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- PDB-6nhj: Atomic structures and deletion mutant reveal different capsid-bin... -

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Basic information

Entry
Database: PDB / ID: 6nhj
TitleAtomic structures and deletion mutant reveal different capsid-binding patterns and functional significance of tegument protein pp150 in murine and human cytomegaloviruses with implications for therapeutic development
Components
  • Major capsid protein
  • Minor capsid protein
  • Small capsomere-interacting protein
  • Tegument protein
  • Triplex capsid protein 2
KeywordsVIRUS / cryoEM / beta-herpesvirus / murine cytomegalovirus / human cytomegalovirus / pp150 / pUL32 / pM32 / bacterial artificial chromosome-based mutagenesis
Function / homology
Function and homology information


T=16 icosahedral viral capsid / viral capsid assembly / viral capsid / host cell nucleus / structural molecule activity / viral process / DNA binding
Herpesvirus capsid shell protein VP19C / Herpesvirus capsid shell protein 1 / Small capsid protein of Herpesviridae / Herpes virus major capsid protein / Herpesvirus large structural phosphoprotein UL32 / Herpesvirus VP23 like capsid protein / Small capsid protein, Herpesviridae / Herpesvirus UL11/UL32 / Herpesvirus major capsid protein, upper domain superfamily / Herpesvirus capsid protein 2 / Herpesvirus major capsid protein
Major capsid protein / Tegument protein / Minor capsid protein / Small capsomere-interacting protein / Triplex capsid protein 2
Biological speciesMurine cytomegalovirus (Murine cytomegalovirus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5 Å
AuthorsLiu, W. / Dai, X.H. / Jih, J. / Chan, K. / Trang, P. / Yu, X.K. / Balogun, R. / Mei, Y. / Liu, F.Y. / Zhou, Z.H.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Dental and Craniofacial Research (NIH/NIDCR)DE025567, DE023935, and DE025462 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI069015 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM071940 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1S10RR23057 United States
National Science Foundation (NSF, United States)DMR-1548924 United States
CitationJournal: PLoS Pathog. / Year: 2019
Title: Atomic structures and deletion mutant reveal different capsid-binding patterns and functional significance of tegument protein pp150 in murine and human cytomegaloviruses with implications for ...Title: Atomic structures and deletion mutant reveal different capsid-binding patterns and functional significance of tegument protein pp150 in murine and human cytomegaloviruses with implications for therapeutic development.
Authors: Wei Liu / Xinghong Dai / Jonathan Jih / Karen Chan / Phong Trang / Xuekui Yu / Rilwan Balogun / Ye Mei / Fenyong Liu / Z Hong Zhou /
Abstract: Cytomegalovirus (CMV) infection causes birth defects and life-threatening complications in immunosuppressed patients. Lack of vaccine and need for more effective drugs have driven widespread ongoing ...Cytomegalovirus (CMV) infection causes birth defects and life-threatening complications in immunosuppressed patients. Lack of vaccine and need for more effective drugs have driven widespread ongoing therapeutic development efforts against human CMV (HCMV), mostly using murine CMV (MCMV) as the model system for preclinical animal tests. The recent publication (Yu et al., 2017, DOI: 10.1126/science.aam6892) of an atomic model for HCMV capsid with associated tegument protein pp150 has infused impetus for rational design of novel vaccines and drugs, but the absence of high-resolution structural data on MCMV remains a significant knowledge gap in such development efforts. Here, by cryoEM with sub-particle reconstruction method, we have obtained the first atomic structure of MCMV capsid with associated pp150. Surprisingly, the capsid-binding patterns of pp150 differ between HCMV and MCMV despite their highly similar capsid structures. In MCMV, pp150 is absent on triplex Tc and exists as a "Λ"-shaped dimer on other triplexes, leading to only 260 groups of two pp150 subunits per capsid in contrast to 320 groups of three pp150 subunits each in a "Δ"-shaped fortifying configuration. Many more amino acids contribute to pp150-pp150 interactions in MCMV than in HCMV, making MCMV pp150 dimer inflexible thus incompatible to instigate triplex Tc-binding as observed in HCMV. While pp150 is essential in HCMV, our pp150-deletion mutant of MCMV remained viable though with attenuated infectivity and exhibiting defects in retaining viral genome. These results thus invalidate targeting pp150, but lend support to targeting capsid proteins, when using MCMV as a model for HCMV pathogenesis and therapeutic studies.
Validation Report
SummaryFull reportAbout validation report
History
DepositionDec 22, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 6, 2019Provider: repository / Type: Initial release
Revision 1.1Nov 27, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Structure visualization

