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Fast and accurate resonance assignment of small-to-large proteins by combining automated and manual approaches.

Niklasson M, Ahlner A, Andresen C, Marsh JA, Lundström P - PLoS Comput. Biol. (2015)

Bottom Line: Unfortunately, the manual assignment of residues is tedious and time-consuming, and can represent a significant bottleneck for further characterization.Furthermore, while automated approaches have been developed, they are often limited in their accuracy, particularly for larger proteins.Here, we address this by introducing the software COMPASS, which, by combining automated resonance assignment with manual intervention, is able to achieve accuracy approaching that from manual assignments at greatly accelerated speeds.

View Article: PubMed Central - PubMed

Affiliation: Division of Biomolecular Technology, Department of Physics, Chemistry and Biology, Linköping University, Linköping, Sweden.

ABSTRACT
The process of resonance assignment is fundamental to most NMR studies of protein structure and dynamics. Unfortunately, the manual assignment of residues is tedious and time-consuming, and can represent a significant bottleneck for further characterization. Furthermore, while automated approaches have been developed, they are often limited in their accuracy, particularly for larger proteins. Here, we address this by introducing the software COMPASS, which, by combining automated resonance assignment with manual intervention, is able to achieve accuracy approaching that from manual assignments at greatly accelerated speeds. Moreover, by including the option to compensate for isotope shift effects in deuterated proteins, COMPASS is far more accurate for larger proteins than existing automated methods. COMPASS is an open-source project licensed under GNU General Public License and is available for download from http://www.liu.se/forskning/foass/tidigare-foass/patrik-lundstrom/software?l=en. Source code and binaries for Linux, Mac OS X and Microsoft Windows are available.

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Assignment completeness for the proteins SAP97 PDZ-2, TPMT*1 and EphB2 JMS-KD.Solid black areas represent assigned regions. The secondary structure as determined from the crystal structures [30], [31], [34] with α-helices and β-strands represented as rectangles and arrows, respectively, are shown above the diagrams. The numbers refer to the residue numbers of the N-terminal and C-terminal residues.
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pcbi-1004022-g008: Assignment completeness for the proteins SAP97 PDZ-2, TPMT*1 and EphB2 JMS-KD.Solid black areas represent assigned regions. The secondary structure as determined from the crystal structures [30], [31], [34] with α-helices and β-strands represented as rectangles and arrows, respectively, are shown above the diagrams. The numbers refer to the residue numbers of the N-terminal and C-terminal residues.

Mentions: The performance of COMPASS was evaluated by using it to assign the backbone of six proteins with different characteristics. The proteins were Abp1p SH3 domain [24], E140Q Tr2C [25], CDPK3 CLD(B) [26], SAP97 PDZ-2 [27], TPMT*1 [28] and EphB2 JMS-KD [29] that differ in size as well as secondary structure composition. More details about the proteins, sample conditions and NMR experiments can be found in Supporting Information. The completeness and accuracy of the assignments were primarily gauged by comparison with careful manual analysis. By completeness we mean two different things: 1) the fraction of peaks in the submitted peak lists that could be assigned and 2) the fraction of assigned residues of the protein. Completeness according to both these definitions is presented in Table 1, where Peaks refers to the percentage of assigned peaks in the supplied peak lists and NH refers to the percentage of assigned amide groups in the proteins. For four small proteins of 59–120 residues, the completeness was 91%–100% regarding fraction of assigned peaks and for all proteins essentially all visible backbone amide groups could be assigned. The low number of 82.6% assigned backbone amide groups for SAP97 PDZ-2 is due to the presence of N-terminal and C-terminal histidine tags included in the calculation. As shown in Fig. 8, all backbone amides except ones from these unstructured tails could be assigned. The assignments were almost as or as complete as ones obtained by manual analysis and we did not detect any assignment inconsistencies between the two approaches. We also tested COMPASS for more challenging systems involving larger proteins and incomplete data sets. The protein TPMT*1 comprises 232 residues and yields spectra where a large fraction of the peaks are broadened beyond detection. The percentage of peaks that could be assigned was 80% and the percentage of assigned backbone amide groups was 63%. Once again, this may sound low but from Fig. 8 it is obvious that unassigned residues cluster to specific regions. We analyzed the structural context of these regions and with few exceptions they mapped to the active site and cofactor binding regions of the protein [30], suggesting that these are highly flexible and broadened beyond detection due to rapid amide proton exchange with solvent. This is not surprising considering the relatively high pH (7.3) and the fact that the crystal structure suggests these regions are largely unstructured. A time-consuming manual analysis only improved the percentage of assigned backbone amides to 68%, again showing that COMPASS almost yields as complete assignments as manual methods. We also assigned the 312-residue EphB2 JMS-KD to check performance for larger, deuterated proteins. With the aid of COMPASS we were able to assign 94.6% of the peaks and 79.0% of the backbone amide groups, where unassigned residues largely were confined to the activation segment that is invisible in the crystal structure of the protein [31]. As for TPMT*1, manual analysis only improved the results marginally. We found no bias in success rate towards α-helical or β-strand structural elements.


