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- EMDB-36311: Cryo-EM Structure of 3-axis block of AAV9P31-Car4 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-36311
TitleCryo-EM Structure of 3-axis block of AAV9P31-Car4 complex
Map data
Sample
  • Complex: 3-fold axes block of AAV9P31 and Car4 complex aat 2.28 angstrom
    • Protein or peptide: Carbonic anhydrase 4
    • Protein or peptide: Capsid protein VP1
  • Ligand: ZINC ION
Keywordsreceptor / aav9p31 / Carbonic anhydrases iv / Block-based reconstruction / VIRUS / VIRAL PROTEIN-LYASE complex
Function / homology
Function and homology information


Erythrocytes take up carbon dioxide and release oxygen / Erythrocytes take up oxygen and release carbon dioxide / Reversible hydration of carbon dioxide / regulation of pH / bicarbonate transport / T=1 icosahedral viral capsid / transport vesicle membrane / endoplasmic reticulum-Golgi intermediate compartment / rough endoplasmic reticulum / secretory granule membrane ...Erythrocytes take up carbon dioxide and release oxygen / Erythrocytes take up oxygen and release carbon dioxide / Reversible hydration of carbon dioxide / regulation of pH / bicarbonate transport / T=1 icosahedral viral capsid / transport vesicle membrane / endoplasmic reticulum-Golgi intermediate compartment / rough endoplasmic reticulum / secretory granule membrane / carbonic anhydrase / carbonate dehydratase activity / carbon dioxide transport / trans-Golgi network / brush border membrane / apical plasma membrane / external side of plasma membrane / perinuclear region of cytoplasm / structural molecule activity / extracellular exosome / zinc ion binding / membrane / plasma membrane
Similarity search - Function
Carbonic anhydrase, CA4/CA15 / 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 / Carbonic anhydrase, alpha-class, conserved site / Alpha-carbonic anhydrases signature. / Carbonic anhydrase, alpha-class ...Carbonic anhydrase, CA4/CA15 / 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 / Carbonic anhydrase, alpha-class, conserved site / Alpha-carbonic anhydrases signature. / Carbonic anhydrase, alpha-class / Alpha carbonic anhydrase domain / Alpha carbonic anhydrase domain superfamily / Eukaryotic-type carbonic anhydrase / Alpha-carbonic anhydrases profile. / Eukaryotic-type carbonic anhydrase
Similarity search - Domain/homology
Carbonic anhydrase 4 / Capsid protein VP1
Similarity search - Component
Biological speciesMus musculus (house mouse) / Adeno-associated virus 9
Methodsingle particle reconstruction / cryo EM / Resolution: 2.28 Å
AuthorsZhang R / Liu Y / Lou Z
Funding support China, 3 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31971126 China
National Natural Science Foundation of China (NSFC)U20A20135 China
National Natural Science Foundation of China (NSFC)32301011 China
CitationJournal: PLoS Pathog / Year: 2024
Title: Structural basis of the recognition of adeno-associated virus by the neurological system-related receptor carbonic anhydrase IV.
Authors: Ran Zhang / Yixiao Liu / Fengxi Yu / Guangxue Xu / Lili Li / Baobin Li / Zhiyong Lou /
Abstract: Carbonic anhydrase IV (Car4) is a newly identified receptor that allows adeno-associated virus (AAV) 9P31 to cross the blood-brain barrier and achieve efficient infection in the central nervous ...Carbonic anhydrase IV (Car4) is a newly identified receptor that allows adeno-associated virus (AAV) 9P31 to cross the blood-brain barrier and achieve efficient infection in the central nervous system (CNS) in mouse models. However, the molecular mechanism by which engineered AAV capsids with 7-mer insertion in the variable region (VR) VIII recognize these novel cellular receptors is unknown. Here we report the cryo-EM structures of AAV9P31 and its complex with Mus musculus Car4 at atomic resolution by utilizing the block-based reconstruction (BBR) method. The structures demonstrated that Car4 binds to the protrusions at 3-fold axes of the capsid. The inserted 7-mer extends into a hydrophobic region near the catalytic center of Car4 to form stable interactions. Mutagenesis studies also identified the key residues in Car4 responsible for the AAV9P31 interaction. These findings provide new insights into the novel receptor recognition mechanism of AAV generated by directed evolution and highlight the application of the BBR method to studying the virus-receptor molecular mechanism.
History
DepositionMay 26, 2023-
Header (metadata) releaseJan 31, 2024-
Map releaseJan 31, 2024-
UpdateNov 20, 2024-
Current statusNov 20, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_36311.png

