Carlo (MC) simulations.  Here,  is a probability of fragment k to occupy position s;' k = 1,….,N_f. ;