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- EMDB-8100: Structure of the R432A variant of Adeno-associated virus type 2 VLP -

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Basic information

Entry
Database: EMDB / ID: EMD-8100
TitleStructure of the R432A variant of Adeno-associated virus type 2 VLP
Map dataNone
Sample
  • Virus: Adeno-associated virus - 2
    • Protein or peptide: Capsid protein VP1
KeywordsAdeno-associated virus / R432A / gene therapy / icosahedral / dependoparvovirus / VIRUS LIKE PARTICLE
Function / homology
Function and homology information


permeabilization of host organelle membrane involved in viral entry into host cell / symbiont entry into host cell via permeabilization of inner membrane / host cell nucleolus / T=1 icosahedral viral capsid / clathrin-dependent endocytosis of virus by host cell / structural molecule activity / virion attachment to host cell
Similarity search - Function
Phospholipase A2-like domain / Phospholipase A2-like domain / Parvovirus coat protein VP2 / Parvovirus coat protein VP1/VP2 / Parvovirus coat protein VP2 / Capsid/spike protein, ssDNA virus
Similarity search - Domain/homology
Biological speciesAdeno-associated virus - 2
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsDrouin LM / Lins B
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM33050 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL51811 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI1081961 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32-GM008799 United States
CitationJournal: J Virol / Year: 2016
Title: Cryo-electron Microscopy Reconstruction and Stability Studies of the Wild Type and the R432A Variant of Adeno-associated Virus Type 2 Reveal that Capsid Structural Stability Is a Major Factor in Genome Packaging.
Authors: Lauren M Drouin / Bridget Lins / Maria Janssen / Antonette Bennett / Paul Chipman / Robert McKenna / Weijun Chen / Nicholas Muzyczka / Giovanni Cardone / Timothy S Baker / Mavis Agbandje-McKenna /
Abstract: The adeno-associated viruses (AAV) are promising therapeutic gene delivery vectors and better understanding of their capsid assembly and genome packaging mechanism is needed for improved vector ...The adeno-associated viruses (AAV) are promising therapeutic gene delivery vectors and better understanding of their capsid assembly and genome packaging mechanism is needed for improved vector production. Empty AAV capsids assemble in the nucleus prior to genome packaging by virally encoded Rep proteins. To elucidate the capsid determinants of this process, structural differences between wild-type (wt) AAV2 and a packaging deficient variant, AAV2-R432A, were examined using cryo-electron microscopy and three-dimensional image reconstruction both at an ∼5.0-Å resolution (medium) and also at 3.8- and 3.7-Å resolutions (high), respectively. The high resolution structures showed that removal of the arginine side chain in AAV2-R432A eliminated hydrogen bonding interactions, resulting in altered intramolecular and intermolecular interactions propagated from under the 3-fold axis toward the 5-fold channel. Consistent with these observations, differential scanning calorimetry showed an ∼10°C decrease in thermal stability for AAV2-R432A compared to wt-AAV2. In addition, the medium resolution structures revealed differences in the juxtaposition of the less ordered, N-terminal region of their capsid proteins, VP1/2/3. A structural rearrangement in AAV2-R432A repositioned the βA strand region under the icosahedral 2-fold axis rather than antiparallel to the βB strand, eliminating many intramolecular interactions. Thus, a single amino acid substitution can significantly alter the AAV capsid integrity to the extent of reducing its stability and possibly rendering it unable to tolerate the stress of genome packaging. Furthermore, the data show that the 2-, 3-, and 5-fold regions of the capsid contributed to producing the packaging defect and highlight a tight connection between the entire capsid in maintaining packaging efficiency.
IMPORTANCE: The mechanism of AAV genome packaging is still poorly understood, particularly with respect to the capsid determinants of the required capsid-Rep interaction. Understanding this mechanism ...IMPORTANCE: The mechanism of AAV genome packaging is still poorly understood, particularly with respect to the capsid determinants of the required capsid-Rep interaction. Understanding this mechanism may aid in the improvement of AAV packaging efficiency, which is currently ∼1:10 (10%) genome packaged to empty capsid in vector preparations. This report identifies regions of the AAV capsid that play roles in genome packaging and that may be important for Rep recognition. It also demonstrates the need to maintain capsid stability for the success of this process. This information is important for efforts to improve AAV genome packaging and will also inform the engineering of AAV capsid variants for improved tropism, specific tissue targeting, and host antibody escape by defining amino acids that cannot be altered without detriment to infectious vector production.
History
DepositionMar 9, 2016-
Header (metadata) releaseJul 13, 2016-
Map releaseJul 20, 2016-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.9
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 2.9
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5ipk
  • Surface level: 2.9
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5ipk
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8100.png

Downloads & links

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Map

FileDownload / File: emd_8100.map.gz / Format: CCP4 / Size: 135.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 2.9 / Movie #1: 2.9
Minimum - Maximum-6.1753774 - 14.965730000000001
Average (Standard dev.)-0.000000001858625 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions329329329
Spacing329329329
CellA=B=C: 361.9 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z329329329
origin x/y/z0.0000.0000.000
length x/y/z361.900361.900361.900
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ329329329
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS329329329
D min/max/mean-6.17514.966-0.000

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Supplemental data

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Sample components

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Entire : Adeno-associated virus - 2

EntireName: Adeno-associated virus - 2
Components
  • Virus: Adeno-associated virus - 2
    • Protein or peptide: Capsid protein VP1

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Supramolecule #1: Adeno-associated virus - 2

SupramoleculeName: Adeno-associated virus - 2 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all / NCBI-ID: 10804 / Sci species name: Adeno-associated virus - 2 / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Capsid protein VP1

MacromoleculeName: Capsid protein VP1 / type: protein_or_peptide / ID: 1 / Number of copies: 60 / Enantiomer: LEVO
Source (natural)Organism: Adeno-associated virus - 2
Molecular weightTheoretical: 81.945234 KDa
Recombinant expressionOrganism: unidentified baculovirus
SequenceString: MAADGYLPDW LEDTLSEGIR QWWKLKPGPP PPKPAERHKD DSRGLVLPGY KYLGPFNGLD KGEPVNEADA AALEHDKAYD RQLDSGDNP YLKYNHADAE FQERLKEDTS FGGNLGRAVF QAKKRVLEPL GLVEEPVKTA PGKKRPVEHS PVEPDSSSGT G KAGQQPAR ...String:
MAADGYLPDW LEDTLSEGIR QWWKLKPGPP PPKPAERHKD DSRGLVLPGY KYLGPFNGLD KGEPVNEADA AALEHDKAYD RQLDSGDNP YLKYNHADAE FQERLKEDTS FGGNLGRAVF QAKKRVLEPL GLVEEPVKTA PGKKRPVEHS PVEPDSSSGT G KAGQQPAR KRLNFGQTGD ADSVPDPQPL GQPPAAPSGL GTNTMATGSG APMADNNEGA DGVGNSSGNW HCDSTWMGDR VI TTSTRTW ALPTYNNHLY KQISSQSGAS NDNHYFGYST PWGYFDFNRF HCHFSPRDWQ RLINNNWGFR PKRLNFKLFN IQV KEVTQN DGTTTIANNL TSTVQVFTDS EYQLPYVLGS AHQGCLPPFP ADVFMVPQYG YLTLNNGSQA VGRSSFYCLE YFPS QMLRT GNNFTFSYTF EDVPFHSSYA HSQSLDALMN PLIDQYLYYL SRTNTPSGTT TQSRLQFSQA GASDIRDQSR NWLPG PCYR QQRVSKTSAD NNNSEYSWTG ATKYHLNGRD SLVNPGPAMA SHKDDEEKFF PQSGVLIFGK QGSEKTNVDI EKVMIT DEE EIRTTNPVAT EQYGSVSTNL QRGNRQAATA DVNTQGVLPG MVWQDRDVYL QGPIWAKIPH TDGHFHPSPL MGGFGLK HP PPQILIKNTP VPANPSTTFS AAKFASFITQ YSTGQVSVEI EWELQKENSK RWNPEIQYTS NYNKSVNVDF TVDTNGVY S EPRPIGTRYL TRNL

UniProtKB: Capsid protein VP1

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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

Concentration1 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
50.0 mMHEPESHEPES
100.0 mMNaClSodium chloridesodium chloride
1.0 mMCaCl2calcium chloride
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER
Details: Grid was blotted for 5 seconds before plunge freezing..

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 56924 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.26 mm / Nominal defocus max: 3.2 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN
TemperatureMin: 90.0 K / Max: 97.0 K
Image recordingFilm or detector model: KODAK SO-163 FILM / Number grids imaged: 1 / Number real images: 49 / Average exposure time: 0.8 sec. / Average electron dose: 20.0 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 19457
Startup modelType of model: NONE
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Auto3DEM (ver. v4.05) / Number images used: 19457

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