building partial atomic model of cpv from cryoem maps
DESCRIPTION
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. Reoviridae : a large family of dsRNA viruses. No recognizable sequence homology across difference genera. - PowerPoint PPT PresentationTRANSCRIPT
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