Movie
  • Biological unit as complete icosahedral assembly
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  • Biological unit as icosahedral pentamer
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  • Biological unit as icosahedral 23 hexamer
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Major capsid protein
B: Major capsid protein
C: Major capsid protein
D: Major capsid protein
E: Major capsid protein
F: Major capsid protein
G: Major capsid protein
H: Major capsid protein
I: Major capsid protein
k: Major capsid protein
l: Major capsid protein
m: Major capsid protein
n: Major capsid protein
o: Major capsid protein
p: Major capsid protein
q: Major capsid protein
2: Tegument protein
3: Tegument protein
e: Tegument protein
h: Tegument protein
f: Tegument protein
i: Tegument protein
g: Tegument protein
j: Tegument protein
J: Small capsomere-interacting protein
K: Small capsomere-interacting protein
L: Small capsomere-interacting protein
M: Small capsomere-interacting protein
N: Small capsomere-interacting protein
O: Small capsomere-interacting protein
P: Small capsomere-interacting protein
Q: Small capsomere-interacting protein
R: Small capsomere-interacting protein
r: Small capsomere-interacting protein
s: Small capsomere-interacting protein
t: Small capsomere-interacting protein
u: Small capsomere-interacting protein
v: Small capsomere-interacting protein
w: Small capsomere-interacting protein
x: Small capsomere-interacting protein
y: Minor capsid protein
S: Minor capsid protein
T: Minor capsid protein
U: Minor capsid protein
V: Minor capsid protein
z: Triplex capsid protein 2
W: Triplex capsid protein 2
X: Triplex capsid protein 2
Y: Triplex capsid protein 2
Z: Triplex capsid protein 2
1: Triplex capsid protein 2
a: Triplex capsid protein 2
b: Triplex capsid protein 2
c: Triplex capsid protein 2
d: Triplex capsid protein 2


Theoretical massNumber of molelcules
Total (without water)3,727,30055
Polymers3,727,30055
Non-polymers00
Water0
1
A: Major capsid protein
B: Major capsid protein
C: Major capsid protein
D: Major capsid protein
E: Major capsid protein
F: Major capsid protein
G: Major capsid protein
H: Major capsid protein
I: Major capsid protein
k: Major capsid protein
l: Major capsid protein
m: Major capsid protein
n: Major capsid protein
o: Major capsid protein
p: Major capsid protein
q: Major capsid protein
2: Tegument protein
3: Tegument protein
e: Tegument protein
h: Tegument protein
f: Tegument protein
i: Tegument protein
g: Tegument protein
j: Tegument protein
J: Small capsomere-interacting protein
K: Small capsomere-interacting protein
L: Small capsomere-interacting protein
M: Small capsomere-interacting protein
N: Small capsomere-interacting protein
O: Small capsomere-interacting protein
P: Small capsomere-interacting protein
Q: Small capsomere-interacting protein
R: Small capsomere-interacting protein
r: Small capsomere-interacting protein
s: Small capsomere-interacting protein
t: Small capsomere-interacting protein
u: Small capsomere-interacting protein
v: Small capsomere-interacting protein
w: Small capsomere-interacting protein
x: Small capsomere-interacting protein
y: Minor capsid protein
S: Minor capsid protein
T: Minor capsid protein
U: Minor capsid protein
V: Minor capsid protein
z: Triplex capsid protein 2
W: Triplex capsid protein 2
X: Triplex capsid protein 2
Y: Triplex capsid protein 2
Z: Triplex capsid protein 2
1: Triplex capsid protein 2
a: Triplex capsid protein 2
b: Triplex capsid protein 2
c: Triplex capsid protein 2
d: Triplex capsid protein 2
x 60


  • complete icosahedral assembly
  • 224 MDa, 3300 polymers
Theoretical massNumber of molelcules
Total (without water)223,638,0253300
Polymers223,638,0253300
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation60
2


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
point symmetry operation1
3
A: Major capsid protein
B: Major capsid protein
C: Major capsid protein
D: Major capsid protein
E: Major capsid protein
F: Major capsid protein
G: Major capsid protein
H: Major capsid protein
I: Major capsid protein
k: Major capsid protein
l: Major capsid protein
m: Major capsid protein
n: Major capsid protein
o: Major capsid protein
p: Major capsid protein
q: Major capsid protein
2: Tegument protein
3: Tegument protein
e: Tegument protein
h: Tegument protein
f: Tegument protein
i: Tegument protein
g: Tegument protein
j: Tegument protein
J: Small capsomere-interacting protein
K: Small capsomere-interacting protein
L: Small capsomere-interacting protein
M: Small capsomere-interacting protein
N: Small capsomere-interacting protein
O: Small capsomere-interacting protein
P: Small capsomere-interacting protein
Q: Small capsomere-interacting protein
R: Small capsomere-interacting protein
r: Small capsomere-interacting protein
s: Small capsomere-interacting protein
t: Small capsomere-interacting protein
u: Small capsomere-interacting protein
v: Small capsomere-interacting protein
w: Small capsomere-interacting protein
x: Small capsomere-interacting protein
y: Minor capsid protein
S: Minor capsid protein
T: Minor capsid protein
U: Minor capsid protein
V: Minor capsid protein
z: Triplex capsid protein 2
W: Triplex capsid protein 2
X: Triplex capsid protein 2
Y: Triplex capsid protein 2
Z: Triplex capsid protein 2
1: Triplex capsid protein 2
a: Triplex capsid protein 2
b: Triplex capsid protein 2
c: Triplex capsid protein 2
d: Triplex capsid protein 2
x 5


