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Yorodumi- EMDB-2638: Cryo-electron microscopy of tubular arrays of HIV-1 Gag resolves ... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-2638 | |||||||||
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Title | Cryo-electron microscopy of tubular arrays of HIV-1 Gag resolves structures essential for immature virus assembly. | |||||||||
Map data | Reconstruction of HIV-1 CANC immature-like tubular particles | |||||||||
Sample |
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Keywords | HIV-1 / capsid / SP1 / helical reconstruction | |||||||||
Function / homology | gag protein p24 N-terminal domain / viral process / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / Retrovirus capsid, C-terminal / viral capsid / Retrovirus capsid, N-terminal / Gag protein Function and homology information | |||||||||
Biological species | Human immunodeficiency virus 1 | |||||||||
Method | helical reconstruction / cryo EM / Resolution: 9.4 Å | |||||||||
Authors | Bharat TAM / Castillo-Menendez LR / Hagen WH / Lux V / Igonet S / Schorb M / Schur FKM / Krauesslich HG / Briggs JAG | |||||||||
Citation | Journal: Proc Natl Acad Sci U S A / Year: 2014 Title: Cryo-electron microscopy of tubular arrays of HIV-1 Gag resolves structures essential for immature virus assembly. Authors: Tanmay A M Bharat / Luis R Castillo Menendez / Wim J H Hagen / Vanda Lux / Sebastien Igonet / Martin Schorb / Florian K M Schur / Hans-Georg Kräusslich / John A G Briggs / Abstract: The assembly of HIV-1 is mediated by oligomerization of the major structural polyprotein, Gag, into a hexameric protein lattice at the plasma membrane of the infected cell. This leads to budding and ...The assembly of HIV-1 is mediated by oligomerization of the major structural polyprotein, Gag, into a hexameric protein lattice at the plasma membrane of the infected cell. This leads to budding and release of progeny immature virus particles. Subsequent proteolytic cleavage of Gag triggers rearrangement of the particles to form mature infectious virions. Obtaining a structural model of the assembled lattice of Gag within immature virus particles is necessary to understand the interactions that mediate assembly of HIV-1 particles in the infected cell, and to describe the substrate that is subsequently cleaved by the viral protease. An 8-Å resolution structure of an immature virus-like tubular array assembled from a Gag-derived protein of the related retrovirus Mason-Pfizer monkey virus (M-PMV) has previously been reported, and a model for the arrangement of the HIV-1 capsid (CA) domains has been generated based on homology to this structure. Here we have assembled tubular arrays of a HIV-1 Gag-derived protein with an immature-like arrangement of the C-terminal CA domains and have solved their structure by using hybrid cryo-EM and tomography analysis. The structure reveals the arrangement of the C-terminal domain of CA within an immature-like HIV-1 Gag lattice, and provides, to our knowledge, the first high-resolution view of the region immediately downstream of CA, which is essential for assembly, and is significantly different from the respective region in M-PMV. Our results reveal a hollow column of density for this region in HIV-1 that is compatible with the presence of a six-helix bundle at this position. | |||||||||
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_2638.map.gz | 3.5 MB | EMDB map data format | |
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Header (meta data) | emd-2638-v30.xml emd-2638.xml | 12.7 KB 12.7 KB | Display Display | EMDB header |
Images | EMD-2638-image_for_EMDB1_500x500.jpg | 77.7 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-2638 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2638 | HTTPS FTP |
-Validation report
Summary document | emd_2638_validation.pdf.gz | 301.1 KB | Display | EMDB validaton report |
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Full document | emd_2638_full_validation.pdf.gz | 300.2 KB | Display | |
Data in XML | emd_2638_validation.xml.gz | 5.6 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2638 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2638 | HTTPS FTP |
-Related structure data
Related structure data | 4d1kMC 4cocC 4copC 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_2638.map.gz / Format: CCP4 / Size: 3.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of HIV-1 CANC immature-like tubular particles | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.53 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 : HIV-1 CANC Y169L/S protein assembled with 73mer DNA into tubular ...