Downloads & links

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Map

FileDownload / File: emd_36311.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 160 pix.
= 134.928 Å		0.84 Å/pix.
x 160 pix.
= 134.928 Å		0.84 Å/pix.
x 160 pix.
= 134.928 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8433 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.01
Minimum - Maximum-0.01405853 - 0.03826549
Average (Standard dev.)-0.00016429197 (±0.0033116597)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 134.928 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_36311_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_36311_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : 3-fold axes block of AAV9P31 and Car4 complex aat 2.28 angstrom

EntireName: 3-fold axes block of AAV9P31 and Car4 complex aat 2.28 angstrom
Components
  • Complex: 3-fold axes block of AAV9P31 and Car4 complex aat 2.28 angstrom
    • Protein or peptide: Carbonic anhydrase 4
    • Protein or peptide: Capsid protein VP1
  • Ligand: ZINC ION

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Supramolecule #1: 3-fold axes block of AAV9P31 and Car4 complex aat 2.28 angstrom

SupramoleculeName: 3-fold axes block of AAV9P31 and Car4 complex aat 2.28 angstrom
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Carbonic anhydrase 4

MacromoleculeName: Carbonic anhydrase 4 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: carbonic anhydrase
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 29.281367 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: WCYEIQTKDP RSSCLGPEKW PGACKENQQS PINIVTARTK VNPRLTPFIL VGYDQKQQWP IKNNQHTVEM TLGGGACIIG GDLPARYEA VQLHLHWSNG NDNGSEHSID GRHFAMEMHI VHKKLTSSKE DSKDKFAVLA FMIEVGDKVN KGFQPLVEAL P SISKPHST ...String:
WCYEIQTKDP RSSCLGPEKW PGACKENQQS PINIVTARTK VNPRLTPFIL VGYDQKQQWP IKNNQHTVEM TLGGGACIIG GDLPARYEA VQLHLHWSNG NDNGSEHSID GRHFAMEMHI VHKKLTSSKE DSKDKFAVLA FMIEVGDKVN KGFQPLVEAL P SISKPHST STVRESSLQD MLPPSTKMYT YFRYNGSLTT PNCDETVIWT VYKQPIKIHK NQFLEFSKNL YYDEDQKLNM KD NVRPLQP LGKRQVFKS

UniProtKB: Carbonic anhydrase 4

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Macromolecule #2: Capsid protein VP1

MacromoleculeName: Capsid protein VP1 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Adeno-associated virus 9
Molecular weightTheoretical: 59.295277 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DGVGSSSGNW HCDSQWLGDR VITTSTRTWA LPTYNNHLYK QISNSTSGGS SNDNAYFGYS TPWGYFDFNR FHCHFSPRDW QRLINNNWG FRPKRLNFKL FNIQVKEVTD NNGVKTIANN LTSTVQVFTD SDYQLPYVLG SAHEGCLPPF PADVFMIPQY G YLTLNDGS ...String:
DGVGSSSGNW HCDSQWLGDR VITTSTRTWA LPTYNNHLYK QISNSTSGGS SNDNAYFGYS TPWGYFDFNR FHCHFSPRDW QRLINNNWG FRPKRLNFKL FNIQVKEVTD NNGVKTIANN LTSTVQVFTD SDYQLPYVLG SAHEGCLPPF PADVFMIPQY G YLTLNDGS QAVGRSSFYC LEYFPSQMLR TGNNFQFSYE FENVPFHSSY AHSQSLDRLM NPLIDQYLYY LSKTINGSGQ NQ QTLKFSV AGPSNMAVQG RNYIPGPSYR QQRVSTTVTQ NNNSEFAWPG ASSWALNGRN SLMNPGPAMA SHKEGEDRFF PLS GSLIFG KQGTGRDNVD ADKVMITNEE EIKTTNPVAT ESYGQVATNH QSAQWPTSYD AAQAQTGWVQ NQGILPGMVW QDRD VYLQG PIWAKIPHTD GNFHPSPLMG GFGMKHPPPQ ILIKNTPVPA DPPTAFNKDK LNSFITQYST GQVSVEIEWE LQKEN SKRW NPEIQYTSNY YKSNNVEFAV NTEGVYSEPR PIGTRYLTRN L

UniProtKB: Capsid protein VP1

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Macromolecule #3: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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

Concentration2 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMTris-ClTris-Cl
150.0 mMNaClNaCl

Details: 20mM Tris-Cl, pH8.0; 150mM NaCl.
GridModel: Quantifoil R0.6/1 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 280 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.6 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

2znc


Details: and PDB: 7WJW as startup model
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.28 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 13604676
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationNumber classes: 16 / Avg.num./class: 100000

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