Fast and accurate resonance assignment of small-to-large proteins by combining automated and manual approaches.

Niklasson M, Ahlner A, Andresen C, Marsh JA, Lundström P - PLoS Comput. Biol. (2015)

Assignment completeness for the proteins SAP97 PDZ-2, TPMT*1 and EphB2 JMS-KD.Solid black areas represent assigned regions. The secondary structure as determined from the crystal structures [30], [31], [34] with α-helices and β-strands represented as rectangles and arrows, respectively, are shown above the diagrams. The numbers refer to the residue numbers of the N-terminal and C-terminal residues.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4288728&req=5

pcbi-1004022-g008: Assignment completeness for the proteins SAP97 PDZ-2, TPMT*1 and EphB2 JMS-KD.Solid black areas represent assigned regions. The secondary structure as determined from the crystal structures [30], [31], [34] with α-helices and β-strands represented as rectangles and arrows, respectively, are shown above the diagrams. The numbers refer to the residue numbers of the N-terminal and C-terminal residues.
Mentions: The performance of COMPASS was evaluated by using it to assign the backbone of six proteins with different characteristics. The proteins were Abp1p SH3 domain [24], E140Q Tr2C [25], CDPK3 CLD(B) [26], SAP97 PDZ-2 [27], TPMT*1 [28] and EphB2 JMS-KD [29] that differ in size as well as secondary structure composition. More details about the proteins, sample conditions and NMR experiments can be found in Supporting Information. The completeness and accuracy of the assignments were primarily gauged by comparison with careful manual analysis. By completeness we mean two different things: 1) the fraction of peaks in the submitted peak lists that could be assigned and 2) the fraction of assigned residues of the protein. Completeness according to both these definitions is presented in Table 1, where Peaks refers to the percentage of assigned peaks in the supplied peak lists and NH refers to the percentage of assigned amide groups in the proteins. For four small proteins of 59–120 residues, the completeness was 91%–100% regarding fraction of assigned peaks and for all proteins essentially all visible backbone amide groups could be assigned. The low number of 82.6% assigned backbone amide groups for SAP97 PDZ-2 is due to the presence of N-terminal and C-terminal histidine tags included in the calculation. As shown in Fig. 8, all backbone amides except ones from these unstructured tails could be assigned. The assignments were almost as or as complete as ones obtained by manual analysis and we did not detect any assignment inconsistencies between the two approaches. We also tested COMPASS for more challenging systems involving larger proteins and incomplete data sets. The protein TPMT*1 comprises 232 residues and yields spectra where a large fraction of the peaks are broadened beyond detection. The percentage of peaks that could be assigned was 80% and the percentage of assigned backbone amide groups was 63%. Once again, this may sound low but from Fig. 8 it is obvious that unassigned residues cluster to specific regions. We analyzed the structural context of these regions and with few exceptions they mapped to the active site and cofactor binding regions of the protein [30], suggesting that these are highly flexible and broadened beyond detection due to rapid amide proton exchange with solvent. This is not surprising considering the relatively high pH (7.3) and the fact that the crystal structure suggests these regions are largely unstructured. A time-consuming manual analysis only improved the percentage of assigned backbone amides to 68%, again showing that COMPASS almost yields as complete assignments as manual methods. We also assigned the 312-residue EphB2 JMS-KD to check performance for larger, deuterated proteins. With the aid of COMPASS we were able to assign 94.6% of the peaks and 79.0% of the backbone amide groups, where unassigned residues largely were confined to the activation segment that is invisible in the crystal structure of the protein [31]. As for TPMT*1, manual analysis only improved the results marginally. We found no bias in success rate towards α-helical or β-strand structural elements.

Bottom Line: Unfortunately, the manual assignment of residues is tedious and time-consuming, and can represent a significant bottleneck for further characterization.Furthermore, while automated approaches have been developed, they are often limited in their accuracy, particularly for larger proteins.Here, we address this by introducing the software COMPASS, which, by combining automated resonance assignment with manual intervention, is able to achieve accuracy approaching that from manual assignments at greatly accelerated speeds.

View Article: PubMed Central - PubMed

Affiliation: Division of Biomolecular Technology, Department of Physics, Chemistry and Biology, Linköping University, Linköping, Sweden.

ABSTRACT
The process of resonance assignment is fundamental to most NMR studies of protein structure and dynamics. Unfortunately, the manual assignment of residues is tedious and time-consuming, and can represent a significant bottleneck for further characterization. Furthermore, while automated approaches have been developed, they are often limited in their accuracy, particularly for larger proteins. Here, we address this by introducing the software COMPASS, which, by combining automated resonance assignment with manual intervention, is able to achieve accuracy approaching that from manual assignments at greatly accelerated speeds. Moreover, by including the option to compensate for isotope shift effects in deuterated proteins, COMPASS is far more accurate for larger proteins than existing automated methods. COMPASS is an open-source project licensed under GNU General Public License and is available for download from http://www.liu.se/forskning/foass/tidigare-foass/patrik-lundstrom/software?l=en. Source code and binaries for Linux, Mac OS X and Microsoft Windows are available.

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