  • icosahedral pentamer
  • 18.6 MDa, 275 polymers
Theoretical massNumber of molelcules
Total (without water)18,636,502275
Polymers18,636,502275
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation5
4
A: Major capsid protein
B: Major capsid protein
C: Major capsid protein
D: Major capsid protein
E: Major capsid protein
F: Major capsid protein
G: Major capsid protein
H: Major capsid protein
I: Major capsid protein
k: Major capsid protein
l: Major capsid protein
m: Major capsid protein
n: Major capsid protein
o: Major capsid protein
p: Major capsid protein
q: Major capsid protein
2: Tegument protein
3: Tegument protein
e: Tegument protein
h: Tegument protein
f: Tegument protein
i: Tegument protein
g: Tegument protein
j: Tegument protein
J: Small capsomere-interacting protein
K: Small capsomere-interacting protein
L: Small capsomere-interacting protein
M: Small capsomere-interacting protein
N: Small capsomere-interacting protein
O: Small capsomere-interacting protein
P: Small capsomere-interacting protein
Q: Small capsomere-interacting protein
R: Small capsomere-interacting protein
r: Small capsomere-interacting protein
s: Small capsomere-interacting protein
t: Small capsomere-interacting protein
u: Small capsomere-interacting protein
v: Small capsomere-interacting protein
w: Small capsomere-interacting protein
x: Small capsomere-interacting protein
y: Minor capsid protein
S: Minor capsid protein
T: Minor capsid protein
U: Minor capsid protein
V: Minor capsid protein
z: Triplex capsid protein 2
W: Triplex capsid protein 2
X: Triplex capsid protein 2
Y: Triplex capsid protein 2
Z: Triplex capsid protein 2
1: Triplex capsid protein 2
a: Triplex capsid protein 2
b: Triplex capsid protein 2
c: Triplex capsid protein 2
d: Triplex capsid protein 2
x 6


  • icosahedral 23 hexamer
  • 22.4 MDa, 330 polymers
Theoretical massNumber of molelcules
Total (without water)22,363,803330
Polymers22,363,803330
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation6
5


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: I (icosahedral))

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Components

#1: Protein
Major capsid protein / MCP


Mass: 151673.031 Da / Num. of mol.: 16 / Source method: isolated from a natural source / Source: (natural) Murine cytomegalovirus (strain Smith) / Strain: Smith / References: UniProt: A0A1S5YGR4
#2: Protein
Tegument protein


Mass: 78749.961 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Source: (natural) Murine cytomegalovirus (strain Smith) / Strain: Smith / References: UniProt: D3XDM2
#3: Protein
Small capsomere-interacting protein


Mass: 9845.198 Da / Num. of mol.: 16 / Source method: isolated from a natural source / Source: (natural) Murine cytomegalovirus (strain Smith) / Strain: Smith / References: UniProt: D3XDN6
#4: Protein
Minor capsid protein


Mass: 33281.289 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Source: (natural) Murine cytomegalovirus (strain Smith) / Strain: Smith / References: UniProt: D3XDN4
#5: Protein
Triplex capsid protein 2


Mass: 34660.262 Da / Num. of mol.: 10 / Source method: isolated from a natural source / Source: (natural) Murine cytomegalovirus (strain Smith) / Strain: Smith / References: UniProt: D3XDR2

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Murid herpesvirus 1 (strain Smith) / Type: VIRUS / Entity ID: 1, 2, 3, 4, 5 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Murid herpesvirus 1 (strain Smith)
Details of virusEmpty: NO / Enveloped: YES / Isolate: STRAIN / Type: VIRION
Buffer solutionpH: 7.4 / Details: PBS buffer, PH=7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in.
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Details: The grids were manually plunged into the Ethane.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 47000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 1000 nm / Calibrated defocus max: 3000 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 25 e/Å2 / Film or detector model: AGFA SCIENTA FILM / Num. of real images: 2200
Image scansScanner model: NIKON SUPER COOLSCAN 9000

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Processing

EM software
IDNameCategory
4CTFFINDCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 58254
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 47982 / Symmetry type: POINT
Atomic model buildingB value: 200 / Protocol: OTHER / Space: REAL

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