Improving RNA Crystallographic Models Using Rosetta. Fang-Chieh Chou (Das Lab) For RosettaCon 2011

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1 Improving RNA Crystallographic Models Using Rosetta Fang-Chieh Chou (Das Lab) For RosettaCon 211

2 The number RNA crystal structures increased explosively after the first Ribosome crystal structure is solved in 2 ( ). However multiple errors exist in the current RNA crystal structures, which can be revealed in Molprobity. Chen et al. (21) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Cryst D66:12-21 Ban N, Nissen P, Hansen J, Moore P, Steitz T (2). The complete atomic structure of the large ribosomal subunit at 2.4 Angstrom resolution. Science 289 (5481): 95-2

3 Determination of X-ray Structure Figure from Wikipedia

4 Limits of Crystallography Position of atoms become ambiguous when the resolution is low (especially for RNA sugar ring). The majority of the current RNA structure has a resolution below 2.5 Å, and they may contain multiple errors. Keating KS & Pyle AM (21) Semiautomated model building for RNA crystallography using a directed rotameric approach. Proc. Natl. Acad. Sci. USA 17(18):

5 Current Status of RNA Model Improvement Tools RNABC RCrane Wang X, et al. (28) RNABC: forward kinematics to reduce all-atom steric clashes in RNA backbone. J. Math. Biol. 56(1): Keating KS & Pyle AM (21) Semiautomated model building for RNA crystallography using a directed rotameric approach. Proc. Natl. Acad. Sci. USA 17(18):

6 Tools in Rosetta: Electron Density Scoring and Stepwise Assembly (SWA) DiMaio F, Tyka MD, Baker ML, Chiu W, & Baker D (29) Refinement of Protein Structures into Low-Resolution Density Maps Using Rosetta. J. Mol. Biol. 392(1):

7 Tools in Rosetta: Electron Density Scoring and Stepwise Assembly (SWA) DiMaio F, Tyka MD, Baker ML, Chiu W, & Baker D (29) Refinement of Protein Structures into Low-Resolution Density Maps Using Rosetta. J. Mol. Biol. 392(1):

8 Tools in Rosetta: Electron Density Scoring and Stepwise Assembly (SWA) DiMaio F, Tyka MD, Baker ML, Chiu W, & Baker D (29) Refinement of Protein Structures into Low-Resolution Density Maps Using Rosetta. J. Mol. Biol. 392(1):

9 PHENIX: Structure Refinement Tool Adams PD, et al. (21) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Cryst. D 66(2):

10 Overall Strategy Rosetta Structure Refinement Step Iterate if necessary Refine the starting PDB in PHENIX Electron density guided whole structure minimization Rebuild problematic nucleotides one at a time using Stepwise Assembly Final whole structure minimization Final PHENIX refinement

11 Improvement of Backbone 1U8D (1.95 Å ) 2GDI (2.5 Å ) Red: PDB, Green: Rosetta/PHENIX

12 Improvement of Clashes PDB Rosetta/PHENIX From 3IWN(3.2 Å )

13 PDB PHENIX Rcrane/PHENIX Rosetta/PHENIX 15 test cases are remodeled Clash Score Wrong Pucker Bad Conformer Bad Bond (%) Bad Angle (%)

14 Wrong Puckers 15 test cases are remodeled PDB PHENIX Rcrane/PHENIX Rosetta/PHENIX PDB Clash Score Wrong Pucker Bad Conformer Bad Bond (%) Bad Angle (%) Resolution (Å )

15 Wrong Puckers 15 test cases are remodeled PDB PHENIX Rcrane/PHENIX Rosetta/PHENIX PDB PHENIX Clash Score Wrong Pucker Bad Conformer Bad Bond (%) Bad Angle (%) Resolution (Å )

16 Wrong Puckers 15 test cases are remodeled PDB PHENIX Rcrane/PHENIX Rosetta/PHENIX PDB PHENIX Rcrane/PHENIX Clash Score Wrong Pucker Bad Conformer Bad Bond (%) Bad Angle (%) Resolution (Å )

17 Wrong Puckers 15 test cases are remodeled 3 PDB PHENIX Rcrane/PHENIX Rosetta/PHENIX PDB PHENIX Rcrane/PHENIX Rosetta/PHENIX Clash Score Wrong Pucker Bad Conformer Bad Bond (%) Bad Angle (%) Resolution (Å )

18 13 out 15 cases in Rosetta/PHENIX have equal or better R free than in PHENIX PHENIX Rcrane/PHENIX Rosetta/PHENIX Average Rfree Drop Median Rfree Drop Average R Drop Median R Drop

19 Rfree 13 out 15 cases in Rosetta/PHENIX have equal or better R free than in PHENIX PHENIX Rcrane/PHENIX Rosetta/PHENIX Average Rfree Drop Median Rfree Drop Average R Drop Median R Drop PDB Resolution (Å )

20 Rfree 13 out 15 cases in Rosetta/PHENIX have equal or better R free than in PHENIX PHENIX Rcrane/PHENIX Rosetta/PHENIX Average Rfree Drop Median Rfree Drop Average R Drop Median R Drop.31 PDB PHENIX Resolution (Å )

21 Rfree 13 out 15 cases in Rosetta/PHENIX have equal or better R free than in PHENIX PHENIX Rcrane/PHENIX Rosetta/PHENIX Average Rfree Drop Median Rfree Drop Average R Drop Median R Drop PDB PHENIX Rcrane/PHENIX Resolution (Å )

22 Rfree 13 out 15 cases in Rosetta/PHENIX have equal or better R free than in PHENIX PHENIX Rcrane/PHENIX Rosetta/PHENIX Average Rfree Drop Median Rfree Drop Average R Drop Median R Drop PDB PHENIX Rcrane/PHENIX Rosetta/PHENIX Resolution (Å )

23 We compared the similarity of sugar puckers and backbone conformers for the two chains of PDB with two copies in the structure and two PDBs of the same sequence by different groups. The two aptamer stems of glycine riboswitch (3P49) are also compared. Average Pucker/Conformer Similarity PDB PHENIX RCrane/PHENIX Rosetta/PHENIX Pucker Similarity (%) Conformer Similarity (%)

24 Summary Rosetta/PHENIX improves RNA crystallographic models. Consistent reduction of clashes and bad geometry (Molprobity) Lower or maintain R free in 13 out of 15 cases Reasonable computational cost. Less than one day to fix a ~17 residues RNA in a single core PC. We plan to add it into the next release of Rosetta and also set up a server to host this code after we publish it.

25 Acknowledgement Rhiju Das, my advisor. Parin Sripakdeevong, for providing the SWA code and helping me with all the Rosetta questions. All other members in the Das lab for their advices. Bio-X² cluster at Stanford for the computational resource. Rosetta community for making the code publicly available.

26 Thank you for your attention!