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- PDB-6nxe: Cryo-EM Reconstruction of Protease-Activateable Adeno-Associated ... -

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Basic information

Entry
Database: PDB / ID: 6nxe
TitleCryo-EM Reconstruction of Protease-Activateable Adeno-Associated Virus 9 (AAV9-L001)
ComponentsCapsid protein VP1
KeywordsVIRUS / AAV / activatable / gene therapy / provector
Function / homologyParvovirus coat protein VP2 / Parvovirus coat protein VP1, N-terminal / Capsid/spike protein, ssDNA virus / Parvovirus coat protein VP1/VP2 / Parvovirus coat protein VP2 / Parvovirus coat protein VP1 / viral capsid / structural molecule activity / Capsid protein VP1
Function and homology information
Specimen sourceAdeno-associated virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.16 Å resolution
AuthorsBennett, A.B. / Agbandje-Mckenna, M.
Funding supportUnited States , 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research InstituteR21HL126053, R01HL138126, and R01CA207497United States
CitationJournal: Mol. Ther. / Year: 2019
Title: Protease-Activatable Adeno-Associated Virus Vector for Gene Delivery to Damaged Heart Tissue.
Authors: Caitlin M Guenther / Mitchell J Brun / Antonette D Bennett / Michelle L Ho / Weitong Chen / Banghe Zhu / Michael Lam / Momona Yamagami / Sunkuk Kwon / Nilakshee Bhattacharya / Duncan Sousa / Annicka C Evans / Julie Voss / Eva M Sevick-Muraca / Mavis Agbandje-McKenna / Junghae Suh
Abstract: Adeno-associated virus (AAV) has emerged as a promising gene delivery vector because of its non-pathogenicity, simple structure and genome, and low immunogenicity compared to other viruses. However, ...Adeno-associated virus (AAV) has emerged as a promising gene delivery vector because of its non-pathogenicity, simple structure and genome, and low immunogenicity compared to other viruses. However, its adoption as a safe and effective delivery vector for certain diseases relies on altering its tropism to deliver transgenes to desired cell populations. To this end, we have developed a protease-activatable AAV vector, named provector, that responds to elevated extracellular protease activity commonly found in diseased tissue microenvironments. The AAV9-based provector is initially inactive, but then it can be switched on by matrix metalloproteinases (MMP)-2 and -9. Cryo-electron microscopy and image reconstruction reveal that the provector capsid is structurally similar to that of AAV9, with a flexible peptide insertion at the top of the 3-fold protrusions. In an in vivo model of myocardial infarction (MI), the provector is able to deliver transgenes site specifically to high-MMP-activity regions of the damaged heart, with concomitant decreased delivery to many off-target organs, including the liver. The AAV provector may be useful in the future for enhanced delivery of transgenes to sites of cardiac damage.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Feb 8, 2019 / Release: Mar 13, 2019
RevisionDateData content typeGroupCategoryItemProviderType
1.0Mar 13, 2019Structure modelrepositoryInitial release
1.1Mar 20, 2019Structure modelData collection / Database referencescitation / em_image_scans_citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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Assembly

Deposited unit
A: Capsid protein VP1
B: Capsid protein VP1
C: Capsid protein VP1
D: Capsid protein VP1
E: Capsid protein VP1
F: Capsid protein VP1
G: Capsid protein VP1
H: Capsid protein VP1
I: Capsid protein VP1
J: Capsid protein VP1
K: Capsid protein VP1
L: Capsid protein VP1
M: Capsid protein VP1
N: Capsid protein VP1
O: Capsid protein VP1
P: Capsid protein VP1
Q: Capsid protein VP1
R: Capsid protein VP1
S: Capsid protein VP1
T: Capsid protein VP1
U: Capsid protein VP1
V: Capsid protein VP1
W: Capsid protein VP1
X: Capsid protein VP1
Y: Capsid protein VP1
Z: Capsid protein VP1
a: Capsid protein VP1
b: Capsid protein VP1
c: Capsid protein VP1
d: Capsid protein VP1
e: Capsid protein VP1
f: Capsid protein VP1
g: Capsid protein VP1
h: Capsid protein VP1
i: Capsid protein VP1
j: Capsid protein VP1
k: Capsid protein VP1
l: Capsid protein VP1
m: Capsid protein VP1
n: Capsid protein VP1
o: Capsid protein VP1
p: Capsid protein VP1
q: Capsid protein VP1
r: Capsid protein VP1
s: Capsid protein VP1
t: Capsid protein VP1
u: Capsid protein VP1
v: Capsid protein VP1
w: Capsid protein VP1
x: Capsid protein VP1
y: Capsid protein VP1
z: Capsid protein VP1
1: Capsid protein VP1
2: Capsid protein VP1
3: Capsid protein VP1
4: Capsid protein VP1
5: Capsid protein VP1
6: Capsid protein VP1
7: Capsid protein VP1
8: Capsid protein VP1


Theoretical massNumber of molelcules
Total (without water)3,656,35060
Polyers3,656,35060
Non-polymers00
Water0
1


  • idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)958840
ΔGint (kcal/M)-4243
Surface area (Å2)957480

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Components

#1: Protein/peptide ...
Capsid protein VP1 /


Mass: 60939.168 Da / Num. of mol.: 60 / Fragment: UNP residues 219-736 / Source: (gene. exp.) Adeno-associated virus / Gene: cap / Variant: Serotype9-L001 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q6JC22

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Adeno-associated virus / Type: VIRUS / Entity ID: 1 / Source: RECOMBINANT
Source (natural)Organism: Adeno-associated virus / Strain: 9-L001
Source (recombinant)Organism: Homo sapiens (human)
Details of virusEmpty: NO / Enveloped: NO / Virus isolate: SEROTYPE / Virus type: VIRION
Virus shellDiameter: 260 nm / Triangulation number (T number): 1
Buffer solutionpH: 7.4
Buffer componentName: TD Buffer / Formula: PBS-MK
SpecimenConc.: 0.5 mg/ml / Details: The sample was monodisperse. / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 20 e/Å2 / Film or detector model: DIRECT ELECTRON DE-20 (5k x 3k)

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Processing

EM software
IDNameCategory
4Auto3DEMCTF correction
7Cootmodel fitting
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.16 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 114044 / Symmetry type: POINT
Atomic model buildingRef space: REAL
Atomic model buildingPDB-ID: 3UX1
Pdb chain ID: A

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