Journal: Science / Year: 2008 Title: Structure of the immature dengue virus at low pH primes proteolytic maturation. Authors: I-Mei Yu / Wei Zhang / Heather A Holdaway / Long Li / Victor A Kostyuchenko / Paul R Chipman / Richard J Kuhn / Michael G Rossmann / Jue Chen / Abstract: Intracellular cleavage of immature flaviviruses is a critical step in assembly that generates the membrane fusion potential of the E glycoprotein. With cryo-electron microscopy we show that the ...Intracellular cleavage of immature flaviviruses is a critical step in assembly that generates the membrane fusion potential of the E glycoprotein. With cryo-electron microscopy we show that the immature dengue particles undergo a reversible conformational change at low pH that renders them accessible to furin cleavage. At a pH of 6.0, the E proteins are arranged in a herringbone pattern with the pr peptides docked onto the fusion loops, a configuration similar to that of the mature virion. After cleavage, the dissociation of pr is pH-dependent, suggesting that in the acidic environment of the trans-Golgi network pr is retained on the virion to prevent membrane fusion. These results suggest a mechanism by which flaviviruses are processed and stabilized in the host cell secretory pathway.
History
Deposition
Feb 5, 2008
Deposition site: RCSB / Processing site: RCSB
Revision 1.0
Apr 22, 2008
Provider: repository / Type: Initial release
Revision 1.1
Jul 13, 2011
Group: Version format compliance
Revision 1.2
Jul 18, 2018
Group: Data collection / Source and taxonomy / Category: em_image_scans / em_software / entity_src_nat Item: _em_software.image_processing_id / _em_software.name / _entity_src_nat.pdbx_ncbi_taxonomy_id
A: Envelope protein D: Peptide pr B: Envelope protein E: Peptide pr C: Envelope protein F: Peptide pr
x 5
icosahedral pentamer
797 kDa, 30 polymers
Theoretical mass
Number of molelcules
Total (without water)
797,489
30
Polymers
797,489
30
Non-polymers
0
0
Water
0
Type
Name
Symmetry operation
Number
identity operation
1_555
x,y,z
1
point symmetry operation
4
4
A: Envelope protein D: Peptide pr B: Envelope protein E: Peptide pr C: Envelope protein F: Peptide pr
x 6
icosahedral 23 hexamer
957 kDa, 36 polymers
Theoretical mass
Number of molelcules
Total (without water)
956,987
36
Polymers
956,987
36
Non-polymers
0
0
Water
0
Type
Name
Symmetry operation
Number
identity operation
1_555
x,y,z
1
point symmetry operation
5
5
Idetical with deposited unit in distinct coordinate
icosahedral asymmetric unit, std point frame
Type
Name
Symmetry operation
Number
transform to point frame
1
Symmetry
Point symmetry: (Schoenflies symbol: I (icosahedral))
-
Components
#1: Protein
Envelopeprotein / Coordinate model: Cα atoms only
Mass: 43904.410 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Dengue virus type 2 / Strain: Thailand/PUO-218/1980 / References: UniProt: P18356
#2: Protein
Peptidepr / Coordinate model: Cα atoms only
Mass: 9261.531 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Dengue virus type 2 / Strain: Thailand/PUO-218/1980 / References: UniProt: P18356
Sequence details
THE AUTHORS STATE THAT THE SEQUENCE CONFLICTS ARE DUE TO STRAIN DIFFERENCES. THE PDB ENTRY USED TO ...THE AUTHORS STATE THAT THE SEQUENCE CONFLICTS ARE DUE TO STRAIN DIFFERENCES. THE PDB ENTRY USED TO FIT INTO THE MAP IS A MODEL GENERATED FROM TWO DIFFERENT STRUCTURES.
-
Experimental details
-
Experiment
Experiment
Method: ELECTRON MICROSCOPY
EM experiment
Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction
-
Sample preparation
Component
Name: immature Dengue virus / Type: VIRUS
Buffer solution
pH: 6 Details: The virus was mixed in NTE buffer (10 mM Tris, 120 mM NaCl, and 1 mM EDTA at pH 8) with 50 mM MES, 120 mM NaCl at pH 5.6 to yield a final pH of 6
Specimen
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Vitrification
Cryogen name: ETHANE
-
Electron microscopy imaging
Microscopy
Model: FEI/PHILIPS CM200FEG
Electron gun
Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lens
Mode: BRIGHT FIELD / Nominal magnification: 50000 X / Nominal defocus max: 2900 nm / Nominal defocus min: 1400 nm
Image recording
Electron dose: 17 e/Å2 / Film or detector model: KODAK SO-163 FILM
Radiation
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength
Relative weight: 1
-
Processing
EM software
ID
Name
Category
1
EMfit
modelfitting
2
EM3DR
3Dreconstruction
CTF correction
Details: Both amplitude and phase of CTF are corrected for each boxed particle.
Symmetry
Point symmetry: I (icosahedral)
3D reconstruction
Method: Cross-correlation in real and reciprocal space for orientation determination; Fourier-Bessel method for reconstruction. Resolution: 25 Å / Num. of particles: 231 / Nominal pixel size: 2.8 Å / Actual pixel size: 2.7 Å / Magnification calibration: Grating Replica EM grid / Symmetry type: POINT
Atomic model building
Space: REAL
Refinement step
Cycle: LAST
Protein
Nucleic acid
Ligand
Solvent
Total
Num. atoms
1422
0
0
0
1422
+
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