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- EMDB-5559: Cryo-em map of one molecule of factor VIII light chain from helic... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-5559 | |||||||||
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Title | Cryo-em map of one molecule of factor VIII light chain from helically organized factor VIII light chain molecules bound to lipid nanotubes | |||||||||
![]() | Cropped volume from helical reconstruction of membrane-bound Factor vIII light chain bound to single bilayer lipid nanotubes (EMD-5540) with a Gaussian filter of 3.0 applied | |||||||||
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Function / homology | ![]() Defective F8 accelerates dissociation of the A2 domain / Defective F8 binding to the cell membrane / Defective F8 secretion / Gamma carboxylation, hypusinylation, hydroxylation, and arylsulfatase activation / Defective F8 sulfation at Y1699 / Defective F8 binding to von Willebrand factor / ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() Similarity search - Function | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | helical reconstruction / ![]() | |||||||||
![]() | Stoilova-McPhie S / Lynch GC / Ludtke S / Pettitt BM | |||||||||
![]() | ![]() Title: Domain organization of membrane-bound factor VIII. Authors: Svetla Stoilova-McPhie / Gillian C Lynch / Steven Ludtke / B Montgomery Pettitt / ![]() Abstract: Factor VIII (FVIII) is the blood coagulation protein which when defective or deficient causes for hemophilia A, a severe hereditary bleeding disorder. Activated FVIII (FVIIIa) is the cofactor to the ...Factor VIII (FVIII) is the blood coagulation protein which when defective or deficient causes for hemophilia A, a severe hereditary bleeding disorder. Activated FVIII (FVIIIa) is the cofactor to the serine protease factor IXa (FIXa) within the membrane-bound Tenase complex, responsible for amplifying its proteolytic activity more than 100,000 times, necessary for normal clot formation. FVIII is composed of two noncovalently linked peptide chains: a light chain (LC) holding the membrane interaction sites and a heavy chain (HC) holding the main FIXa interaction sites. The interplay between the light and heavy chains (HCs) in the membrane-bound state is critical for the biological efficiency of FVIII. Here, we present our cryo-electron microscopy (EM) and structure analysis studies of human FVIII-LC, when helically assembled onto negatively charged single lipid bilayer nanotubes. The resolved FVIII-LC membrane-bound structure supports aspects of our previously proposed FVIII structure from membrane-bound two-dimensional (2D) crystals, such as only the C2 domain interacts directly with the membrane. The LC is oriented differently in the FVIII membrane-bound helical and 2D crystal structures based on EM data, and the existing X-ray structures. This flexibility of the FVIII-LC domain organization in different states is discussed in the light of the FVIIIa-FIXa complex assembly and function. | |||||||||
History |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 289.1 KB | ![]() | |
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Header (meta data) | ![]() ![]() | 12.2 KB 12.2 KB | Display Display | ![]() |
Images | ![]() | 30 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 3j2sMC ![]() 5540C ![]() 3j2qC M: atomic model generated by this map C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Cropped volume from helical reconstruction of membrane-bound Factor vIII light chain bound to single bilayer lipid nanotubes (EMD-5540) with a Gaussian filter of 3.0 applied | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.9 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Cropped volume corresponding to 2x1 factor VIII light chain molec...
Entire | Name: Cropped volume corresponding to 2x1 factor VIII light chain molecules from EMD-5540 |
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Components |
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-Supramolecule #1000: Cropped volume corresponding to 2x1 factor VIII light chain molec...
Supramolecule | Name: Cropped volume corresponding to 2x1 factor VIII light chain molecules from EMD-5540 type: sample / ID: 1000 Oligomeric state: One molecule selected from 15 molecules organized around a 120-Angstrom lipid nanotube Number unique components: 1 |
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Molecular weight | Experimental: 80 KDa / Theoretical: 80 KDa / Method: SDS-PAGE |
-Macromolecule #1: blood coagulation Factor VIII light chain
Macromolecule | Name: blood coagulation Factor VIII light chain / type: protein_or_peptide / ID: 1 / Name.synonym: Hemophilia factor light chain A Details: 15 molecules organized helically around a 120-Angstrom lipid nanotube Oligomeric state: 15 subunits helically organized onto lipid nanotube with a length of 114 Angstrom Recombinant expression: Yes |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Experimental: 80 KDa / Theoretical: 80 KDa |
Recombinant expression | Organism: ![]() ![]() ![]() |
Sequence | UniProtKB: Coagulation factor VIII |
-Experimental details
-Structure determination
Method | ![]() |
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![]() | helical reconstruction |
Aggregation state | helical array |
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Sample preparation
Concentration | 1 mg/mL |
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Buffer | pH: 7.4 / Details: 20 mM Tris-HCl, 150 mM NaCl, 5mM CaCl2 |
Grid | Details: 300 mesh R2x2 Quantifoil grids |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 95 K / Instrument: FEI VITROBOT MARK III / Method: Blot 3.5 seconds before plunging |
Details | The protein was mixed in 1:1 w/w ratio with lipid nanotubes solution |
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Electron microscopy
Microscope | JEOL 2010F |
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Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | Calibrated magnification: 52000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD![]() |
Sample stage | Specimen holder model: GATAN LIQUID NITROGEN |
Temperature | Min: 93 K / Max: 103 K / Average: 99 K |
Alignment procedure | Legacy - Astigmatism: corrected at 400,000 times magnification |
Date | Apr 2, 2010 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 69 / Average electron dose: 16 e/Å2 / Details: Each image was acquired for 1 second. |
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Image processing
CTF correction | Details: particle stacks for each micrograph were corrected for CTF (only phase correction) |
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Final reconstruction | Applied symmetry - Helical parameters - Δz: 7.6 Å Applied symmetry - Helical parameters - Δ&Phi: 0.5 ° Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 15.0 Å / Resolution method: OTHER / Software - Name: IHRS, SPIDER, EMAN2 Details: The final 3D reconstructions was calculated from a set of 2043 helical segments cut off from the selected helical tubes at 256 x 256 pixels with 10% overlap. |
Details | IHRSR, SPIDER and EMAN2 |
-Atomic model buiding 1
Initial model | PDB ID: Chain - Chain ID: B |
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Software | Name: UCSF-Chimera, VMD |
Details | The 3CDZ chain B coordinates were fitted flexibly within the 3D map with the 'fit to volume' option of the UCSF Chimera software. |
Refinement | Space: REAL / Protocol: FLEXIBLE FIT / Target criteria: optimal fit |
Output model | ![]() PDB-3j2s: |