Protein Structure Determination Using NMR Restraints BCMB/CHEM 8190

Transcription

1 Protein Structure Determination Using NMR Restraints BCMB/CHEM 8190

2 Programs for NMR Based Structure Determination CNS - Brunger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.; Gros, P.; Grosse-Kunstleve, R. W.; Jiang, J. S.; Kuszewski, J.; Nilges, M.; Pannu, N. S.; Read, R. J.; Rice, L. M.; Simonson, T.; Warren, G. L. Acta Cryst. D 1998, 54, 905. XPLOR-NIH - Schwieters, C. D.; Kuszewski, J. J.; Tjandra, N.; Clore, G. M. J. Magn. Reson. 2003, 160, 65. DYANA - Guntert, P.; Mumenthaler, C.; Wuthrich, K. J. Mol. Biol. 1997, 273, 283. ARIA - Linge JP, Habeck M, Rieping W, et al. Bioinformatics 2003, 19, JAN

3 NOE data from 2D and 3D experiments are a primary source of information I cp = C{exp(-ρT) (1 exp(-2σt)} ρ = 2W 1 + W 2 + W 0, σ = (W 2 -W 0 ) I cp di cp /dt 1/r 6 T

4 NOESY Spectrum of ACP

5 Potential NOE Interactions In an Idealized α-helix Some can be used as a distance calibration

6 Long range NOEs (sidechain to sidechain) are among the most important in structure determination

7 Getting an Initial Structure - Embedding Metric Matrix producing an approximate fold from an extended chain View as a set of products of vectors in N dimensional hyperspace W. Braun, Quart. Rev. Biophys. 19, (1987) M = r i r j 0 r j r i D ij r i r j can be written in terms of distances D 2 ij = r i 2 + r j 2-2r i r j

8 Solving for positions in Cartesian space Fill in matrix with inter-atom distances some from NOEs a lot from covalent geometry - Diagonalize M; λ = A M A -1 ; M = A -1 λ A A diagonal matrix corresponds to vectors in real space Only 3 λ should be finite and equal (r i r i finite only for x x, etc) r i r i = Σ k λ k A -1 ik A jk = A -1 j1 A i1 +A -1 j2 A i2 +A -1 j3 A i3 = x i x i +y i y i +z i z i Hence, elements of A are x,y,z coordinates of atoms In practice often use upper and lower bounds and fill in matrix by random number selection within bounds Solution is only approximate

9 Structures Using Error Functions and Simulated Annealing E = E bond + E vdw + E angle E NMR E NMR = Σ I (r obs r trial ) I 2 (or use r min,max for r obs ) x new = x old + t v x = x old + t a x dt, y new = a x = F x /m= - (1/m) de/dx + a rand (T), a y = T t

10 20 NMR Structures of DnaJ

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21 Validation of Structures R factor for NOEs: n ~ 1/6 R = Σ NOEs [(I obs ) n (I calc ) n ] / Σ NOEs (I obs ) n Other statistics: rmsd of backbone and all atoms. NOE violations Molecular energy Procheck output

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24 Structure Refinement Using RDCs Write RDCs in principal alignment frame: D = (D a /r 3 ){(3cos 2 θ 1)/r 3 + (3/2)Rsin 2 θcos(2φ)} Write error function in terms of D meas and D calc E RDC = (D meas D calc ) 2 Seek minimum in E RDC to refine structure Need to float alignment axes during search

25 REsidual Dipolar Coupling Analysis Tool (REDCAT) Valafar, H., & J.H. Prestegard (2004), J. Mag. Res. 167: Dosset, Hus, Marion & Blackledge (2001), JBNMR, 20: Given a proposed structure and RDCs, calculates order tensor solutions. Finds best order tensor solution. Gives principal elements and Euler angles. Back-calculates RDCs. Estimates errors and helps identify problematic data.

26 Access Prepare Input From File menu

27 Input from file menu is pdb file and rdc list HEADER METAL BINDING PROTEIN 1BQ8.pdb ATOM 1 N MET A ATOM 2 CA MET A ATOM 3 C MET A ATOM 4 O MET A # Pf-Fe-Rubredoxin residues 2-54 # H-N RDCs from field induced orientation

28 Loaded Coordinates and Couplings

29 A List of Possible Solutions is Generated by Monte Carlo Sampling

30 Problematic Data Identification by Numbers of Rejections Caused

31 Error Analysis with Error < 1.0 Green: Acceptable error, Red: Small indicated error, Gray: Excluded from analysis.

32 Best Solution After the Adjustment of Errors