Abstract
Determining the structure of a protein by any method requires various contributions from experimental and computational sides. In a recent study, high-density cross-linking/mass spectrometry (HD-CLMS) data in combination with ab initio structure prediction determined the structure of human serum albumin (HSA) domains, with an RMSD to X-ray structure of up to 2.5 Å, or 3.4 Å in the context of blood serum. This paper reports the blind test on the readiness of this technology through the help of Critical Assessment of protein Structure Prediction (CASP). We identified between 201-381 unique residue pairs at an estimated 5% FDR (at link level albeit with missing site assignment precision evaluation), for four target proteins. HD-CLMS proved reliable once crystal structures were released. However, improvements in structure prediction using cross-link data were slight. We identified two reasons for this. Spread of cross-links along the protein sequence and the tightness of the spatial constraints must be improved. However, for the selected targets even ideal contact data derived from crystal structures did not allow modellers to arrive at the observed structure. Consequently, the progress of HD-CLMS in conjunction with computational modeling methods as a structure determination method, depends on advances on both arms of this hybrid approach.
Highlights
Cross-linking/mass spectrometry (CLMS) is a well-established, low-resolution technique for revealing protein interactions in protein complexes and studying protein conformational changes[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16]
The resulting increased data density opens up the possibility of determining tertiary protein structure, which we demonstrated by recapitulating the domain structures of human serum albumin (HSA) in purified form (RMSD to crystal structure of 2.5 Å) and in its native environment, blood serum (RMSD to crystal structure 3.4 Å)[19]
1) we analyzed whether the blindly generated cross-links fit the crystal structures; 2) we analyzed if Critical Assessment of protein Structure Prediction (CASP) modeling groups were able to utilize the CLMS data in their predictions, which is discussed in detail elsewhere[21]; and 3) we identified technical shortcomings of high-density cross-linking and the blind study approach taken here
Summary
Cross-linking/mass spectrometry (CLMS) is a well-established, low-resolution technique for revealing protein interactions in protein complexes and studying protein conformational changes[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16]. A possible culprit for the low data density is the NHS-ester based cross-linking chemistry used in most studies. These cross-linker reagents predominantly react with lysines, which results in linkage maps that are not sufficiently dense to define the detailed structure of proteins
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