Building Partial Atomic Building Partial Atomic Model of CPV from CryoEM Model of CPV from CryoEM

MapsMaps

Hong Zhou, Ph.D.Hong Zhou, Ph.D.The University of Texas - Medical School at HoustonThe University of Texas - Medical School at Houston

CPVCPV: Cytoplasmic polyhedrosis virus: Cytoplasmic polyhedrosis virus

6/10/2006, Italy 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

no

cryoEM

no

no

no

Reoviridae: a large family of dsRNA viruses

OrthoreovirusOrthoreovirus

RotavirusRotavirus

Orbivirus Orbivirus (BTV)

Phytoreovirus Phytoreovirus (rice dwarf (rice dwarf virus)virus)

Cypovirus Cypovirus (CPV)(CPV)

FijivirusFijivirus

ColtivirusColtivirus

AquareovirusAquareovirus

SeadornavirusSeadornavirus

MycoreovirusMycoreovirus

OryzavirusOryzavirus

10

11

10

12

10

10

12

11

12

11

10

8

6

7

12

5

11

12

7

12

10(?)

10

850

800/1050

800

780

590/800

700

800

800

800

800(?)

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 ranges 3D structure # RNA segments

# Structural Proteins

Capsid size (Å)

No recognizable sequence homology across difference generaNo recognizable sequence homology across difference genera

Facts of CPV (cytoplasmic polyhedrosis virus)

Used as a bio-control agent, an Used as a bio-control agent, an environment-friendly pesticide, environment-friendly pesticide, thus widely availablethus widely available

Single-shelledSingle-shelled capsid, yet very capsid, yet very STABLESTABLE

Fully capable of endogenous RNA Fully capable of endogenous RNA transcription, mRNA capping and transcription, mRNA capping and release within intact virusrelease within intact virus

Nice model system for pushing Nice model system for pushing cryoEM toward atomic resolutioncryoEM toward atomic resolution

Structural Organization of the CPV (13 Å) Structural Organization of the CPV (13 Å)

Empty CPVEmpty CPVFull CPVFull CPV

TEC: Transcriptional Enzyme Complex

Turret protein (TP)

Large protrusion protein (LPP)

Capsid shell protein (CSP)

dsRNA

Protein-RNA interactions play major role.Protein-RNA interactions play major role.

Xia, Zhou et al. JBCXia, Zhou et al. JBC

CryoEM Imaging and Evaluation of Data Quality

Tens or even Tens or even hundreds of hundreds of thousands of thousands of particle images particle images may be needed may be needed toward 4-5 toward 4-5 Å Å resolutionresolution

Spatial Frequency (1/Å)Å)

1/4.5Å-1

2x

104

3x

104

4x

104

5x

104

6x

104

7x

104

Ave

rage

d I

nte

nsi

ty

1/4.5 Å1/4.5 Å-1-1

Incoherent average of FTs

300kV FEG, Liquid 300kV FEG, Liquid helium-cooled helium-cooled specimen (4 K) specimen (4 K) ((JEOL3000 @ NCMIJEOL3000 @ NCMI))

Kodak SO163 Kodak SO163 films at 60,000 xfilms at 60,000 x

Liang et al., unpublishedLiang et al., unpublished

http://hub.med.uth.tmc.edu/~hong/IMIRS

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

Summary of data processing Summary of data processing statisticsstatistics

Summary of data processing Summary of data processing statisticsstatistics

Number of focal pairs scanned:Number of focal pairs scanned: >1,000 pairs>1,000 pairsNumber of focal pairs refined:Number of focal pairs refined: 646 pairs646 pairs1.16Å/pixel, 800x800 particle1.16Å/pixel, 800x800 particleNumber of particles processed: Number of particles processed: 135,000 135,000

Defocus ranges: Defocus ranges: 1.9-3.7 1.9-3.7 μμmm and 0.2-1.7 and 0.2-1.7 μμmm

B factor: 100-210 and 40-140 B factor: 100-210 and 40-140 ÅÅ22 respectively respectivelyFinal reconstructionFinal reconstruction

25,70525,705 particle images used, all close-to-focus particle images used, all close-to-focusrefined to 1/3.5 refined to 1/3.5 ÅÅ-1-1 effective resolution 5.2 Åeffective resolution 5.2 Å

Total averaging is about 1.5 million (25,705 x 60)Total averaging is about 1.5 million (25,705 x 60)

Liang et al., unpublishedLiang 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., unpublishedLiang et al., unpublished

Liang et al., unpublishedLiang et al., unpublished

Asymmetric Unit: Molecular Asymmetric Unit: Molecular Interactions Interactions

Asymmetric Unit: Molecular Asymmetric Unit: Molecular Interactions Interactions

180180°°LPP-3LPP-3CSP-BCSP-B

CSP-ACSP-A

TPTP

LPP-5LPP-5

Liang et al., unpublishedLiang et al., unpublished

Structural Basis of Stability: CPV Structure

•Intensive molecular interactions•Molecular clamps

Resolving Strands in Resolving Strands in SheetSheet

Liang et al., unpublishedLiang et al., unpublished

Integrative Model Integrative Model BuildingBuilding

Primary SequencePrimary Sequence

Secondary Structure

Prediction & Profiling

Secondary Structure

Prediction & Profiling

Element Model Model (helices & (helices & sheets)sheets)

Element Model Model (helices & (helices & sheets)sheets)

CryoEM StructureCryoEM Structure

Secondary Structure Analysis (SSEhunter)

Secondary Structure Analysis (SSEhunter)

SkeletonizationSkeletonization

Side-Chain Densities

Side-Chain Densities

Backbone & All-Atom Refinement

(MaxSprout, XBuild)

Backbone & All-Atom Refinement

(MaxSprout, XBuild)

Rotamer Refinement

(Coot, XBuild)

Rotamer Refinement

(Coot, XBuild)

Atomic Model

PDB

Atomic Model

PDB

Profile Anchoring & Topology

Mapping

Profile Anchoring & Topology

Mapping

Cα Model & Model & Refinement Refinement

(Coot)(Coot)

Cα Model & Model & Refinement Refinement

(Coot)(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., unpublishedLiang et al., unpublished

CSPa Modeling: structure-based sequence alignment

Line 0: annotationLine 1: CPV sequenceLine 2: psipred predictionLine 3: profsec prediction

Liang et al., unpublishedLiang et al., unpublished

Model Building

40.0% identity in 25 aalower 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., unpublishedLiang et al., unpublished

Summary & Conclusion

CryoEM reconstruction of CPV to ~5 Å CryoEM reconstruction of CPV to ~5 Å (including data up to 4 Å)(including data up to 4 Å)

Development of an integrative modeling Development of an integrative modeling method method to build partial Cto build partial Cαα models models

Partial CPartial Cαα models of CSP-A and CSP-B and models of CSP-A and CSP-B and implication of their conformational switch in implication of their conformational switch in interacting with RNA and regulating interacting with RNA and regulating endogenous RNA transcriptionendogenous RNA transcription

Secondary structure elements and Secondary structure elements and bulkybulky amino-amino- acid side chains are resolvedacid 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