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Yorodumi- EMDB-5937: Electron cryo-microscopy of the Moloney murine leukemia virus fur... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-5937 | |||||||||
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Title | Electron cryo-microscopy of the Moloney murine leukemia virus furin precursor Env in its isomerization arrested state (IAS) form | |||||||||
Map data | Reconstruction of the furin precursor of the Moloney murine leukemia virus at isomerization arrested intermediate stage (IAS) | |||||||||
Sample |
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Keywords | Furin precursor / Moloney murine leukemia virus / Env maturation / intermediate form | |||||||||
Biological species | Moloney murine leukemia virus | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 23.0 Å | |||||||||
Authors | Sjoberg M / Wu SR / Loving R / Rantalainen K / Lindqvist B / Garoff H | |||||||||
Citation | Journal: Proc Natl Acad Sci U S A / Year: 2014 Title: Furin cleavage of the Moloney murine leukemia virus Env precursor reorganizes the spike structure. Authors: Mathilda Sjöberg / Shang-Rung Wu / Robin Löving / Kimmo Rantalainen / Birgitta Lindqvist / Henrik Garoff / Abstract: The trimeric Moloney murine leukemia virus Env protein matures by two proteolytic cleavages. First, furin cleaves the Env precursor into the surface (SU) and transmembrane (TM) subunits in the cell ...The trimeric Moloney murine leukemia virus Env protein matures by two proteolytic cleavages. First, furin cleaves the Env precursor into the surface (SU) and transmembrane (TM) subunits in the cell and then the viral protease cleaves the R-peptide from TM in new virus. Here we analyzed the structure of the furin precursor, by cryoelectron microscopy. We transfected 293T cells with a furin cleavage site provirus mutant, R466G/K468G, and produced the virus in the presence of amprenavir to also inhibit the R-peptide cleavage. Although Env incorporation into particles was inhibited, enough precursor could be isolated and analyzed by cryoelectron microscopy to yield a 3D structure at 22 Å resolution. This showed an open cage-like structure like that of the R-peptide precursor and the mature Env described before. However, the middle protrusion of the protomeric unit, so prominently pointing out from the side of the more mature forms of the Env, was absent. Instead, there was extra density in the top protrusion. This suggested that the C-terminal SU domain was associated alongside the receptor binding N-terminal SU domain in the furin precursor. This was supported by mapping with a SU C-terminal domain-specific antigen binding fragment. We concluded that furin cleavage not only separates the subunits and liberates the fusion peptide at the end of TM but also allows the C-terminal domain to relocate into a peripheral position. This conformational change might explain how the C-terminal domain of SU gains the potential to undergo disulfide isomerization, an event that facilitates membrane fusion. | |||||||||
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_5937.map.gz | 329.9 KB | EMDB map data format | |
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Header (meta data) | emd-5937-v30.xml emd-5937.xml | 11.7 KB 11.7 KB | Display Display | EMDB header |
Images | 400_5937.gif 80_5937.gif | 22.2 KB 2.5 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-5937 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-5937 | HTTPS FTP |
-Validation report
Summary document | emd_5937_validation.pdf.gz | 79.3 KB | Display | EMDB validaton report |
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Full document | emd_5937_full_validation.pdf.gz | 78.4 KB | Display | |
Data in XML | emd_5937_validation.xml.gz | 495 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5937 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5937 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_5937.map.gz / Format: CCP4 / Size: 422.9 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of the furin precursor of the Moloney murine leukemia virus at isomerization arrested intermediate stage (IAS) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 3.5 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 : Isomerization arrested stage (IAS) of the furin cleavage deficien...
Entire | Name: Isomerization arrested stage (IAS) of the furin cleavage deficient mutant (R466G/K468G) Env of Moloney murine leukemia virus |
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Components |
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-Supramolecule #1000: Isomerization arrested stage (IAS) of the furin cleavage deficien...
Supramolecule | Name: Isomerization arrested stage (IAS) of the furin cleavage deficient mutant (R466G/K468G) Env of Moloney murine leukemia virus type: sample / ID: 1000 Details: Affinity purified, gradient separated protein in 0.05% Triton X-100 Oligomeric state: trimer / Number unique components: 1 |
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Molecular weight | Experimental: 500 KDa / Theoretical: 270 KDa / Method: Estimated from Blue native PAGE |
-Macromolecule #1: gp90
Macromolecule | Name: gp90 / type: protein_or_peptide / ID: 1 / Name.synonym: Furin precursor / Number of copies: 3 / Oligomeric state: Trimer / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: Moloney murine leukemia virus |
Molecular weight | Experimental: 500 KDa / Theoretical: 270 KDa |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.1 mg/mL |
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Buffer | pH: 7.4 / Details: 50 mM HEPES, 100 mM NaCl, 1.8 mM CaCl2, pH 7.4 |
Grid | Details: 400 mesh holey carbon grid, glow discharged |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 77 K / Instrument: FEI VITROBOT MARK II Method: Blotted for 3 seconds before plunging in liquid ethane, followed by transfer into liquid nitrogen. |
-Electron microscopy
Microscope | JEOL 2100F |
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Temperature | Min: 93 K / Max: 96 K / Average: 95 K |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected using online FFT. |
Date | Jan 25, 2013 |
Image recording | Category: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Sampling interval: 3.5 µm / Number real images: 456 / Average electron dose: 9 e/Å2 / Bits/pixel: 14 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 43200 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.5 µm / Nominal magnification: 43200 |
Sample stage | Specimen holder model: GATAN LIQUID NITROGEN |
+Image processing
-Atomic model buiding 1
Initial model | PDB ID: |
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Software | Name: Chimera |
Details | The atomic model for the Mo-RBD was obtained using the atomic structure of the highly homologous F-RBD (Fass et al, 1997) (PDB ID 1AOL) and the SWISS-MODEL protein structure homology-modeling server (accessible through the ExPASy web server). |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |