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- EMDB-26189: Third class of AAV5 bound with PKD1-2 revealed by classification ... -

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Basic information

Entry
Database: EMDB / ID: EMD-26189
TitleThird class of AAV5 bound with PKD1-2 revealed by classification of aligned subtomograms
Map dataThird class of AAV5 bound with PKD1-2 revealed by classification of aligned subtomgrams
Sample
  • Complex: Binary complex of AAV-5 with a two domain fragment of its cellular receptor, AAVR
    • Complex: AAV-5
    • Complex: AAVR
Biological speciesadeno-associated virus 5 / Homo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 25.0 Å
AuthorsHu GQ / Silveria MA / Chapman MS / Stagg SM
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM108753 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM122564 United States
CitationJournal: J Virol / Year: 2022
Title: Adeno-Associated Virus Receptor-Binding: Flexible Domains and Alternative Conformations through Cryo-Electron Tomography of Adeno-Associated Virus 2 (AAV2) and AAV5 Complexes.
Authors: Guiqing Hu / Mark A Silveria / Grant M Zane / Michael S Chapman / Scott M Stagg /
Abstract: Recombinant forms of adeno-associated virus (rAAV) are vectors of choice in the development of treatments for a number of genetic dispositions. Greater understanding of AAV's molecular virology is ...Recombinant forms of adeno-associated virus (rAAV) are vectors of choice in the development of treatments for a number of genetic dispositions. Greater understanding of AAV's molecular virology is needed to underpin needed improvements in efficiency and specificity. Recent advances have included identification of a near-universal entry receptor, AAVR, and structures detected by cryo-electron microscopy (EM) single particle analysis (SPA) that revealed, at high resolution, only the domains of AAVR most tightly bound to AAV. Here, cryogenic electron tomography (cryo-ET) is applied to reveal the neighboring domains of the flexible receptor. For AAV5, where the PKD1 domain is bound strongly, PKD2 is seen in three configurations extending away from the virus. AAV2 binds tightly to the PKD2 domain at a distinct site, and cryo-ET now reveals four configurations of PKD1, all different from that seen in AAV5. The AAV2 receptor complex also shows unmodeled features on the inner surface that appear to be an equilibrium alternate configuration. Other AAV structures start near the 5-fold axis, but now β-strand A is the minor conformer and, for the major conformer, partially ordered N termini near the 2-fold axis join the canonical capsid jellyroll fold at the βA-βB turn. The addition of cryo-ET is revealing unappreciated complexity that is likely relevant to viral entry and to the development of improved gene therapy vectors. With 150 clinical trials for 30 diseases under way, AAV is a leading gene therapy vector. Immunotoxicity at high doses used to overcome inefficient transduction has occasionally proven fatal and highlighted gaps in fundamental virology. AAV enters cells, interacting through distinct sites with different domains of the AAVR receptor, according to AAV clade. Single domains are resolved in structures by cryogenic electron microscopy. Here, the adjoining domains are revealed by cryo-electron tomography of AAV2 and AAV5 complexes. They are in flexible configurations interacting minimally with AAV, despite measurable dependence of AAV2 transduction on both domains.
History
DepositionFeb 14, 2022-
Header (metadata) releaseJun 1, 2022-
Map releaseJun 1, 2022-
UpdateAug 10, 2022-
Current statusAug 10, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26189.png

Downloads & links

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Map

FileDownload / File: emd_26189.map.gz / Format: CCP4 / Size: 356.4 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThird class of AAV5 bound with PKD1-2 revealed by classification of aligned subtomgrams
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
5.48 Å/pix.
x 45 pix.
= 246.6 Å		5.48 Å/pix.
x 45 pix.
= 246.6 Å		5.48 Å/pix.
x 45 pix.
= 246.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 5.48 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.16
Minimum - Maximum-0.68523735 - 0.8300359
Average (Standard dev.)0.020254653 (±0.09188395)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-22-22-22
Dimensions454545
Spacing454545
CellA=B=C: 246.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Binary complex of AAV-5 with a two domain fragment of its cellula...

EntireName: Binary complex of AAV-5 with a two domain fragment of its cellular receptor, AAVR
Components
  • Complex: Binary complex of AAV-5 with a two domain fragment of its cellular receptor, AAVR
    • Complex: AAV-5
    • Complex: AAVR

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Supramolecule #1: Binary complex of AAV-5 with a two domain fragment of its cellula...

SupramoleculeName: Binary complex of AAV-5 with a two domain fragment of its cellular receptor, AAVR
type: complex / Chimera: Yes / ID: 1 / Parent: 0
Molecular weightExperimental: 82 KDa

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Supramolecule #2: AAV-5

SupramoleculeName: AAV-5 / type: complex / Chimera: Yes / ID: 2 / Parent: 1
Source (natural)Organism: adeno-associated virus 5
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
Molecular weightTheoretical: 60 KDa

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Supramolecule #3: AAVR

SupramoleculeName: AAVR / type: complex / Chimera: Yes / ID: 3 / Parent: 1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 22 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
Component:
ConcentrationNameFormula
25.0 mMGibco HEPES
150.0 mMsodium chlorideNaCl
GridModel: PELCO Ultrathin Carbon with Lacey Carbon / Support film - Material: CARBON / Support film - topology: LACEY / Support film - Film thickness: 3.0 nm
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
Details: 4ul of AAV-2 at concentration 0.2 mg/ml was was applied on grid and incubated for 1.5 minutes. The grid was gently blotted on the side with filter paper, and another 4ul of PKD1-2 at ...Details: 4ul of AAV-2 at concentration 0.2 mg/ml was was applied on grid and incubated for 1.5 minutes. The grid was gently blotted on the side with filter paper, and another 4ul of PKD1-2 at concentration 1.5 mg/ml was applied and allowed to incubate for another 1.5 minutes followed by plunge-freeze using a Vitrobot Mark IV..
Details2 ul of 6.5 uM AAV5 VLP was added to the grid and given 2 minutes to adhere. Sample was then wicked and 2 ul of 33 uM PKD1-2 was added. Grids were then plunge frozen using an FEI Vitrobot Mark IV with a blot force of 4, time of 2 sec, temperature of 25 C, and at 100 percent humidity.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Average electron dose: 100.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 5.0 µm / Calibrated defocus min: 5.0 µm / Calibrated magnification: 33000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 5.0 µm / Nominal magnification: 33000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber classes used: 3 / Applied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: EMAN (ver. 2) / Number subtomograms used: 5100
ExtractionNumber tomograms: 1 / Number images used: 5100 / Software - Name: EMAN (ver. 2)
CTF correctionSoftware - Name: EMAN (ver. 2)
Final angle assignmentType: OTHER

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