Protein Structure determination by NMR

1. Integration of Fast Data Collection and Automated Probabilistic Assignment for Protein NMR SpectroscopyArash Bahrami

2. Protein Structure determination by NMR Sample Preparation Data collection Peak Picking Backbone resonance assignment Sidechain resonance assignment Secondary structure determination NOE data collection and assignment Structure calculation and refinement 2 On the average 1-4 months 80k$ per structure 1 3 Automation in NMR • Individual software packages have been developed for each part but no integrated tool is available for the whole process. • Integration needs interaction of individual components • Probabilistic frameworkcan provides robust interaction of components

3. Individual tools developed in CESG and NMRFAM • PISTACHIO (Automated resonance assignment) • PECAN (Secondary structure determination) • MANI-LACS (Reference correction and outlier detection) • HIFI-NMR (Fast and adaptive NMR data collection) • HIFI-C (Adaptive determination of NMR couplings) 1 Hamid R. Eghbalnia, Arash Bahrami, Liya Wang, Amir Assadi, and John L. Markley (2005) J. Biomol. NMR, 32(3):219-233. 2 Hamid R. Eghbalnia, Liya Wang, Arash Bahrami, Amir Assadi, and John L. Markley (2005) J. Biomol. NMR, 32(1):71-81. 3 Liya Wang, Hamid R. Eghbalnia, Arash Bahrami, and John L. Markley (2005) J. Biomol. NMR, 32(1):13-22. 4 Hamid R. Eghbalnia, Arash Bahrami, Marco Tonelli, Klaus Hallenga, and John L. Markley (2005) J. Am. Chem. Soc., 127(36) 12528 – 12536. 5 Gabriel Cornilescu, Arash Bahrami,Marco Tonelli, John L. Markley, Hamid R. Eghbalnia. (2007) J. Biomol. NMR, 38(4):341-351.

4. PISTACHIO PISTACHIO is a probabilistic method for backbone and sidechain assignment. The input to PISTACHIO can be a any subset of following NMR experiments: • HSQC • HNCO • CBCA(CO)NH • HNCACB • HN(CO)CACB • HNCA • HN(CO)CA • HN(CA)CO • HN(CO)(CA)CB • HN(CA)CB • C(CO)NH • HBHA(CO)NH • H(CCO)NH • HCCH-TOCSY

5. Helix Extended PECAN PECAN optimizes a combination of information sources to yield energetic descriptions of secondary structure and constructs a probabilistic description wherein each residue is assigned a probability of belonging to a designated state (e.g. helix, sheet, etc.). PECAN is available at: http://www.bija.nmrfam.wisc.edu/PECAN

6. LACS MANI-LACS3 (Linear Analysis of Chemical Shifts for reference correction and outlier detection) can detect potential outliers using linear analysis of chemical shifts. An outlier may be the result of miss assignment of chemical shifts. MANI-LACS reports probabilities for the presence of outliers. MANI-LACS is available at: http://www.bija.nmrfam.wisc.edu/MANI-LACS/

7. HIFI-NMR: High-Resolution Iterative Frequency Identification for NMR Tilted-plane reduced dimensionality data collection that employs on-the-fly peak identification, spectral modeling, and selection of the next data plane to be collected. 2D planes of 3D CBCA(CO)NNH experiment collected on 800 MHz Varian Inova spectrometer

8. predicted chemical shift distribution orthogonal planes assign a probability of a peak being in a given voxel, p 0° 90° probability color map dispersion function, fq (p), measures the dispersion of the putative peaks on the selected tilted plane find a tilt angle that maximizes a dispersion function fq (p) collect tilted plane Has the last tilted plane added new information ??? X° NO peak list Simplified Description of the HIFI NMR Approach YES

9. HIFI application to automated backbone assignments

10. HIFI–C: A Fast and Robust Method for Determining NMR Couplings from Adaptive 3D to 2D Projections • Correlation and RMSD comparison of couplings collected by HIFI-C and 3D. Agreement between the two was within experimental error. • GB3 protein (R = 99.8%, rmsd = 0.03 Hz). The total data collection times were 1.7 h for HIFI-C and 7.9 h for 3D. • PRP24-12 protein (R = 94.0%, rmsd = 0.25 Hz). The total data collection times were 14.6 h for HIFI-C and 44.1 h for 3D .

11. Back to Automation Steps in NMR Proteomics HIFI-NMR PISTACHIO MANI-LACS PECAN HIFI-C

12. Redesign the Individual Tools to Provide Robust Probabilistic Interaction: PINE PINE PISTACHIO MANI-LACS PECAN

13. General Overview of Probabilistic Network Defined by PINE

14. Spin System Generation Network Amino Acid Typing Network

16. PINE Web Server

17. PINE Server Statistics Total Number of jobs submitted since July 2006: 1175 jobs

18. Iterative HCCH-TOCSY assignment HBHA(CO)NH HCCH-TOCSY C(CO)NH H (CCO)NH

19. PINE, HIFI and Time Saving in NMR Proteomics

20. On going project: Integration of HIFI and PINE Fast data collection and peak identification HIFI-NMR PINE Referencing and outlier check Secondary structure determination Automated assignment MANI-LACS PECAN PISTACHIO

21. Probabilistic Analysis of Spectra in HIFI (B) HNCA (HC plane) 1024 zero filling; 0.45 delay in sine window function (A) HNCA (HC plane) 512 zero filling; 0.15 delay in sine window function (D) Probabilistic peak lists are generated for every plane based on different parameter settings and peaks volume. (C) Difference between spectra (A) and (B)

22. On Fly Spin System Generation in HIFI

23. On Fly Spin System Generation in HIFI

24. On Fly Spin System Generation in HIFI

25. On Fly Spin System Generation in HIFI

26. On Fly Spin System Generation in HIFI

27. On Fly Spin System Generation in HIFI

28. On Fly Spin System Generation in HIFI

29. Collect N15-HSQC Predicted chemical shift distribution Spectra Analysis Generate probabilistic peak list 0° Collect the most sensitive orthogonal plane X° Derive the initial probabilistic spin systems Spectra Analysis: Generate probabilistic peak list Update the probabilistic spin system PINE Derive the latest assignment and secondary structure collect the optimal tilted or orthogonal plane Is the spin system network quality good enough for the assignment process? YES Are the assignment and secondary structure complete? NO NO YES Find the optimum experiment and tilted angle The optimum is the plane that maximizes the information regarding the ambiguous or missing position in spin systems considering latest state of chemical shift assignment. Report the final peak lists, chemical shift assignments, and secondary structure

30. Fast data collection and peak identification HIFI-NMR PINE Referencing and outlier check Secondary structure determination Automated assignment MANI-LACS PECAN PISTACHIO NOESY Assignment

31. Acknowledgements All CESG member providing data: • Claudia Cornilescu • Shanteri Singh • Jikui Song • Brian Volkman • Francis Peterson • John Markley • Hamid Eghbalnia • Marco Tonelli • Ziqi Dai • Gabriel Cornislescu • Klaus Hallenga • Milo Westler • Liya Wang • Eldon Ulrich