Building Partial Atomic Model of CPV from CryoEM Maps

1. Building Partial Atomic Model of CPV from CryoEM Maps Hong Zhou, Ph.D. The University of Texas - Medical School at Houston CPV: Cytoplasmic polyhedrosis virus 6/10/2006, Italy

2. Reoviridae: a large family of dsRNA viruses No recognizable sequence homology across difference genera

3. Facts of CPV(cytoplasmic polyhedrosis virus) • Used as a bio-control agent, an environment-friendly pesticide, thus widely available • Single-shelledcapsid, yet very STABLE • Fully capable of endogenous RNA transcription, mRNA capping and release within intact virus • Nice model system for pushing cryoEM toward atomic resolution

4. Structural Organization of the CPV (13 Å) Empty CPV Full CPV Turret protein (TP) dsRNA Capsid shell protein (CSP) Large protrusion protein (LPP) Xia, Zhou et al. JBC TEC: Transcriptional Enzyme Complex Protein-RNA interactions play major role.

5. 7x104 6x104 1/4.5 Å-1 Averaged Intensity 5x104 4x104 3x104 1/4.5Å-1 2x104 Spatial Frequency (1/Å) Incoherent average of FTs CryoEM Imaging and Evaluation of Data Quality • 300kV FEG, Liquid helium-cooled specimen (4 K) (JEOL3000 @ NCMI) • Kodak SO163 films at 60,000 x • Tens or even hundreds of thousands of particle images may be needed toward 4-5 Å resolution Liang et al., unpublished

6. Data Processing & Reconstruction by IMIRS: an integrative and modular approach http://hub.med.uth.tmc.edu/~hong/IMIRS

7. Summary of data processing statistics • Number of focal pairs scanned: >1,000 pairs • Number of focal pairs refined: 646 pairs • 1.16Å/pixel, 800x800 particle • Number of particles processed: 135,000 • Defocus ranges: 1.9-3.7 μm and 0.2-1.7 μm • B factor: 100-210 and 40-140 Å2 respectively • Final reconstruction 25,705 particle images used, all close-to-focus refined to 1/3.5 Å-1 effective resolution 5.2 Å • Total averaging is about 1.5 million (25,705 x 60) Liang et al., unpublished

8. CPV Structure at 5.9 Å: Overall Organization 120 copies of Capsid Shell Protein (CSP), colored by red and purple. 120 copies of Large Protrusion Protein (LPP), colored by green and yellow. 60 copies of Turret Protein (TP), colored by blue. Liang et al., unpublished

9. Liang et al., unpublished

10. Asymmetric Unit: Molecular Interactions TP LPP-5 CSP-A LPP-3 180° CSP-B Liang et al., unpublished

11. Intensive molecular interactions • Molecular clamps Structural Basis of Stability: CPV Structure

12. Resolving Strands in  Sheet Liang et al., unpublished

13. Primary Sequence CryoEM Structure Secondary Structure Analysis (SSEhunter) Side-Chain Densities Secondary Structure Prediction & Profiling Atomic Model PDB Rotamer Refinement (Coot, XBuild) Element Model (helices & sheets) Skeletonization Backbone & All-Atom Refinement (MaxSprout, XBuild) Profile Anchoring & Topology Mapping Cα Model & Refinement (Coot) Integrative Model Building • Motivation: bottom-up approach (O, MAID, X-Build etc) NOT readily applicable to moderate resolution cryoEM maps • Our approach: top-down and integrates all available knowledge Liang et al., unpublished

14. Line 0: annotation Line 1: CPV sequence Line 2: psipred prediction Line 3: profsec prediction CSPa Modeling: structure-based sequence alignment Liang et al., unpublished

15. 40.0% identity in 25 aa lower QADFIQTSDAVRQLRALMPTLSTSQ : :.: : .::. : .:. .: .: 1EJ6B QRDMI-TCEAVQTLVTLVAQISETQ Model Building 21.0% identity in 119 aa lower_ RFNGVRIMYLTDDDPDPDFVPDVPEGYVA--VQYAHRLFSSS---LANKRN-----RVTY :.. . :.: . . .:..: . .: :: . ..:. .. :. :: PDBFIN RMTQLAIQYQQYNGRTFNVIPEMPGSVIADCVQLTAEVFNHEYNLFGIARGDIIIGRVQS lower_ TH------PPTGMAYPSPTGRPHVHMTINE------RAGMSKLVADNIIASV-IKSNWV :: :: ... : : ::. . : . .. :. : ...::. PDBFIN THLWSPLAPPPDLVFDRDT--PGVHIFGRDCRISFGMNGAAPMIRDETGMMVPFEGNWI 1EJ6

16. Secondary structure profile instead of sequence alignment Conserved helices are anchor points Skeleton used as “road maps” to connect neighboring helices Illustration of Approach: Modeling CSP-A Liang et al., unpublished

17. Summary & Conclusion • CryoEM reconstruction of CPV to ~5 Å (including data up to 4 Å) • Secondary structure elements and bulky amino- acid side chains are resolved • Development of an integrative modeling method to build partial Cα models • Partial Cαmodels of CSP-A and CSP-B and implication of their conformational switch in interacting with RNA and regulating endogenous RNA transcription

18. Acknowledgements University of Texas Medical School at Houston Yuyao (Mario) Liang, Xue-Kui Yu, Hua Tsen Baylor College of Medicine Wah Chiu, Joanita Jakana, Matthew Baker Zhongshan University, China Jing -Qiang Zhang