A new approach to evaluate protein structure (3D)-sequence (1D) compatibility has been developed. This approach uses four functions: side-chain packing, solvation, hydrogen-bonding, and local structure functions. The side-chain packing function takes into account not only inter-residue distance but also inter-residue contact and angle. Parameters defining the functions were statistically derived from 101 proteins with known 3D structures. The functions are combined to give a score quantifying the 3D-1D compatibility. The ability to identify the native structure of a protein among a large number of incorrect structures was tested. For 69 out of the 70 proteins examined, the native structures were successfully identified. Particularly, the side-chain packing function showed clear improvement over our previous function. The only unsuccessful case was observed for cytochrome c3 containing four haems; such prosthetic groups were ignored in the calculation. In addition to the above functions, two procedures for removing membrane-spanning regions and estimating electrostatic interactions were also adopted. A sequence for which membrane-spanning regions were predicted was considered incompatible with a structure irrespective of the compatibility score. A method to calculate the electrostatic interaction energy was developed and used for the detection of electrostatically undesirable interactions between residues of a sequence threaded onto a structure. These procedures greatly help to remove false positives in the 3D-1D compatibility search.