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

X-ray & cryoEM (Harrison/Baker) cryoEM & X-ray (Chiu/Prasad/Harrison) X-ray & cryoEM (Stuart/Prasad/Hewat) cryoEM & X-ray (Zhou/Chiu/Tsukihara) cryoEM (Zhou/Stuart) no cryoEM no Reoviridae: a large family of dsRNA virusesOrthoreovirusRotavirus Orbivirus Orbivirus (BTV) Phytoreovirus (rice dwarf virus) Cypovirus (CPV) Fijivirus Coltivirus Aquareovirus Seadornavirus Mycoreovirus Oryzavirus (?) / / (?) 700 Mammals Mammals/birds Mammals/insect vectors Plants/insect vectors Insects Plant/insect vectors Mammals,invertebrate Bony fish, crustaceans Mammals fungus Plant,invertebrate Genus Host ranges3D structure# RNA segments # Structural Proteins Capsid size (Å) No recognizable sequence homology across difference genera

Facts of CPV (cytoplasmic polyhedrosis virus)  Used as a bio-control agent, an environment-friendly pesticide, thus widely available  Single-shelled capsid, 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

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

CryoEM Imaging and Evaluation of Data Quality  Tens or even hundreds of thousands of particle images may be needed toward 4-5 Å resolution Å) Spatial Frequency (1/Å) 1/4.5Å -1 2x10 4 3x10 4 4x10 4 5x10 4 6x10 4 7x10 4 Averaged Intensity 1/4.5 Å -1 Incoherent average of FTs  300kV FEG, Liquid helium-cooled specimen (4 K) ( NCMI )  Kodak SO163 films at 60,000 x Liang et al., unpublished

Data Processing & Reconstruction by IMIRS: an integrative and modular approach

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: μm and μm  B factor: and Å 2 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refined to 1/3.5 Å -1 effective resolution 5.2 Å  Total averaging is about 1.5 million (25,705 x 60) Liang et al., unpublished

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

Asymmetric Unit: Molecular Interactions 180° LPP-3 CSP-B CSP-A TP LPP-5 Liang et al., unpublished

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

Resolving Strands in  Sheet Liang et al., unpublished

Integrative Model Building Primary Sequence Secondary Structure Prediction & Profiling Model (helices & sheets) Element Model (helices & sheets) CryoEM Structure Secondary Structure Analysis (SSEhunter) Skeletonization Side-Chain Densities Backbone & All- Atom Refinement (MaxSprout, XBuild) Rotamer Refinement (Coot, XBuild) Atomic Model PDB Atomic Model PDB Profile Anchoring & Topology Mapping Model & Refinement (Coot) Cα Model & Refinement (Coot)   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

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

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

 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

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

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