Entire | Name: HIV-1 CANC Y169L/S protein assembled with 73mer DNA into tubular particles |
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Components |
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-Supramolecule #1000: HIV-1 CANC Y169L/S protein assembled with 73mer DNA into tubular ...
Supramolecule | Name: HIV-1 CANC Y169L/S protein assembled with 73mer DNA into tubular particles type: sample / ID: 1000 / Details: Helical tubular crystals / Oligomeric state: helical / Number unique components: 1 |
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-Macromolecule #1: Human immunodeficiency virus 1 Gag
Macromolecule | Name: Human immunodeficiency virus 1 Gag / type: protein_or_peptide / ID: 1 / Name.synonym: HIV-1 Gag Details: 10nm protein-A conjugated gold particles were added to the sample. Protein construct consists of CA to NC domains with mutation in position Y169. Oligomeric state: Helical / Recombinant expression: Yes |
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Source (natural) | Organism: Human immunodeficiency virus 1 / synonym: HIV-1 |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | UniProtKB: Gag protein |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | helical reconstruction |
Aggregation state | helical array |
-Sample preparation
Concentration | 2 mg/mL |
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Buffer | pH: 6 / Details: 30 mM MES, 1 mM EDTA, 1 mM DTT, 0.5 M NaCl |
Grid | Details: 300 mesh C-Flat copper grids were glow discharged for 20 seconds |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: HOMEMADE PLUNGER |
Details | Protein stock solutions were dialyzed for 2 h at 4 degree Celsius against the assembly buffer (50 mM Tris HCl pH 7.5) in the presence of nucleic acid |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Date | Dec 25, 2012 |
Image recording | Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Number real images: 82 / Average electron dose: 23 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: -0.0045 µm / Nominal defocus min: -0.001 µm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Details | Helical reconstruction was carried out as described in Sachse et al, J. Mol. Biol. (2007), based on symmetries determined by sub-tomogram averaging as described in Bharat et al Nature (2012). Averaging of the asymmetric unit was carried out in 3D using methods described in Briggs et al PNAS (2009). |
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Final reconstruction | Applied symmetry - Helical parameters - Δz: 1.92 Å Applied symmetry - Helical parameters - Δ&Phi: 11.07 ° Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric) Resolution.type: BY AUTHOR / Resolution: 9.4 Å / Resolution method: OTHER / Software - Name: Spider, AV3 Details: Resolution is calculated using the independently aligned and averaged data sets (gold standard technique) |
-Atomic model buiding 1
Initial model | PDB ID: |
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Software | Name: Chimera, MDFF |
Details | For generating flexible fits of HIV-1 CA, six copies each of the PDB files corresponding to the CA-NTD (PDB 2JPR), and CA-CTD (CA-CTD Y169S, PDB 4COP) were rigid-body fitted into the cryoEM map using UCSF Chimera). The rigid-body fitted structures of the CA-NTD and CA-CTD that corresponded to the same Gag molecule were joined together manually in Coot. This rigid body fit of all proteins was refined using the molecular dynamics flexible fitting (MDFF) package. Secondary structure elements in the input PDB file were constrained during the simulation. The simulation was conducted in an explicit solvent model with periodic boundary conditions. |
Refinement | Space: REAL / Protocol: FLEXIBLE FIT |
Output model | PDB-4d1k: |
-Atomic model buiding 2
Initial model | PDB ID: |
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Software | Name: Chimera, MDFF |
Details | For generating flexible fits of HIV-1 CA, six copies each of the PDB files corresponding to the CA-NTD (PDB 2JPR), and CA-CTD (CA-CTD Y169S, PDB 4COP) were rigid-body fitted into the cryoEM map using UCSF Chimera). The rigid-body fitted structures of the CA-NTD and CA-CTD that corresponded to the same Gag molecule were joined together manually in Coot. This rigid body fit of all proteins was refined using the molecular dynamics flexible fitting (MDFF) package. Secondary structure elements in the input PDB file were constrained during the simulation. The simulation was conducted in an explicit solvent model with periodic boundary conditions. |
Refinement | Space: REAL / Protocol: FLEXIBLE FIT |
Output model | PDB-4d1k: |