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- EMDB-5511: CryoEM Visualization of an Adenovirus Capsid-Incorporated HIV Antigen -

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Basic information

Entry
Database: EMDB / ID: EMD-5511
TitleCryoEM Visualization of an Adenovirus Capsid-Incorporated HIV Antigen
Map data3D reconstruction of an adenovirus vector engineered for a vaccine application
Sample
  • Sample: Ad vector with a capsid-incorporated HIV epitope based on the membrane proximal ectodomain region (MPER) of gp41
  • Virus: Human adenovirus 5
KeywordscryoEM / viral vaccine vector / adenovirus / HIV / gp41 / MPER
Biological speciesHuman adenovirus 5
Methodsingle particle reconstruction / cryo EM / Resolution: 8.7 Å
AuthorsFlatt JW / Fox TL / Makarova N / Blackwell JL / Dmitriev IP / Kashentseva EA / Curiel DT / Stewart PL
CitationJournal: PLoS One / Year: 2012
Title: CryoEM visualization of an adenovirus capsid-incorporated HIV antigen.
Authors: Justin W Flatt / Tara L Fox / Natalia Makarova / Jerry L Blackwell / Igor P Dmitriev / Elena A Kashentseva / David T Curiel / Phoebe L Stewart /
Abstract: Adenoviral (Ad) vectors show promise as platforms for vaccine applications against infectious diseases including HIV. However, the requirements for eliciting protective neutralizing antibody and ...Adenoviral (Ad) vectors show promise as platforms for vaccine applications against infectious diseases including HIV. However, the requirements for eliciting protective neutralizing antibody and cellular immune responses against HIV remain a major challenge. In a novel approach to generate 2F5- and 4E10-like antibodies, we engineered an Ad vector with the HIV membrane proximal ectodomain region (MPER) epitope displayed on the hypervariable region 2 (HVR2) of the viral hexon capsid, instead of expressed as a transgene. The structure and flexibility of MPER epitopes, and the structural context of these epitopes within viral vectors, play important roles in the induced host immune responses. In this regard, understanding the critical factors for epitope presentation would facilitate optimization strategies for developing viral vaccine vectors. Therefore we undertook a cryoEM structural study of this Ad vector, which was previously shown to elicit MPER-specific humoral immune responses. A subnanometer resolution cryoEM structure was analyzed with guided molecular dynamics simulations. Due to the arrangement of hexons within the Ad capsid, there are twelve unique environments for the inserted peptide that lead to a variety of conformations for MPER, including individual α-helices, interacting α-helices, and partially extended forms. This finding is consistent with the known conformational flexibility of MPER. The presence of an extended form, or an induced extended form, is supported by interaction of this vector with the human HIV monoclonal antibody 2F5, which recognizes 14 extended amino acids within MPER. These results demonstrate that the Ad capsid influences epitope structure, flexibility and accessibility, all of which affect the host immune response. In summary, this cryoEM structural study provided a means to visualize an epitope presented on an engineered viral vector and suggested modifications for the next generation of Ad vectors with capsid-incorporated HIV epitopes.
History
DepositionOct 3, 2012-
Header (metadata) releaseOct 24, 2012-
Map releaseNov 28, 2012-
UpdateNov 28, 2012-
Current statusNov 28, 2012Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.044
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.044
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5511.jpg

Downloads & links

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Map

FileDownload / File: emd_5511.map.gz / Format: CCP4 / Size: 976.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of an adenovirus vector engineered for a vaccine application
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.25 Å/pix.
x 640 pix.
= 1440. Å		2.25 Å/pix.
x 640 pix.
= 1440. Å		2.25 Å/pix.
x 640 pix.
= 1440. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.25 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.044 / Movie #1: 0.044
Minimum - Maximum-0.25064996 - 0.38825402
Average (Standard dev.)-0.06938177 (±0.05499396)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-320-320-320
Dimensions640640640
Spacing640640640
CellA=B=C: 1440.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.252.252.25
M x/y/z640640640
origin x/y/z0.0000.0000.000
length x/y/z1440.0001440.0001440.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-5029166
NX/NY/NZ106122134
MAP C/R/S123
start NC/NR/NS-320-320-320
NC/NR/NS640640640
D min/max/mean-0.2510.388-0.069

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Supplemental data

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Sample components

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Entire : Ad vector with a capsid-incorporated HIV epitope based on the mem...

EntireName: Ad vector with a capsid-incorporated HIV epitope based on the membrane proximal ectodomain region (MPER) of gp41
Components
  • Sample: Ad vector with a capsid-incorporated HIV epitope based on the membrane proximal ectodomain region (MPER) of gp41
  • Virus: Human adenovirus 5

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Supramolecule #1000: Ad vector with a capsid-incorporated HIV epitope based on the mem...

SupramoleculeName: Ad vector with a capsid-incorporated HIV epitope based on the membrane proximal ectodomain region (MPER) of gp41
type: sample / ID: 1000 / Number unique components: 1
Molecular weightTheoretical: 150 MDa

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Supramolecule #1: Human adenovirus 5

SupramoleculeName: Human adenovirus 5 / type: virus / ID: 1
Details: recombinant type 5 adenovirus vector, Ad-HVR2-GP41-L15, generated with a 24 amino acid MPER insertion within hypervariable region 2 (HVR2) of the hexon capsid protein
NCBI-ID: 28285 / Sci species name: Human adenovirus 5 / Database: NCBI / Virus type: VIRION / Virus isolate: OTHER / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Homo sapiens (human) / synonym: VERTEBRATES
Molecular weightTheoretical: 150 MDa
Virus shellShell ID: 1 / Name: Capsid / Diameter: 1170 Å / T number (triangulation number): 25

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.2 mg/mL
BufferpH: 7.6 / Details: 50 mM Tris, 150 mM NaCl, 2 mM CaCl2, 2 mM MgCl2
GridDetails: Quantifoil R2/4 holey carbon grids, glow discharged
VitrificationCryogen name: ETHANE / Chamber humidity: 30 % / Chamber temperature: 90 K / Instrument: HOMEMADE PLUNGER / Method: Blot for 4 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F30
TemperatureAverage: 90 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 300,000 times magnification
Legacy - Electron beam tilt params: 0
DateJun 1, 2011
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 5025 / Average electron dose: 20 e/Å2 / Bits/pixel: 16
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 400000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.26 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 310000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

DetailsThe particles were selected with in-house script and processed using Frealign
CTF correctionDetails: Each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.7 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Frealign / Number images used: 1306

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