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Yorodumi- EMDB-5540: 3D membrane-bound structure of FVIII bound to single lipid bilaye... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-5540 | |||||||||
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Title | 3D membrane-bound structure of FVIII bound to single lipid bilayer nanotubes | |||||||||
Map data | Helical reconstruction of membrane-bound Factor vIII light chain bound to single bilayer lipid nanotubes | |||||||||
Sample |
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Keywords | Coagulation factor VIII / Cryo-electron microscopy / Membrane-bound organization / Molecular modeling / Protein-lipid interactions / Hemophilia A | |||||||||
Function / homology | Function and homology information Defective F8 accelerates dissociation of the A2 domain / Defective F8 binding to the cell membrane / Defective F8 secretion / Defective F8 sulfation at Y1699 / Gamma carboxylation, hypusinylation, hydroxylation, and arylsulfatase activation / Defective F8 binding to von Willebrand factor / blood coagulation, intrinsic pathway / Cargo concentration in the ER / Defective factor IX causes thrombophilia / Defective cofactor function of FVIIIa variant ...Defective F8 accelerates dissociation of the A2 domain / Defective F8 binding to the cell membrane / Defective F8 secretion / Defective F8 sulfation at Y1699 / Gamma carboxylation, hypusinylation, hydroxylation, and arylsulfatase activation / Defective F8 binding to von Willebrand factor / blood coagulation, intrinsic pathway / Cargo concentration in the ER / Defective factor IX causes thrombophilia / Defective cofactor function of FVIIIa variant / Defective F9 variant does not activate FX / COPII-mediated vesicle transport / COPII-coated ER to Golgi transport vesicle / Defective F8 cleavage by thrombin / Common Pathway of Fibrin Clot Formation / Intrinsic Pathway of Fibrin Clot Formation / endoplasmic reticulum-Golgi intermediate compartment membrane / platelet alpha granule lumen / acute-phase response / Golgi lumen / blood coagulation / Platelet degranulation / oxidoreductase activity / copper ion binding / endoplasmic reticulum lumen / extracellular space / extracellular region / plasma membrane Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | helical reconstruction / cryo EM / Resolution: 15.0 Å | |||||||||
Authors | Stoilova-McPhie S / Lynch GC / Ludtke S / Pettitt BM | |||||||||
Citation | Journal: Biopolymers / Year: 2013 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 |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_5540.map.gz | 48.5 MB | EMDB map data format | |
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Header (meta data) | emd-5540-v30.xml emd-5540.xml | 11.6 KB 11.6 KB | Display Display | EMDB header |
Images | emd_5540_1.jpg | 91.3 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-5540 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-5540 | HTTPS FTP |
-Related 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_5540.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Helical reconstruction of membrane-bound Factor vIII light chain bound to single bilayer lipid nanotubes | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 3 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 : Membrane-bound structure of human Factor VIII light chain helical...
Entire | Name: Membrane-bound structure of human Factor VIII light chain helically organized onto single bilayer lipid nanotubes |
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Components |
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-Supramolecule #1000: Membrane-bound structure of human Factor VIII light chain helical...
Supramolecule | Name: Membrane-bound structure of human Factor VIII light chain helically organized onto single bilayer lipid nanotubes type: sample / ID: 1000 / Oligomeric state: 7.5 molecules per 57 Angstrom rise / Number unique components: 96 |
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Molecular weight | Experimental: 89 KDa / Theoretical: 90 KDa / Method: Gel electrophoresis |
-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 / Number of copies: 96 / Oligomeric state: helical / Recombinant expression: Yes |
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Source (natural) | Organism: Homo sapiens (human) / synonym: human / Location in cell: blood plasma |
Molecular weight | Experimental: 89 KDa / Theoretical: 90 KDa |
Recombinant expression | Organism: Cricetulus griseus (Chinese hamster) / Recombinant cell: CHO |
Sequence | UniProtKB: Coagulation factor VIII |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | helical reconstruction |
Aggregation state | helical array |
-Sample preparation
Concentration | 1 mg/mL |
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Buffer | pH: 7.4 / Details: 20 mM Tris-HCl 150 mM NaCl, 20 mM EDTA |
Grid | Details: 300 mesh R2x2 Quantifoil grids |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 106 K / Instrument: FEI VITROBOT MARK III / Method: Blot for 4.5 seconds before plunging |
Details | The protein was mixed in 1:1 w/w ratio with lipid nanotubes solution |
-Electron microscopy
Microscope | JEOL 2010F |
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Temperature | Min: 90 K / Max: 100 K / Average: 99 K |
Alignment procedure | Legacy - Astigmatism: corrected at 400,000 times magnification |
Date | Jul 7, 2009 |
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. |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 52000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: -4.4 µm / Nominal defocus min: -0.7 µm / Nominal magnification: 52000 |
Sample stage | Specimen holder model: GATAN LIQUID NITROGEN |
-Image processing
Details | The 2D analysis was performed with EMAN2 and the helical reconstruction with the IHRSR algorithm |
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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: EMAN2, IHRSR 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. |
CTF correction | Details: EMAN2, only phase correction |
-Atomic model buiding 1
Initial model | PDB ID: Chain - Chain ID: B |
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Software | Name: Chimera |
Details | Protocol: fit in map |
Refinement | Space: REAL / Target criteria: optimal fit |