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Yorodumi- EMDB-5681: Protein-Ligand Interactions by Electron Microscopy: a Disaccharid... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-5681 | |||||||||
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Title | Protein-Ligand Interactions by Electron Microscopy: a Disaccharide receptor analog complex of Adeno-Associated Virus | |||||||||
Map data | Reconstruction of Adeno-Associated Virus DJ | |||||||||
Sample |
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Keywords | Adeno-Associated Virus / contrast transfer function / heparin-binding domain / heparan sulfate proteoglycan / 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 / virion attachment to host cell / structural molecule activity Similarity search - Function | |||||||||
Biological species | Adeno-associated virus | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 4.8 Å | |||||||||
Authors | Xie Q / Spilman M / Meyer N / Lerch T / Stagg S / Chapman M | |||||||||
Citation | Journal: J Struct Biol / Year: 2013 Title: Electron microscopy analysis of a disaccharide analog complex reveals receptor interactions of adeno-associated virus. Authors: Qing Xie / Michael Spilman / Nancy L Meyer / Thomas F Lerch / Scott M Stagg / Michael S Chapman / Abstract: Mechanistic studies of macromolecular complexes often feature X-ray structures of complexes with bound ligands. The attachment of adeno-associated virus (AAV) to cell surface glycosaminoglycans (GAGs) ...Mechanistic studies of macromolecular complexes often feature X-ray structures of complexes with bound ligands. The attachment of adeno-associated virus (AAV) to cell surface glycosaminoglycans (GAGs) is an example that has not proven amenable to crystallography, because the binding of GAG analogs disrupts lattice contacts. The interactions of AAV with GAGs are of interest in mediating the cell specificity of AAV-based gene therapy vectors. Previous electron microscopy led to differing conclusions on the exact binding site and the existence of large ligand-induced conformational changes in the virus. Conformational changes are expected during cell entry, but it has remained unclear whether the electron microscopy provided evidence of their induction by GAG-binding. Taking advantage of automated data collection, careful processing and new methods of structure refinement, the structure of AAV-DJ complexed with sucrose octasulfate is determined by electron microscopy difference map analysis to 4.8Å resolution. At this higher resolution, individual sulfate groups are discernible, providing a stereochemical validation of map interpretation, and highlighting interactions with two surface arginines that have been implicated in genetic studies. Conformational changes induced by the SOS are modest and limited to the loop most directly interacting with the ligand. While the resolution attainable will depend on sample order and other factors, there are an increasing number of macromolecular complexes that can be studied by cryo-electron microscopy at resolutions beyond 5Å, for which the approaches used here could be used to characterize the binding of inhibitors and other small molecule effectors when crystallography is not tractable. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_5681.map.gz | 68.9 MB | EMDB map data format | |
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Header (meta data) | emd-5681-v30.xml emd-5681.xml | 11.7 KB 11.7 KB | Display Display | EMDB header |
Images | emd_5681_1.jpg | 409.2 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-5681 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-5681 | HTTPS FTP |
-Validation report
Summary document | emd_5681_validation.pdf.gz | 404 KB | Display | EMDB validaton report |
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Full document | emd_5681_full_validation.pdf.gz | 403.5 KB | Display | |
Data in XML | emd_5681_validation.xml.gz | 6.5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5681 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5681 | HTTPS FTP |
-Related structure data
Related structure data | 3j4pMC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_5681.map.gz / Format: CCP4 / Size: 75 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of Adeno-Associated Virus DJ | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.3067 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Recombinant Adeno-Associated virus DJ with Sucrose Octasulfate
Entire | Name: Recombinant Adeno-Associated virus DJ with Sucrose Octasulfate |
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Components |
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-Supramolecule #1000: Recombinant Adeno-Associated virus DJ with Sucrose Octasulfate
Supramolecule | Name: Recombinant Adeno-Associated virus DJ with Sucrose Octasulfate type: sample / ID: 1000 Oligomeric state: one virus subunit binds to one molecule of sucrose octasulfate Number unique components: 2 |
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Molecular weight | Experimental: 3.7 MDa / Theoretical: 3.7 MDa |
-Supramolecule #1: Adeno-associated virus
Supramolecule | Name: Adeno-associated virus / type: virus / ID: 1 / Name.synonym: Recombinant Adeno-Associated Virus DJ, AAVDJ / NCBI-ID: 272636 / Sci species name: Adeno-associated virus / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: SEROTYPE / Virus enveloped: No / Virus empty: Yes Syn species name: Recombinant Adeno-Associated Virus DJ, AAVDJ Sci species serotype: DJ |
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Host (natural) | Organism: Homo sapiens (human) / synonym: VERTEBRATES |
Host system | Organism: Spodoptera frugiperda (fall armyworm) / Recombinant strain: sf9 / Recombinant plasmid: pAVDJ |
Molecular weight | Experimental: 3.7 MDa / Theoretical: 3.7 MDa |
Virus shell | Shell ID: 1 / Diameter: 275 Å / T number (triangulation number): 1 |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1.5 mg/mL |
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Buffer | pH: 6.8 / Details: 10 mM Tris, 125 mM NaCl, 1 mM MgCl2 |
Grid | Details: 400 mesh carbon-coated grids (C-flat) glow-discharged for 5 seconds (Gatan Model 950) |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV Method: Both sides of the grid were blotted for 2.5 seconds. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Temperature | Average: 94 K |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 120,000 times magnification |
Date | Apr 15, 2013 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 5207 / Average electron dose: 15 e/Å2 |
Electron beam | Acceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 120000 |
Sample stage | Specimen holder: Liquid nitrogen cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Details | Particles were processed semi-automatically using Appion for picking, contrast transfer function (CTF) estimation, and stack making. Particles were selected automatically using template matching. |
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CTF correction | Details: CTF correction of each particle |
Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN / Details: Final map was amplitude corrected using embfactor. / Number images used: 45000 |
Final angle assignment | Details: Frealign convention |