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Yorodumi- EMDB-5559: Cryo-em map of one molecule of factor VIII light chain from helic... -
+Open data
-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 | |||||||||
Map data | 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 | |||||||||
Sample |
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Keywords | membrane binding / factor VIII light chain / helical organization / cryo-EM | |||||||||
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_5559.map.gz | 289.1 KB | EMDB map data format | |
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Header (meta data) | emd-5559-v30.xml emd-5559.xml | 12.2 KB 12.2 KB | Display Display | EMDB header |
Images | emd_5559_1.jpg | 30 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-5559 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-5559 | HTTPS FTP |
-Validation report
Summary document | emd_5559_validation.pdf.gz | 266.7 KB | Display | EMDB validaton report |
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Full document | emd_5559_full_validation.pdf.gz | 266.2 KB | Display | |
Data in XML | emd_5559_validation.xml.gz | 4.2 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5559 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5559 | HTTPS FTP |
-Related structure data
Related structure data | 3j2sMC 5540C 3j2qC 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_5559.map.gz / Format: CCP4 / Size: 330.1 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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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 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. generated in cubic-lattice coordinate | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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
-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: Homo sapiens (human) / synonym: human / Location in cell: blood plasma |
Molecular weight | Experimental: 80 KDa / Theoretical: 80 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, 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 |
-Electron microscopy
Microscope | JEOL 2010F |
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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. |
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 | IHRSR, SPIDER and EMAN2 |
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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. |
CTF correction | Details: particle stacks for each micrograph were corrected for CTF (only phase correction) |
-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: |